key: cord-006860-a3b8hyyr
authors: nan
title: 40th Annual Meeting of the GTH (Gesellschaft für Thrombose- und Hämostaseforschung)
date: 1996
journal: Ann Hematol
DOI: 10.1007/bf00641048
sha: 
doc_id: 6860
cord_uid: a3b8hyyr

nan

The variable molecular weight (MW) of vWF is due to differences in the number of subunits comprising the protein. It is assumed that endothelial cells secrete large polymeric forms of vWF and that smaller species arise from proteolytic cleavage. vWF has two main properties: it stabilizes factor VIII protecting it from inactivation by activated protein C or factor Xa, and it mediates platelet adhesion to subendothelium of the damaged blood vessel wall. Each vWF subunit contains binding sites for collagen and for platelet giycoproteins GP Ib and GP IIb/I~a. Multiple interactions of the multivalent vWF lead to extremely strong binding of platelets to subendothelial surface, that is capable of resisting high wall shear rate in the circulating blood. Only the largest multimers are hemostatically active. Lack of the largest vWF multimers was observed in patients with yon WiUebrand disease type 2A. Unusually large molecular forms of vWF were found in patients with thrombotic thrombocytopenlc purpura. Proteolytic enzyme(s) may be involved in the physiologic regulation of the polymeric size of vWF and thus play an important role in the pathogenesis of vWF abnormalities in some patients with congenital or acquired disorders of hemostasis. We have purified (-10,000-fold) from human plasma a vWF degrading proteas¢ using affinity chromatography and gel filtration. The proteolytic activity was associated with a high MW protein (Mr -300 kD). vWF was resistant against the protease in a physiologic buffer but became degraded at low salt concentration or in the presence of 1 M urea. Proteolytic activity had a pH optimum at g-9 and was not inhibited by serine protease inhibitors or sulfl~ydryl reagents. Inhibition by chelating agents was best reversed by barium ions, The observed properties of the vWF degrading enzyme differ from those of all hitherto described pretenses. Analysis of cleaved vWF showed that the peptide bond 842Tyr-g43Met had been cleaved -the same bond that has been proposed to be cleaved in rive. The endothelium releases the vasodilator nitric oxide (NO) and the vasoconstrictor endothelin (ET)-I. NO is formed from L-arginine via the activity of constitutive nitric oxide synthase (cNOS or eNOS). An inducible form of NOS (iNOS) is activated by cytokines. NO activates guanylyl cyclase in vascular smooth muscle and platelets, leading to the formation of cGMP which induces relaxation or platelet inhibition, respectively. In vessels, NO is responsible for endothelium-dependent relaxations; in vivo it exerts a vasodilator tone which can be enhanced by shear forces and receptor-operated agonists such as acetylcholine, bradykinin, thrombin, ATP and ADP. Infusion of NO-inhibitors in vivo leads to vasoconstriction and increases in blood pressure and oral administration to hypertension in the rat. Within the endothelium, NO inhibits ET gene expression and release of the peptide via cGMP. Hence, NO-induced hypertension is associated with increased plasma ET levels. ET, a 21-amino acid peptide, has potent vasoconstrictor properties via ETA-and in part ETB-raceptors on vascular smooth muscle. In endothelial cells, ET activates ETB-receptors linked to NO and prostacyclin formation. Under basal conditions, little ET is formed, but is increased by thrombin, angiotensin II, arginine vasopressin, cytokines and ox-LDL. ET antagonists have been developed and allow to study the effects of ET in vivo. ET and NO most likely play an important role in disease states such as hypertension, atherosclerosis, coronary artery disease, heart failure, pulmonary hypertension and subarachnoid hemorrhage. Clinical trials to further define their role in these disease states are now under way. In summary, the endothelium is an important regulator of vascular tone and structure in vitro and in vivo. In disease states, their interaction is imbalanced leading to enhanced vasoconstriction, thrombus formation and structural changes of the blood vessel wall. Pharmacological tools aiming to inhibit those changes are now being developed.

J.M. Harlan, R.K. W/nn, S. Sharar, and N. Vedder Universit3, of Washington, Seattle, Washington Ischemia-reperfusion injury has been implicated in the pathogenesis of a wide variety of efinical disorders. [n preclinical models, tissue damage .clearly occurs during ischemia, but, parado.,dcally, may be exacerbated during reperfusion.

This reperfusion injury appears to involve activation of the intlammato~, cascade with generation of complement 'components, lipoxygenase products, and chemokines as proximal mediators and neutrophils as final effectors of vascular and tissue damage. We have examined the role of leukocyte adhesion in reperfusion injury in two models -the rabbit ear as a model of isolated organ injury and hemorrhagic shock and resuscitation in the rabbit and primate as a model of traumatic shock and multiple organ failure. Data regarding the efficacy, timing, and safety of leukocyte adhesion blockade using selectin-or integrindirected reagents in these models w/ll be presented. The current status of anti-adhesion therapy in other pre-clinical models and early clinical trials will be re~4ewed. An amidolytic assay for the determination of activated protein C (APC)-resistant factor Va (FVa) has been developed. This assay measures the cofactor activity of FVa in diluted plasma samples via the rate of thrombin formation. The APC response is calculated from two FV determinations: one performed in the presence (APC-FV) and one in the absence of recombinant APC. The APC-FV activity is expressed as percentage of the initial FV activity and indicates the sensitivity of FVa to APC. Normal ranges were established by analysing plasma samples of 100 healthy individuals and an APC-FV activity above 60% was found to be indicative for APC-resistance (APC-R). In a control group of 34 patients the APC-R assay gave abnormal results in 15 patients. DNA analysis confirmed heterozygous FV R506Q mutation in all 15 patients and confirmed the non-carrier status in all of the 19 patients yielding normal results. An aPTT-based APC-R assay performed on the same group of patients showed abnormal results in two of the non-carrier patients. One of these patients was diagnosed as positive for lupus anticoagulant, whereas the reason for the wrong positive result in the second patient remains unclear. Eleven patients were analyzed before start of oral anticoagulation and during oral anticoagulant treatment. Comparison of the assay results demonstrate a correlation of 96% indicating that the assay is independent of the activities of vitamin K-dependent clotting factors. The APC-R amidolytic assays allows specific and sensitive detection of FVa-resistant to APC. The assay is performable in plasma samples of all persons in whom the diagnosis of APC-R may be indicated. In patients treated with oral anticoagulants or showing other clotting abnormalities affecting the aPTT the APC-R amidolytic assay is helpful to establish the diagnosis of APC-R. Dept of Pediatrics, University Hospitals Kiel and Mtinster, Germany Resistance to activated protein C (APCR), in the majority of cases associated with the Arg 506 Gin point mutation in the factor V gene is present in more than 50 % of patients < 60 years of age with unexplained thrombophilia. To determine to what exteut this relativdy common gene mutation affects the risk of thromboembolie events in infants and children, its occurrence was investigated in a population of children with unexplained venous or arterial thromboernbotism: thrombosis of the central nervous system (CNS, n=4), vena portae (n=4), deep vein thrombosis (n=4), vena caval occlusion (n=4), neonatal renal venous thrombosis (RVI'; n--3), neonatal stroke (n=14), stroke (n=3), arteria femoralis ocdusion (n=2). Four ont of these 38 patients showed a positive history of unexplained familial thrombophilia. APCR was measured in an activated thromboplastin time (aFIT) according to Dahlbtick. The results were expressed as APC-ratios: Clotting time obtained using the APC/Caci2 -solution divided by dotting time obtained with CaC12. Concerning the special properties of the childhood hemostatic system, infants and children with APCR < 2 were considered to be APC-resistent only when the results were confirmed in a 1: l 1 dilution with factor V deficient plasma (Instrumentation Laboratory Munich, Germany). Plasma of 268 healthy children served as controls. The Arg 506 Gin mutation of the factor V gene was assayed by amplification of the DNA samples by PCR followed by digestion of the amplified products with the restriction enzyme MUl I. Results were confirmed by SSCP -analysis or by direct sequencing of DNA from patients with APCR. Consistent with the aP'IT based method 10 out of 19 children with venous (V) thrombosis and eight out of 19 patients with arterial (A) vascular insults showed the common factor V mutation. Additional coagulation defects (autithrombin, protein C type I, enhanced antiphospbolipid IgG, enhanced lipoprotein (a)) were found in 30% (V) and 28% (A). Furthermore, we diagnosed exogenlc reasons (septicemia, postpastal asphyxia, fetopathia diabetica, central line and steroid/asparaginase administration) in six out of 10 (V) and three out of 8 (A) children with thrombosis and APCR. All four patients with a positive family history of thrombophilia (mothers only !) showed the common factor V mutation Arg 506 Gin. In the control group the prevalence of APCR was 5.1%. The high incidence of additional exengenic factors in children with APCR confirm literature data of previously described inherited coagulation disorders during infancy and childhood: An acquired risk of thromboembolic disorders masks the coagulation deficiency in the majority of patients with an inherited prethrombotic state. Furthermore, the incidence of 42 % APC resistant children with arterial insults in this study challenge the view that APCR is associated with venous but not with arterial thrombo-st8.

12 ACTIVATED PROTEIN C RESISTANCE AND PLASMINOGEN DEFICIENCY IN A FAMILY WITH THROMBOPHILIA M, Zttger 1 , F. Demarmels Biasiutti 1, Ch. Mannhalter 2, M. Furlan 1 , B.~e 1 1Httmatologisches Zentrallabor der Universit~t, Inselspital, CH-3010 Bern 2Klinisches Institut fur medizinische und ehemische Labordiagnostik, Universit~t Wien, A-1090 Wien Several hereditary defects of the proteins regulating blood coagulation have been associated with familial thrombophilia. Since the recent discovery of activated protein C (APC) resistance due to the factor V Rf06Q mutation as a highly prevalent hereditary risk factor for venous thromboembolism (TEl, evidence is accumulating that familial thrombophilia may be due to a combination of genetic defects. Thus, protein C-or protein S-deficient patients having suffered from TE seem to be more likely to carry the factor V R506Q mutation than expected from its allelic frequency in the population. We report a family (see figure) in which plasminogen deficiency (0.60 U/ml) had been found in the propositus having had twice postoperative deep vein thrombosis (DVT) at ages of 29 and 31 yrs, respectively, as well as in 5 family members (0.53-0.69 U/ml). Out of these 5 plasminogen deficient individuals, only the propositus' daughter had suffered from recurrent DVT at age <20yrs. Reinvestigation of this family in 1995 showed factor V R506Q mutation in the propositus, his daughter, an asymptomatic sister and a brother with postoperative pulmonary embolism (PE). His father had had postoperative PE; he is deceased and could not be examined.

~, [] Plasminogen deficiency ~ ,rll Factor V Rf06Q mutation /~ Propositus ~¢ bistory of DVT and/or PE e superficial phlebitis J2~,,~ not investigated "1" deceased Even though this family is small for establishing unequivocal association of TE with known defects, the two most severely affected individuals with recurrent TE at ages <30yrs had combined plasminogen deficiency and APC resistance whereas those with isolated plasminogen deficiency were asymptomatic. These data support the concept of multigenie interactions leading to familial thrombophilia. Resistance to activated protein C (APC resistance) is the most common dsk factor for venous thrombosis (VT). In most cases APC resistance is caused by a single point mutation at position Arg 506 in the factor V gene (factor V Leiden). While ample data in hetarozygous patients have been published, reports in homozygous patients are limited. We studied 29 patients (12 males [m] , 17 females It]) in whom a homozygous mutation had been verified by DNA analysis. The median age at the time of the study was 38.8 years (y) (range 9-83 y). Twenty-five patients had experienced VT (10 m, 15 f). Four patients were discovered dunng family studies and were asymptomatic, three were children (between 9 and 13 y) and one patient was a 62 y old man. In males the first thrombosis occurred at a median age of 38 y (range 21-82 y), in females this was at a significantly younger median age of 26 y (range 17-49 y). Twelve of the 15 symptomatic females had taken oral contraceptives (OC, estregen content 0.02-0.ling) for 6 to 150 months (median 71 m) pdor to thrombosis. In 2 women VT occurred during pregnancy, in 1 female it was precipitated by hormone replacement therapy. In contrast, in 8,<10 males the thrombosis happened spontaneously, in 2 males it followed surgery. The sites of thrombosis were DVT in 9 males and 14 females, DVT and pulmonary embolism (PE) in 4 females and 1 male, DVT and caval vein thrombosis in 1 female and superficial thrombophlebitis in 4 males and 1 female. Eight females had at least one pregnancy, in total 11 children and 5 abortions. Two had thrombotic events during pregnancy and 2 after delivery. All homozygous patients showed APC ratios between 1.12 and 1.68 (mean 1.39 + 0.19). Conclusion: Patients with homozygous FV Leiden have similar clinical symptoms as patients with deficiencies of antithrombin-, protein C or protein S deficiency. However, in contrast to these defects a very high dsk dudng oral contraceptive medication leading to an ealier manifestation in females can be observed. Several synthetic (efegatran, argatroban, inogatran and napsagatran) and recombinant (hirudin, PEG-hirudin and hirulog) antithrombin agents are in different stages of nlinical development for cardiovascular and thrombotic indications. While the specificity of these agents for thrombin is a concern, little has been done to study the effects of these agents on other serine proteases involved in coagulation and fibrinolytic processes. Fihrinolytic compromise by site directed thrombin inhibitors has been reported recently (Thromb Res 74(3): 193-205, 1994) . While these agents have been shown to inhibit plasmin and related enzymes, little or no informatienentheir effects onthe generation and functionality of APC is available. SinceAPC plays an increasingly important role both as an antienagulant enzyme, by inhibiting factors V and VIII, end a pro-fibrinelytic enzyme, by stimulating the release of t-PA fi'om endothelial sites, an inhibition of APC may result in both pro-coagulant state and fibrinolytic deficit. Representative thrombin inhibitors (DuP 714 -a prototype boronic acid peptide derivative, efega~an, argatroban, hirulog, hirudin and FEG-hirudin) have been compared for their ability to inhibit APC (American Red Cross). These bionhemically defined studies in which the remaining activity of APC after incubation with a thrombin inhibitor was determined speclrophotometrically with a ehromogenic substrate (S-2288, Pharmacia, Franklin, OH) , demonstrated that DuP 714 and efegatran inhibit APC in a coneentrafinn dependent manner (IC~0=0.75 and 8,4 gM resp~tively), hirnlog inhibits APC weakly (60 p2¢I produced only 25% inhibition), while argatroban and ~ have no anti-APC activities, While hirulog, hirudin and argatroban produced no direct enti-APC activities, it is conceivable that they may inhibit thrombomodnlin-bound thrombin and thus prevent activation of protein C, resulting in a functional APC deficit and failure to improve clinical outcomes despite higher dosage. While initially it was thought that sole targeting of thrombin will provide monospacifin anticoagulant agents devoid of some of the adverse effects observed with heparin, the recent clinical trials clearly suggest that thrombin is not the only determinant of thrombogenesis. Furthermore, potent antithrombin agents such as hirudin, hirulog and peptides, indirectly inhibit the generation of APC, by compromising thrombomodulin-bound thrornhin and such agents as efegalran and DuP 714 also produce direct APC inhibition. Endogenous inhibition of formed APe by thrombin inhibitors may therefore compromise the feedback regulatory funetiens of APC and may lead to thrombotic amplification in fully enticoagulated patients. These studies warrant prcelinlcal assessment of thrombin inhibitors to evaluate their relative inhibitory effects on APC. Poor anticoagulant response to activated protein C (APC resistance) causes a significant portion of deep vein thrombosis (DVT) whereas its association with coronary artery disease (CAD) and myocardial infarction (MD is still controversial. Therefore, we investigated 346 recently hospitalised patients suffering from CAD with or without previous MI. The CAD was proven by coronary angiography. APC resistance was analysed by using the aP'l-I'-based assay COATEST APC Resistance (Chromogenix). Eleven patients showed an APC sensitivity index below 2.0 viewed as APC resistance. Using PCR technology, the factor V mutation causing APC resistance 11691 G "-) A) has been shown in nine of these eleven patients. This represents 2.6 % (9/346), compared to 8,5 % found in healthy blood donors (8/94). One homozygous carrier (male, age 76) was identified (APC sensitivity index 1.4) who suffered from DVT at age 59. Recent angiography demonstrated diffuse CAD, No thrombotic events were reported in his family. In contrast, multiple thrombotic manifestations (DVT, MI, stroke) occurred in the relatives of four heterozygous patients. We conclude that the prevalence of APC resistance is rather low in patients with CAD. Nevertheless, the natural history of coronary manifestation of APC resistance seems to vary, probably depending on the presence and severity of cardiovascular risk factors. Resitanee to activated protein C (APC resistance) is the most cormnon hereditary cause of thrombophilia and significantly linked to factor V Leiden PCR based methods are used to identify the crucial point mutation in the factor V g(me. We designed primers in order to identify factor V Leiden by allele-specific PCR amplification. Amplification specificity for factor V was ensured by the 3'primer FV1001, located at the introng/intronl0 border of the g~ae. One sense and two antisense primers were used in ~vo separate primer mixes specific for factor V ArtS06 (wildtypo) or factor V Otn506 (factor V Leiden) yielding 235bp products each. In each PCR reaction a pair of primers amplifying a fragment of the human growth hormone gene was included, fimctioning as an internal positive amplification control (429bp PCRfragment). After an initial denaturation step 10/.tl samples (100rig genomic DNA) were subjected to 10 two-temperature cycles foUowed by 20 threetemperature cycles. For visualisation 8p3 of the amplification product were run on a 2% agarose gel presmined with ethidittm bromide. The presence or absence of specific PCR amplification allowed defiu/te allele assignment without the need for any postamplifieation specificity step. The in~ernal positive control primers it~cate a sucessf-u/PCR amplification, allowing the assignment of homozygosity. In a prospective study 126 p~e.ients with tlaromboembolic events were analysed using this technique and enmpared with PCR -RFLP according to Bertina et al. The concordance between these techniques was 100 %. In 27 patients a heterozygous factor V OhaS06 mutation was detected, whereas one pa6ent with recurrent thrombcembolism was homoz-ygous. No faLse-positive or false-negative results worn observed in the homozygous as well as hcterozygous samples. In addition in 15 samples identified to carry the point mutation by al/ele-specifin PCR anxplification automatic :~equencing confirmed the heterozygous or homozygous point mutation. Due to its time-and cost-saving features allele-specific amplification should be considered for screening of factor V Leiden. Background: An initial intravenous course of tmfractionated heparin ~ljasted on the basis of the activated partial thromboplastin time is the currmt standard treatment for most patients with venous thrombosis. Low-molecularweight hqmrin pre~a~tious can be administered subcutaneously, once or twice daily, without laboratory monitoring. We compared the relative effic.~y and safety of low-molecular weight heparin versus anfractionated heparin for the initial treatment of deep venous thrombosis.

Methods: English-language reports of randomized trials vtta'e identified th~ a MEDLINE search (1984 through 1995) and a complementary extensive manual search. Reasons for exclusion from the analysis were no hepada dosage adjustments, the lack of um of obj~tive tests for deep venous thrombosis, dos~ranging studies that used higher doses of low-molecularweight heparin than are ctareatly in use, and the failure to provide blind endpoint ~sossmeat. We assessed the incidence of symptomatic recurrent vinous thromboembolic disease, the incidence of clinically ii~t bleeding and mortality.

Results: Twelve of the 21 identified trials satisfied the predetermined criteria. The relative risk reductions for symptomatic thromboembolic complicatious, clinically ~t bleeding, and mortality varied firom 20.60% aad were all statistically significantly in favor of low-molecular-wedght hqtmrin.

Coadusions: Low-mol~ular--weight hoparim administered subcutaneously in fixed doses adjusted for body weight aml without laboratory nmaitori~ =re more effective and safea" tlum adjn~_-dose standard h~. sauce low molec~dar weight hqmrim vary in o~apositiou =ad pharma~ogical Im)fil~ the benefits of each ~ shodd ~tabllsbcd separately.

Unfraetionated hcparin (UH) and low molecular weight heparin (LMWH) are widely used for the prevention and treatment of thrombotic disorders. UH and LMWH induce platelet aggregation in vitro. RGD peptides compete with fibrinogm for the binding to the glycoprotein receptor (GP lib-Ilia) of platelets and inhibit platelet aggregation. To inhibit the heparin-indueed platelet ~tion and prolong the half-life in blood of RGD peptides, we linked Ac-RGDV-SSGGS-Ahx-YK eovalently to LMWH in a ratio 1:1. The peptide is composed of three regions: A. RGD-gives the specificity for the receptor GP lib-Ilia; B..SSGGS-Ahx-is the spacer between carrier and ligand, which should facilitate the intnraetion between the conjugate and the GP lib-Ills receptor; C. -YK arc functional antino acids for iodination (Y) and for covalent attachment (K) to the cattier LMWH. The aggregation achieved with different concentrations of LMWH, LMWH-eonjugate and LMWH/RGD-peptide mixture in a ratio 1:1 was mea.~ared after 20 rain.; maximum aggregation after platelet activation with 10 pM ADP was set equal to 100%. Platelet aggregation in normal human plateletrich eitrated plasma (PRP; 220 000/p.l) was induced by LMWH in a dose ~ndent manner. Heparin can induce antbodies which interact with platelets and endothelial cells. This causes thrombocytopenia and thromboembolic complications. HITpatients do need effective parenteral anticoagulation. We treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory prooven HIT (HIPA-Test) with recombinant (r-)hirudin. As these patients had been preseleeted by their immunological response during heparin treatment and the treatment duration of the study was longer than in any other study using thlrudin, all patient samples were investigated for anti-r-hirudin antibodies Himdin antibodies were screened by a sandwich ELISA using r-hirudin fixed to the solid phase as antigen. All plasma samples were screened for anti-Hirudin antibodies of the IgG class, but solar only a suset of samples for lgE anti-r-himdin antibodies. 38 of 82 patients (46.3%) developed anti-hirudin antibodies of the IgG class. Anti-hirudin antibodies were not detectable not before 6 days of r-hirudin administration Solar no IgE anti-hirudin antibodies were found. None of the patients devdoped thromboeytopenia or allergic symptoms. However, in a subset of patients the anti-hirudin antibodies enhanced the anticoagulatory effect of r-hirudin. In 5 patients the Hirudin dosage had to be decreased by 2-3 fold to maintain a stable aPTT level, in 3 patients, despite stable r-hirudin maintenance dose the aPTT increased to values >100 see.. During the study 4 patients with anti-hirudin antibodies had to be reexposed to a second course of r-hirudin for parenteral anticoaguhtion None of these patients developed any allergic reaction. In conclusion we found a high proportion of anti-hirudin antibodies in HATpatients treated with r-hirudin for more than 7 days. These antibodies seem to have minor clinical relevance in regard to allergic reactions. However, one has to consider that these antibodies may influence the pharmacokinetics of rhimdin and thereby enhance its antieoagulatory potency. Therefore, aPTT must be monitored closely in patients receiying r-hirudin for more than 5 days A major concern in the use of hirndin, the most potent and specific thrombin inhibitor, is the risk of bleeding associated with the potential effect of this drug on hemostasis, particularly when the antithrombotic therapy is combined with invasivo procedures, fibrinolytic treatment, or patient's predisposition to abnormal bleeding. Thus, availability of an antagonist to hirudin would be essential for instant neutralization of the antithrombotie action. However, thueh a hirudin antagonist is unknown in nature. To prepare an antagonist to hirudin, a mutant derivative of human prothrombin, in which active site aspartate at position 419 is replaced by an asparagine, has been designed, expressed in recombinant chinese hamster ovary cells, and purified to homogeneity. D419N-prothrombin was converted to the related molecules D419N-meizothrombin and D419N-thrombin by limited proteolysis by E. carinatue venom and O. scvutellatus venom, respectively. Both D419N-thrombin and D419Nmeizothrombin exhibited no thrombin activity and titration resulted no detection of the active site. However, binding to solid phase immobilized hirudin and fluorescence studies confirmed that the binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins, hi vitro examinations showed that D419N-thrombin and D419Nmeizothrombin bind to immobilized hirudin, neutralize hirudin as well as in the purified system and in human blood plasma and re-activate the thrombin-hirudin complex. Animal model studies confirmed that D419Nthrombin and D419N-meizothromi.,in act as hirudin antagonist in blood cireulatlon without detectable effects on the coagulation system. While i.v. injections of hirudin in mice resulted in an increase in partial thromboplastin time, thrombin time and anti-thrombin potential, additional injections of D419N-thrombin and D419N-meizothrombin resulted in a normalization of these coagulation parameters. Elevation of plasma homocysteine is a hereditary disorder of methionine metabolism associated with a high risk of arterial vascular disease. However, as yet relatively little attention has been directed towards the association between hyperhomocysteinemia and juvenile venous thromboembolism (VTE). Consequently the aim of our study was to evaluate the prevalence of hyperhomocysteinemia (hyper-HCYS) and juvenile VTE. Patients: 85 patients (29 men, median age 42 ys; 56 women, median age 35 ys) who had at least one verified episode of VTE before the age of 45 ys were investigated in regard to their total plasma HCYS levels. None of the patients had renal or liver dysfunction or evidence of any autoimmune or neoplastic disease. Methods: Plasma total homocysteine levels were determined by HPLC with fluorescence detection. Hyperhomocysteinemia was defined as HCYS levels exceeding the upper limit of the normal range obtained in our laboratory from 80 healthy control subjects (40 males, median age 25 ys, HCYS 95% CI: 2.02-5.67 pmol/l; 40 females, median age 27.5 ys, HCYS 95% CI: 2.99-5.40 ,gruel/l). Resuits: 13 out of 85 patients had hyper-HCYS, giving a prevalence of 15.3 %. Of these 13 patients, 9 were male and 4 female, indicating that the relation between elevated plasma HCYS levels and VTE may not be as strong in woman as in men. Discussion: According to previous reports, our study shows that there is a high prevalence of hyper-HCYS in patients with juvenile VTE. However, the mechanisms by which hyper-HCYS can provoke VTE and whether HCYS is an exclusive risk factor or if it contributes to other existing predispositions, possibly working as a trigger factor is unknown yet. Some authors suggest HCYS-iaduced effect on factor V activation or inhibition of thrombomodalin-dependent protein C activation. In addition an influence on thrombocyte aggregation has been postulated. Conclusion: Measurement of HCYS levels may be useful in the evaluation of patients with a history of juvenile venous thromboembolism and could be clinically important as hyper-HCYS is easily corrected by vitamin supplementation. Detailed determination of the pathogenesis of VTE in patients with hyper-HCYS should be the aim of further investigations. A deficiency of one of the coagulation inhibitors antithrombin (AT), protein C (PC) or protein S (PS) and resistance to activated protein C (APC resistance) are established risk factors for venous thromboembolism (VTE). In the majority of patients with APC resistance, the .tug 506 Gin mutation (Factor V Leiden) is present. Whereas deficiencies of one of the coagulation inhibitors are rare in the normal population, the allele frequency of factor V Leiden is 2-7% in Western Europe. Heterozygous individuals have a 3-7fold, homozygous an 80fold increased risk for VTE The typical clinical features of all abnormalities are deep vein thrombosis, pulmonary embolism, superficial vein thrombosis and thrombosis at unusual sites, like mesenteric vein thrombosis or cerebral vein thrombosis. The thrombotic risk is low during childhood, but increases considerably after the 13th year of age. A retrospective study in adult patients out of families with a symptomatic deficiency of AT, PC or PS revealed that around 30% of surgical interventions and traumas of the lower extremities were complicated by VTE. Therefore, these patients should receive thrombosis prophylaxis al~er surgery and trauma if their age is higher than 13 years. Pregnancy is associated with a very high risk for VTE in individuals with AT deficiency and prophylaxis should be initiated already in the first trimester. After delivery, thrombosis prophylaxis is adviced for all females known to have an abnormality. OC increase the risk, especially in AT deficient and in homozygous factor V Leiden females and are therefore contraindicated in these individuals. OC do also increase the risk for VTE in patients heterozygous for factor V Leiden and females known to have this abnormality should be discouraged from taking OC or should at least be informed on their increased risk. 

University Hospital-', Jerusalem, Israel, Hospital Bcan.iou ~. Paris, France, Increased frequency of thrombocmbolie events have been observed iu patients with B-thalassemia. Our findings of shortened platelet survival and enhanced urinary excretion ofthmmboxanc A: metabolitcs (Blood 77:1749 (Blood 77: , 1991 suggested an increased platclet activation in tbese patients. We also fouud that isolated thalassemie RBC enhance prothronlbin activation, suggesting an increased membrane exposure of procoagulant phospholipids i.e, phasphatidylserine (Am J. Hematol. 44:33, 1993) . We now show that annoxin V, which has a high specificity and affinity for anionic phospholipids inhibits pmthrombm activation by Factor Xa, by binding to thalassemic RBC (IC~, = 0.32 nM). Kerckhoff-Klinik, Bad Nauheim ~, Medizinische Poliklinik Bonn 2, Institut for Immunologie und Transfusionsmedizin Universit~ll Greifswald a Antibody-mediated intravascular platelet activation is believed to be the basis for both arterial and venous thrombosis in patients with HAT. While the development of arterial thrombosis can explained sufficiently by intravascular platelet activation, it is a matter of discussion whether additional risk factors are involved in the pathogenesis of HAT-related venous thrombosis. Since resistance to activated protein C (APC) is the most common inherited risk factor for venous thrombosis described the frequency of APC resistance among a population of 68 HAT patients has been studied. HAT was diagnosed using the heparin-induced platelet aggregation assay and confirmed by the ~4C-serotoninrelease test. The diagnosis of APC resistance was established by two functional assays and genetic analysis. At time of diagnosis of HAT, 38 patients showed venous thromboembolic complications. Among these, 24 were found positive for APC-resistance. Pulmonary embolism was diagnosed in 18 HAT patients, 14 of them were APC resistance positive. None of the 18 HAT patients who showed exclusively thrombocytopenia were APC resistance positive. Early oral anticoagulation (OA) was initiated in 7 patients after the diagnosis of HAT has been established. Six of these patients developed serious thrombotic complications including skin necrosis. These results demonstrate that APC resistance is an additional and common risk factor for the development of HAT-related venous thrombosis. Early initiation of OA during an acute episode of HAT dramatically increases the risk of thrombosis. Therefore, OA in HAT patients should be initiated only after platelet counts have been returned to baseline levels and effective parenteral anticoagulation is achieved.

CONTROLLED TRIALS FOR PRIMARY AND SECONDARY PREVENTION OF STROKE G. de (3aetano, C. Cerletti and V. Bertel~ Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy This presentation will review the antithrombotic treatments to prevent ischemic stroke that have been evaluated in controlled clinical trials. In two studies of aspirin therapy for pdmary prevention in male physicians there was no reduction in the incidence of stroke, while that of first myocardial infarction was significantly lowered. Similar results were obtained in a prospective study in a large cohort of women taking aspirin daily. The incidence of vascular death was not modified by aspirin in any of these trials. This is possibly due .to an excess of strokes associated to aspirin treatment: indeed the four vascular events avoided in 1000 US physicians under aspirin prevention for five years would result from five myocardial infarction and one vascular death avoided and two additional strokes occurred. Oral anticoagulant therapy decreases the relative risk of stroke in patients with non valvular atrial fibrillation. Warfarin appears to be superior to aspirin, but the latter drug is a useful alternative when long-term anticoagulant therapy cannot be administered. A metanalysis of about 150 trials and over 100,000 patients with different vascular diseases treated with aspirin (at different doses) and/or other platelet inhibitors showed 25% overall reduction of vascular events including stroke. The optimal dose of aspirin for secondary stroke prevention could not be established. In patients with previous minor strokes or TIA there was 22% reduction of vascular events and 23% of non fatal strokes. The avoidance of nine strokes of any cause among the 38 expected in 1000 patients at risk would result from the sum of 10 ischemic events avoided and a haemorrhagic one occurred in excess. Ticlopidine was reported to reduce the risk of stroke in two large tdals (one in patients with major stroke), but there is no evidence that it is better or safer than aspirin. We compared the effect of the direct specific thrombin inhibitors, napsagatran (NA) and rec. hirudin (rH) with unfractionated heparin (UH) on the further growth of preformed thrombi. As a model of thrombogenesls, an annular perfusion chamber exposing rabbit aortic subendothelium was perfused with native rabbit blood at an arterial wall shear rate (650/s). Fibrin and platelet thrombi were allowed to form during a 10min perfusion period after which the test agents were given iv as a bolus and a continuous infusion (3 and 10pg/kg/min, n=6) and the perfusion continued for 20min. The control groups were perfused for 10 or 30 rain (n=6). Fibrin deposited and platelet thrombi formed on subendothelium were evaluated by microscopic morphometry. The % surface coverage with fibrin was not reduced in the drug-treated groups since fibrin deposition was similar in the 10 and 30 min control groups (39+8% and 335:6%, respectively, mean:l:SEM). Platelet thrombus area (TA) in the control groups increased from 24+7pm2/pm after 10min to 97+32pm2/lim after 30rain perfusion. NA at 1011g/kg/min reduced TA by 94% to values (6+_2ptm2/ ~m) lower than those of the 10min control group whereas rH at this dose reduced TA by 70% (30-J:141.tm2/I.tm). UH at both doses was ineffective. These findings show that in contrast to UH the direct thrombin inhibitors NA and rH inhibit the growth of preexisting thrombi. These results could be explained by the higher potency of NA and rH as compared to UH for inhibiting clotbound thrombin (Gast et al., Blood Coagul Fibrinol 1994, 5.' 879-87) and suggest that thrombus-bound thrombin is an important modulator of platelet thrombus growth and/or stability in this thrombosis model. Platelet adhesion -the initial event of thrombosis -is believed to be completely prevented by intact endothelium. We challenged this theory by superfusing intact human umbilical vein endothelial monolayers with activated human platelet rich plasma utilizing the Stagnation Point Flow Adhesio-Aggregometer (SPAA). The SPAA provides flow mediated contact of platelets with the superfused surface. Heparinized (3.5 -4.0 U/ml) platelet rich plasma (PRP) was obtained from healthy volunteers and activated by addition of adenosine diphosphate (ADP 2"10 -6 M). Platelet deposition was recorded on-line by video as well as by measuring scattered light. Fixed samples were examined by phase contrast and electron microscopy, Inhibition experiments were performed with either the tetrapeptide RGDS, the non-peptide GPIIb/llla-inhibitor Ro-43-8857 or a monoclonal antibody directed against the GPIlb/llla complex. Stimulation with ADP prompted platelets to adhere to intact endothelium single or as microaggregates of a diameter of up to 100 micrometer. Adhesion was dependent upon convective transport resulting in platelet collision with the endothelial monolayer. Infusion of RGDS or Ro-43-8857 into the flowing, ADP-stimulated PRP completely prevented platelet adhesion to the endothelium as well as subsequent aggregation. When the inhibitor inflow was stopped while ADP stimulation persisted, adhesion and aggregation occurred immediately. Re-establishing the inflow of the inhibitors -with still continued ADP stimulation -led to disintegration of the adhering aggregates. When PRP preincubated with the monoclonal antibody against GPllb/llla was superfused, platelet adhesion to the endothelium and aggregation were irreversibly blocked. Our results suggest that convective transport and stimulation of platelets are prerequisites to overcome endothelial thromboresistance and that subsequent platelet adhesion to the endothelium is mediated via the platelet GPIlb/llla receptor complex.

PREVENT THROMBUS FORMATION AFFER PTCA I.P. 8tepanova t, G.V. Bashkov 2, l.P.Kapralova, 2 S.P. Domogatsky ~ Cardiology Research Center t and National Haematology Scientific Cettter 2 Russian Academy of Medical Sciences, Moscow, Russia Percutaneous transluminal coronary angioplasty (PTCA) results in atheroselerotie plague rapture, vascular wall damage and thrombogenic collagen exposure. Subendothelial collagen type I-lll is a very ~rong agonist of platelet-dependent thrombus formation in arteries. The anlithrombotic action of rabbit polyclonal antibodies to rat collagen type I-ill and their chemically synthetized conjugate with monoclonals to human recombinant two-chain/one-chain urokinase type plasminogen activator (rtcu-PA/rseu-PA), cross reacting with rat tcu-PA/scu-PA was studied both an in vifro and in vivo. Anticollagen antibodies and bispecific conjugate inhibited human platelet adhesion, aggregation and formation of thrombi-like ~ructures induced by rat collagen immobilized with the polystiroi surface in a condition mimics the high shear rate in the large elastic-type arteries.

The short-term treatment of the collagen-soaked silk thread by the collagen antibodies suppressed the platelet-dependent thrombus formation in the arterio-venous shunt in rats by 56_+4 % (P<0.001) as well as by the bispecific conjugate (44_+4%, P<0.001). The treatment of collagen-adsorbed conjugate by rtcu-PA did not increase the autithrombotic effect of bifunctional antibodies.

The present date suggest, that the local administration of the anticollagen antibodies at the site of atherosclerotic plague rapture may tm the efficient tool for prophylaxis of platelet-dependent thrombus formation in arteries after PTCA. Increased levels of certain hemostatic factors have been shown to be related to an increased risk of cardiovascular events. Hypercoagulability is suggested to predispose to arterial thrombosis and thereby to participate in atherogcncsis. We therefore assessed fibrinogen, prothrombin fragment 1+2 (FI+2) and yon Willebrand factor (vWf) antigen in 80 consecutive patients (aged 59+5 years) with known coronary artery disease (CAD) who all underwent coronary angiography. The extent of coronary artery disease was quantified according to modified criteria of the American Heart association (total, proximal and distal "score"). Furthermore the intima-media thickness (IMT) was determined in the carotid and femoral arteries by standardized ultrasonographie measurement, vWf antigen was found to correlate positively with the total and proximal coronary score (r=029, p<0.05 and r=O.36, p< 0.01). While FI+2 showed no correlation with the coronary scores, it was significantly correlated with the IMT values in the carotid arteries (r=0.27. p< 0.05). After differentiating tertiles of the parameters patients belonging to the upper tertile of FI+2 concentrations had significantly higher IMT values of the carotid and femoral arteries (0.81_+0.11 mm vs. 0.72+_0.13 mm in the lower tertile, p<0.05:1.38_+0.44 mm vs. I. 15-+0.25 ram, p=0.05) whereas in patients belonging to the upper tertile of vWf antigen concentrations the proximal coronary artery score was significantly higher (!.71-+0.59 vs. 1.31+ 0.92 in the lower fertile, p<0.01). Fro correlation of fibrinogen concentrations and extent of CAD or IMT values of the carotid and femoral arteries could be demonstrated. In conclusion procoagnlatory mechanisms as indicated by elevated concentrations of yon Willehrand factor antigen and FI+2 may be contributing factors in atherogenesis. We have previously shown that PGEI is a potent inhibitor of PDGF-ioducod proliferation of vascniar smooth muscle cells (VSCM) and inhibits DNA replication by a cAMP-related mechanism (Grol~er et al, 1994) . The present study investigates of whether or not this aatimitogeni¢ activity of PGEt can be amplified by trapidil, a compound that has been shown recently to inhibit the incidence of restenosis of hmnan coronary arteries subsequenmt to PTCA (Maresta et al. 1994) , VSMC were prepared from coronary arteries of adult bovine hearts, passagod and kept under standard tissue culture conditions. Cells of passage 4-9 wore incubated in serumfree medium for 24h in the presence of indomethacin (3 p.M). Addition of PDGF-BB (10 ng/ml) under these conditions stimulated DNA-replication as assessed from 'Hthymidin lncm'poration, by 3.-4laid above control level.

Trapidil at 10 idvl caused a minor reduction of PDGF-induced mitogenesis whereas 10t) of the compound resulted in a marked reduction of DNA replication by 69% (P < 0.05, n = 4). PGEI at 0.3 nM diminished the incorporation rate by t 1% while the simultaneous administration of both PGED and trapidil (100 idyll caused a significantly stronger response as seen from n reduction of ~H-thymidine incorporation rate by 82% (P < 0.05, n = 4). As a possible mechanism of action, trapidil might have inhibited phosphodiesterases. To establish this, we measured the cAMP-depcudont proteinkiaasc (PK) A activity in cell homogenates. Trapfdil increased the basal FKA-activity from 19% tO 31% of the maximum reSpOnse while the response to PGEt (10 aM) amounted to 55%. Coincubation of PGEI with trapidil caused a 65% stimulation of PKA activity, sugesting a small though detectable inhibition of VSCM phosphodiesterases by trapidil at anttmitogenic concentrations. Essentially similar results wore obtained when thrombin was used as the mitogenic agent.

The data demonstrate a significant antimitogenic effect of trapidil at p.molar concentrations that are in the range of plasma levels after therapeutic administration of the compound in rive. At these concentratrations, PGEt induced inhibition of mitogenesis is markedly enhanced by trapidil. Inc. I~enna, and ~Cenlral Itematnlogy Laboroto~. , University Hospital of Bern Pibrinogen (Fg), yon Willebrand factor antigen (vWF) and tissue-type plasminogeu activator antigen (l-PAl have recently been shown In be independent risk factors for subsequent coronary events In patients with angina pectoris (NEJM 1995; 332:635) Although PAUl antigen has also been proposed as a risk factor, conclusive dam showing its predictive value is still lacking. Furthermore, we have recently shown in a study investigating 200 survivors of myocardial infarction that not only are Fg, t-PA and PAI-I significantly increased in these patients when compared to a heahhy conlro[ group, but PCI activity is also elevated (7hrornb. tfaemost. 1995;73:1137 abst.) , hi order to obtain cut.off points for the individual parameters, frequenCy histogram plotl; were transformed into straight line cumulative frequency (probit) plots (Thromb I/aemost. 1995;74:718) . The cut-off valu~ for the four parameters were determined as follows: Fg at 2.7 g/L, t-PA at 8.7 ng/mL, PAl-I at 25 ng/mL and PC[ at 126% of a normal pooled plasma. Utilising there cut.off points it was then possible to determine the accumulative discriminatow effectiveness of the parameters. When Fg w;qs employed alone as the discriminatow factor, it was observed that 47% (941200) of the coronary heart dir, ease (CHD) group eilher had the cul..off value or were below it aud 29% (29199) of the normal group were above the cut-off value, thus, resulting in 47% false ne$atives and 29% false positives. When a second additional risk factor, t-PA wa_~ introduced, the number of false negatives dropped to 21% [i.e. 79% (158/200) had two, risk factors elevated] and the number of false positives to 13% To investigate whether a third parameter could discriminate further, PAI-I antigen was used to analyse the rcnudning false positives and negatives. An additional 4% could be detected, resuhing in 83% of the CHD group having three risk factors elevated. Similarly, the number of normal aubjecta with three parameters elevated dropped by 4% to 9% Furthermore. when a fourth parameter was introduced, namely PCI, it was round to discriminate a further 8% in the CHD group, thereby increasing tile di~riminalion to 91%. The number of false positives dropped to 4%, Additionally, determination of PCI increased the discrimination of patienta having had multiple infarctions from 88°/= when thrce parameters were mcasured to 100%. From these results it can be concloded that determination of fibrinogen levels alone is not sumcicnt to separate patients from controls as t-PA adds significant discrimination. PAI-I antigen which correlated strongly with t-PA did not significantly increase the discriminatory potential of both Fg and I-PA. However, by employing PCI as a fourth paramctcr, virtually complete separation between the CHD and normal groups as well as rurthcr recoguitiou of' patients having had multiple infarctions could be obtained. To test the hypothesis that oral contraceptives (OC) enhance exercise-induced activation of blood coagulation we examined 11 women (27 + 3 (SD) years, BMI 20.4 + 2.0 kg/m 2, VOzm.. 49 + 7 ml/kg/min) without OC between day 18 and 22 of the menstrual cycle and 10 women (24 + 2 (SD) years, BMI 20,6 ± 1,6 kg/m', VO2max 47 + 7 ml/kg/min) taking OC (150 mg Desogestrel and 30 mg Ethinylestradiol) between day 7 and 21 of drug intake. Prothrombin fragment 1 +2 (PTF1 + 2) and fibrtnopeptide A (FPA) were measured before and after running for one hour on a treadmill at a speed corresponding to the anaerobic threshold. Mean heart rate [191 ± 10 vs. 196 ± 12 min 1) and mean plasma lactate (3.3 ± 1.6 vs. 3.1 + 1.2 mmot/I) wera comparable during exercise between control and OC group, respectively. Results for markers of thrombin and fibrin formation were: PTF1 +2 (nmol/I) FPA (ng/ml) control before 0,66 ± 0.19 1.0 + 0.2 after 0.73 + 0.23 2.2 + 1.2" OC before 0.82 + 0.31 1.0 + 0.2 after 1.11 + 0.48* + 5.8 -+ 6.0* + * p < 0.05 vs. baseline, + p < 0.05 between groups.

We conclude that oral contraception with 150 mg Desogestrel and 30 mg Ethinylestradiol enhances exercise-induced thrombin and fibrin formation, Our data suggest that exercise testing might be useful for evaluating the risk of thrombosis associated with different compositions of OC.

A. Haushofer +, WM. Halbmayer +, J. Radek +, M. Dittel *, R. Spiel *, H Prachar *, J. Mtczoch *, M. Fischer + + Zentraltaboratorium mit Thrombose-und C~rinnungsambulanz -Krankenhaus Lai~: * 4. Medizinische Ab[eilung mtt Ka~liolo$i¢ -Krankenhaus Lainz und Ludwig Bottzmann-lnstitut ftlr Herzinfarktforsohung, Wien Fifty-one patients (age 61.6 ± 9.2a; 34 m / 171) implanted with 74 coronary stems 03 Palm~-Schatz, 27 Gianturco-Roubin, 14 Micro Stcnts) received a now antithrombotic treatment using a combination of Ti¢lopidine (TIC) 2 ×250 mg/d for 28 days and acetyl salicylic acid (ASA) ZOO mg/d for long-term treatment. Patients (PAT) only received 15000 tU standard hepartn as i.v. bolus immediately before stent implantation (day l ). Side effects and changes in hematological (day I to 7, 14. 28 and 35 [= without T[CIL liver and kidney parameters (day 7, 14, 28, 35) were monitored. Thirty-eight PAT (75%) came for the controls to our del~rtment and were additionally monitored by thromboelastograpy (TEG) and bleeding time (BT) (day 2g and 35). The other PAT were monitored externally, side effects were reported. Thrombin geucration after stenting was monitored from day I to 7 by prothrombin fragment 1+2 (F 1+2) and thrombin-antithrombin-lll-comptex (TAT). "K" of the "lEG decreased (day 28 vs 35; p< 0.0l ). BT prolongation was negatively correlated with the bodysurf ace area (TIC+ASA: p< 0.05, ASA: p< 0.0 l) and showed a reduction after withdrawal of TIC (2 l0 sec, 180/ 300 so: [Median, Quartiles] vs. 120 sec, 881157 sec; p< 0.ix)01). F 1+2 and TAT of day I (blood collection: 0, 2, 4, 6 h after intervention, F 1+2:0.84 nmol/I, 0.68/I.07 nmol/l: TAT: 2.6 pg/1, 2.0/4.6 Ilg/1) were lower compared to day 2 to 7 (F I +2: 1.31 nmol/l, ].08/I .62 nmol/l; TAT: 4.8 pg/I, 3.2/7.2 IJg/1; p< 0.0001). TIC scorns not to be a strong thrombin generation inhibitor. During stenting one PAT (I.9%) sustained a non penetrating MCI and one (1.9%) an ischaemic stroke. TIC+ASA were very effective, only with one PAT (1.9%) stent thrombosis (acute) occurred. Side effects: 8/15.7% gastrointestinal (one lead to hospitalization), 5/9.8% hematomas at the needle site in the groin (one surgical intervention), 5/9.8% leucopcnias (one agranulozytosis with hospitalization), 3/5.9% allergic skin reactions and 2/3.9% increased liver enzymes (GOT, GPT, "pGT, alkaline phosphatase; > 2 × of the J. ). With one PAT with gastrointestinal disturbances and skin reactions TIC had to be withdrawn and treatment was changed to oral anticoagulatlon + ASA. One PAT showed a combination of skin reactions, gastrointestinal distufl~aneas and on day 28 a heavy reaction of the liver enzymes ( J. after 5 weeks). A decrease of the white blood count (day 1:7.19 GH, 6.03/8.2 G/l, day 28:5.46 G/l, 4.63/6.47 G/l; p< 0.000 I) could be observed. The safety of the therapy with TIC+ASA should be elucidated and extensively discussed. The serpins C1 esterase inhibitor (Cllnh), antithrombin III (ATIII), alantitrypsin (slAT), and a2-antiplasmin (aZAP) are known inhibitors of coagulation factor Xla (FXla). Although initial studies suggested al AT to be the main inhibitor of FXla, we recently demonstrated Cllnh to be a predominant inhibitor of FXla in vitro in human plasma (Wuillemin et el., Blood 1995; 85:1517) . The present study was performed to investigate the plasma elimination kinetics of human FXla-FXla inhibitor complexes injected in rats. The amounts of complexes remaining in circulation were measured using ELISAs. The plasma tl/2 of clearance was 98 min for FXla-alAT complexes, whereas it was 19, 1 8, and 1 5 min for FXla-Cllnh, FXla-a2AP, and FXla-ATIII complexes, respectively. Thus, due to this different plasma tl/2, preferentially FXla-alAT complexes may be detected in clinical samples. This was indeed shown in plasma samples from thirteen children with meningococcal septic shock (MSS), a clinical syndrome which is complicated by activation of coagulation, fibrinolytic, and complement systems. FXla-FXla inhibitor complexes were assessed upon admittance to the intensive care unit. FXla-a 1AT complexes were elevated in all patients, FXla-C11nh complexes in nine, FXla-ATIII complexes in one patient, and no elevated FXla-a2AP complexes were found. We conclude from this study that, (1) although C1 Inh is the predominant FXla inhibitor, FXla-alAT complexes may be the best parameter to assess activation of FXI in clinical samples, (2) measuring FXla-FXla inhibitor complexes in patient samples may not help to clarify the relative contribution of the individual serpins to inactivation of FXla in rive, and (3) FXl is activated in patients with meningococcal septic shock. Dudng the coagulation of plasma about 20 % of the (x2AP present is covalently crosslinked to fibrin by factor XIIla (Aoki und Sakata 1980, Thomb. Res. 19: 149-155) . We investigated the binding of azAP by factor XIIla to soluble fibrin (desAABB-fibdno) whose polymerization was inhibited by an isolated fibrin Ddomain named D=,,, (Haverkate and Tiemann 1977, Thromb. Res. 10: 803-812) . D==. is known to have an intact fibrin-polymerization site and is able to block the prolongation of the fibrin protofibrils at an early stage depending on its concentration. Lateral association to fibrin fibers does not take place, since the inhibited protofibnls formed at the conditions used here do not reach a sufficient length (Williams et el. 1981, Biochem. J. 197: 661-668; Hantgan et al. 1983, Ann. N. Y. Acad Sci. 408: 344-365) . Material and method: Soluble desAABB-fibrino was prepared by incubation of (lZtl)-fibrinogen (0.48 mg/ml), D=1o (1.91 mg/ml; molar ratio D==o to fibrin 14:1) and 0.4 U/ml thmmbin for 45 min. Then Q2sl)-c~2AP (16 p.g/ml), faktor ×Ill (2 Ulml) and Ca 2) (5 mmol/I) were added. The crosslinking reaction was stopped at different times of factor XIIla-incubation by adding of urea/EDTAsolution. The suspension was analysed by ultracentrifugation on gradients containing saccharose, urea and SOS. Re~ultl: The elution profiles of the ultrecentifugation-gradients show the formation of cmsslinked fibrin oligomers of increasing size depending on the time of Factor XIIla-action. The crosslinked fibrin polymers contained about 80% of the fibrin initially added. Although Factor XIIla acted well, crosslinking of azAP In the fibrin oligomers could not be observed. Conclusl0n: As we already demonstrated (Kelach et el. 1994, Ann, Hematol. 70 (suppll) : A 60) the crosslinking of azAP to fibnn clots depends on the structure of the fibdn network, especially on the degree of lateral association of the fibrin pmtofibdla. In desAABB-fibrino no lateral association of fibrin protofibnls takes place under the conditions chosen here. Thus it is consistent with our theory that we did not observe any binding of aiAP to the fibrin oligomers of desAABB-fibrino. Human PCI is a non-specific serpin that inhibits several proteases of the coagulation and fibrinolytic systems as well as tissue kallikrein and the sperm protease acrosin. It is synthesized in many organs including liver, pancreas, and testis. The physiological role of PCI has not been defined yet. Recently, we have cloned and sequenced the mouse PCI gene (Zechmeister-Machhart etal., manuscript in prep.) . This enabled us to study PCI gone expression in murino tissues using mouse PCI eDNA and cRNA probes. By Northern blot analysis, mouse PCI taR.HA was exclusively found in the reproductive tract (testis, seminal vesicle, ovary), all other organs analyzed -including the liver were negative for PCI mKNA, indicating that in the mouse PCI is not a plasma protein. To determine which cells of the reproductive tract synthesize PCI, cellular localization was assessed by in situ hybridization of mouse testis and ovary sections. In testis, PCI mRNA was present in the spermstogonia layer and in Leydig cells, while Sertoli cells and peritubular myoid cells were negative. These results are consistent with the immunohistological localization of human PC1 (Laurell et al,, 1992) . In the mouse ovary, stroma cells of the medulla and around the follicles were positive for PCI mRNA. No PCI expression was detected in theca or granulosa cells. We also studied the regulation of mouse PCI gone expression by steroid hormones in vivo. [n mature male mice castration caused an increase in PCI mRNA in seminal vesicles, which was reversible upon the administration of testosterone. In tissues of intact adult male and female mice, PCI mRNA levels decreased after injection of human chorionic gonedotropin (hCG), while in castrated male mice, hCO had no effect on seminal vesicle PCI mRNA. Progesterone and 17-B estradiol decreased ovarian PCI mRNA levels in immature female mice. These data suggest direct down-regulation of mouse PCI by sex steroids. The different tissue specific PCI-geoe expression in men and mice furthermore indicates a different biological role of this serpin in the two species. Ctr. Transgene Technology, Leuven "[' 1-Tissue factor ('IT) is a 47 kDA glycoprotein mainly known a the primary cellular initiator of blood coagulation. Whether TF expression may also play a role in development is unknown, but the lack of spontaneous viable mutations of the TF gene in rive leads to the speculation that its absence may not be compatible with normal embryonic development.

To determine the significance of "IF in ontogenesis, the pattern of TF expression in mouse development was examined and compared to the 'IF distribution in human postlmplantation embryos and fetuses of corresponding gestational age. At early embryonic period of both murine (6.5 and 7.5 pc) and human (stage 5) development there is a strong TF expression in both ectodermal and entodermal cells. "IF decoration was seen during ontogenetic development in tissues such as epidermis, myocardium, bronchial epithelium, and hepatocytes, which express "IF in the adult organism. Surprisingly, during renal development and in adult organism TF expression differs between men and mice. In humans maturing stage glomerali were "IF positive whereas in mice glomeruli were negative and instead epithelia of tubular segments were TF positive. In ncuroepithelial cells there was a striking 'IF expression indicating a possible role of'IF in neumlation. Moreover, there was a robust TF expression in tissues such as skeletal muscle, and pancreas, which do not express in adult. In contrast to TF, its physiologic ligand factor VII was not expressed in early stages of human embryogenesis, but was detectable in fetal liver, The temporal and spatial pattern of TF expression during murine and human development support the hypothesis, that 'IF serves as an important mo~hoJzenic factor darinz embrvozenesis. To serve as an anticoagulant, protein C (PC) must be activated by a complex formed between the enzyme thrombin (T) and its cofactor thrombomodulin (TM). Therefore, downregulation of endothelial cell surface expressed TM, for example, triggered by an inflammatory stimulus, could become a critical factor in effective PC activation. In order to develop a recombinant (r) PC mutant which can be activated independently of the TKM-complex, a peptide sequence including P1-7 in the activation peptide of PC was modified to be identical to the factor Xa (FXa)-cleavage site in prothrombin. The mutant was expressed in HU 293 cells, purified and its anticoagulant properties characterized. Using purified FXa the mutant showed activation rates between 0.02 and 0.13 nMlmin at PC concentrations between 97 and 770 nM, while the rPC wild type was insensitive for FXa activation. The activation reaction is calcium-dependent reaching maximal activation rates at a calcium concentration of 3 mM and was enhanced to 3.8-fold by addition of anionic phospholipids (PL). In contrast to the wild type PC the rPC mutant was insensitive for activation by the T/I-M complex. Addition of the mutant to normal human plasma induces a prolongation of tissue-factor and P-IT-based clotting assays. Using normal human plasma as a source for FXa the the activation rates of the mutant were found 5-fold higher than in the purified system if tissue factor was used to generate FXa. In conclusion, our data demonstrate that the rPC mutant is effectively activated by FXa in a purified as well as in a plasma system. Interestingly, the activation rates are enhanced in the presence of PL and normal human plasma. Fudher studies should clarify the potential use of this mutant as a novel anticoagulant. Thrombln plays a pivotal role in thrombotic events. The time course of thrombln concentration in blood or plasma after activation is of special Interest to answer a variety of questions. With a chromogenic assay developed by Hemker et el. [Thromb. Haemostas, 70, 617, 1993] it became possible to measure the generation of thrombin in activated plasma continuously. Inhibitors of clotting enzymes which are to be developed as anticoagulants should be able to inhibit thrombin generation or to Immediately block generated thrombin. We have used a test based on Hemker's thrombin generation assay to elucidate which potency and specificity an inhibitor of factor Xa needs to efficiently block thrombin generation in human plasma. Thrombin generation after extrinsic (tissue factor) or intrinsic (ellagic acid) activation was followed using the chromogenic substrate H-~Ala-Gly-Arg-pNA (Pentapharm Ltd.). A series of synthetic low molecular weight inhibitors as well as naturally occurring inhibitors of factor Xa with different potency were investigated. Because of the inhibition of activated factor X the generation of thrombin in plasma Is delayed and the amount of the generated thrombin is reduced. The concentrations which cause a 50 % inhibition of thrombin generation (ICso) correlate with the K~ values of the inhibitors. Low molecular weight inhibitors with K~ values of about 20 nmol/I inhibit the generation of thrombin after extrinsic activation with ICso in micromolar range. After activation of the intrinsic pathway tenfold lower concentrations are effective. The strongest inhibitory activity after extrinsic as well as intrinsic activation is shown by recombinant tick anticoagulant peptide (r-TAP) with IC~o of 0.049 pmol/I (axtdqsic) and 0.037 pmo/I (intdnsic). In the compadson of synthetic low molecular weight inhibitors of thrombin end factor Xa which have similar K= values for the inhibition of the respective enzyme (lowest I<1 20 nmol/I), factor Xa inhibitors are less effective tn the thrombin generation assay. In contrast, the highly potent Xa inhibitor r-TAP shows a stronger inhibition of thrombin generation than the tight binding thrombin inhibitor hirudin. Background: Resistance to degradation of coagulation factor V by activated protein C is associated with a point mutation in which adenine is substituted for guanine at nucleotide 1691 in the gene coding for factor V. To date this specifc mutation appears to be the most common inherited abnormality which predisposes patients to venous thrombosis. For this reason a reliable, fast and automatable system for the diagnosis of the described point mutation is required. The conventional methods used to identify the mutation are based on allele-specific restriction enzyme site analysis or direct sequencing. These methods have disadvantages for a large scale DNA diagnosis, which include the need for electrophoresis or a high cost and time consumption. Methods: An alternative strategy of DNA diagnosis, the allele-specific oligonucleotide ligation assay, was adapted for the diagnosis of tile point mutation of factor V. Following PCR amplification of the target DNA, tile procedure was performed completely automatically on a robotic workstation with an integrated ELISA reader using a 96-well microtiter plate. Allelespecific restriction enzyme site analysis was performed to confirm the genotypes. Results: In 10 patients with the mutation and in 20 individuals without the mutation the genotypes determined with the conventional allele-specific restriction enzyme site analysis were in 100% concordance with the ELISAbased oligonucleotide ligation assay. Discussion: The PCK-oligonucleotide ligation assay applied as automated detection system for the identification of the coagul;mon factor V point mutation allows tile rapid, reliable, and large scale analysis of patients at risk for thrombosis. Resistance to the asticoa=m~lant activity of activated protein C (APC resistance) has emerged as the most con'anon inherited thrombophilic state. Patients lreterozygous for factor V Leiden are more likely to suffer from thromboembolie events than controls. This risk is even more pronounced in homozygotes. Due to the low sensitivity and speeifity of most coagulation tests some investigators suggest to examine patients for the presence of Factor V Leiden mutation by PCR-based methods. Re e~tly we presented an APTT-based functional test (acceleria inactivation test AIT): 1:20 diluted plasma (50BI) is mixed with factor V deficient plasma (50~tl) and APTT reagent (501.d), incubated at 37°C and then coagulation is induced by CaCI2 and A.PC (50~d). Using a standard curve, the clotting time (see) is transferred in per cent accelerin inactivation (%AI). Using this test, the widely used APC-ratio as well as PCR-based factor V Leiden detection (confirmed by direct sequencing) we prospectively studied 172 consecutive patients with thromboembolic events. Patients without the factor V mntation eonsitently showed more flazm 50% AL With the exception of one patient with severe factor deficiencies (including factor V) due to hepatic failure and heterozygous for factor V-Leiden resulting in 62*/. AI, there was a complete concordance between the PCRbased method and dysaseelerinemia detected by AIT. Due to these result a specifity and sensitivity of AIT above 95 % was calculated. Furthermore, a clear discrimination could be obsoved beween 34 heterozygotes (20%<AI<50%) and 8 (incl. 5 samples obtained from Jeaa) homozygotes (AI<20%). In contrast A.PC-ratio resulted in a great overlap between patients homozygous for the wildtype , hcterozgous or homozygous for factor V Leiden. In addition we confirmed thc di.~eulties nsing the APC-raUo for patients trader anticoagulation, or with different types of other d~rombophilie defects such as lupus anticoagulant or protein S deficiency. Due to the time and cost-saving featttres the/kiT should be considered for the rapid detection of APC resistance without the use of PCR-based techniques. In summary we found, well in accordance with previous reports, that APCR due to the FV-Leiden mutation is • highly prevalent defect in VTE pts. Pts with the FV-Leiden mutation were eharacterised by a higher incidence of VTE's without recognised prothrombotic risk factors and more frequent positive family history; no higher incidence of recurrent VTE's or other prothrombotie coagulation defects was found; the age at initial manifestation of thrombophitia was comparable.

ACQUIRED A recently identified mechanism for famdial thrombophilia is characterized by a poor anticoagulant response to actavated protein C (APC), It is now welt recoginzed to be due to a genetic defect in the factor V gene, which results in the syn-,thesis of an abnormal factor V molecule and to be a frequent risk factor for thromb.osis, however APC resistenee can also be found in certain acquired conditions. Since pregnancy ~s known to be associated with a hypercongulable state resulting in a prevalance of thrombosis up to 1% and thromboembolic events, we determined the prevalence of APC resistance during the course of pregnancy at the City Hoslfital Ruesselsbeim. Germany. Blood samples from pregnant woman were drawn at mmester 1, 2, 3 and at term. Besides the routine coagulatton screening, protein C and VIII:C, the APC resistance were evaluated. A modified APTT method utilizing human plasma fracnonated APC (Amencan Red Cross. Bethesda, Maryland, ~USA) was used to assay APC resistance. This method relies on the determination of two APTT's in the patient plasma, one in the absence of APC and one in the presence of a fixed amount of APC. Clotting tune was determined after mcubation using the APTT reagent ( We investigated the sensitivity of various species towards activated, human protein C (APC) and found an impaired response in the order monkey < horse < pig < dog < rabbit. We assume that this effect is mainly caused by differences in the APC cleavage region of factor V, as recently described for a factor V mutation in bumat~s: factor V-Leiden. This was corroborated by the finding that addition of human factor V similar to the factor V added with the animal plasma had only minor influence on test outcome. Coagulation assays dependent on APC, such as for protein C and protein S determination, in the standard APC assay, the more factor V-Leiden specific APC assay and in the PCAT, a new screening assay for the whole protein C system, are based on the inactivation of factor Va via APC. Thus, in all these assays, 5% of rabbit plasma added to human plasma mimicked a APC response as observed for heterozygous carriers of factor V-Leiden and 20% as for homozygous carriers.

We exploited this factor V-Leiden like behaviour to prepare plasma standards for calibration of APC dependent assays by mixing rabbit plasma with human plasma~ As reference for these standards the 0% value was assigned to the clotting time in the absence of APC, while the 100% value was assigned to the clotting time in presence of APC as obtained with a human, normal plasma pool. Then these calibrated standards were used for establishing reference curves. On automated coagulation analyzers, the results obtained were automatically reported in '% of normal' or '% sensitivity'• This not only eases evaluation, more importantly, this procedure also considerably improves the comparability of results obtained with different reagents and instruments. In order to establish the methods for activated protein C resistance and the Faktor V Leiden-Mutation we investigated 204 healthy individuals (128 women, 76 men, median age 37 ys) using the assay "Contest ® APC-resistance" (Chromogenix, Sweden). The mean APC-ratio was 2.65, the cut-off level for low response to APC was calculated as 2.15, 46/128 women were oral-contraceptive users. They showed a slight decreased APC-ratio (mean 2.46). 24 of the 204 individuals (11,8%) had a poor response to APC (mean ratio 1.94) as a APC-resistance. For detecting the factor V gene mutation we a used non commercial own modified easy to handle PCR-based test. In 14 of the 204 individuals (6.9%) the factor V-Leiden mutation could be found as heterozygotes. In this group, the APC-ratio was significant lower (mean 1.87) as in the group with APC-resistanee and without deteetiun of the factor V mutation (mean 2.04). Nevertheless in one individual with beterozygote factor V-Leiden, the APC-ratio was found in the normal range (2.24) , also in a few individuals with very iow APC-ratio the factor V mutation could not he detected. Finally this data suggests a high prevalence of APC-resistance and factor V-Leiden mutation in the population of the southwest of Germany.

Resistance to activated protein C (APC) has been reported to interfere in clotting assays for protein C (PC) and protein S (PS) from various manufacturers, resulting in decreased values or even apparent deficiency (type II') of PC. or IS. In our laboratory, when using functional, aVIT-based assay kits for PC., IS, and APC sensitivity from Behring (Marburg, Germany) on an automatic turbidimctric coagulation analyzer (Fibrintiraer A, Behring), the interference in the IS assay has been observed in a very pronounced manner soon after the introduction of APC sensitivity testing into the standard laboratory procedure for patients with thrumbophilia. Yet we were not aware of an interferenca caused by APC resistance in the PC test. We therefore evaluated our results for PC eed PS with respnet to APC sensitivity status in a retrospective analysis. Exclusion criteria were: oral anticoagulant therapy with vitamin K antagonists, impaired liver function, borderline result in the APC sensitivity test, known analytical intedercnc* other than APC resistance (e.g. elevated factor VIII:C). Resu/ts: Apparant PC activity was slightly (12 %) lower and apparent PS activity was grossly (45 %) lower in APe resistent patients than in patients with normal APC sensitivity: There was no case with a presumed du~l defect of both At~ rnsistaac¢ and PC deficiency. In addition, none of the few patients diagnosed with PC deficiency before the introduction of the APC sensitivity assay turned out to Ix: APC resistent. Much more of a problem is the known sensitivity of the PC test to an elevated factor VIII concentration which is quite a common finding among hospital patients but rarely known in an individual case. By aeccelerating the clotting process, elevated factor VIII can cause apparent PC deficiency that can not be confirmed in a chromogunie PC assay. ,Clearly, when combined with APC resistance, this problem is exspacted to be more severe. On the other hand, the comhination of both APC resistance and apparent IS deficiency was found on a regular basis with many decreased and some bordeflinc values for IS but only few in the lower normal range and virtually none in the upper non~al range. Also, several #eats previously diagnosed with IS deficiency turned out to be PC resistent, and the diagnosis had to be corrected, but dual defects cannot be ruled out. Conclusion: For, the interpretation of PC and/or 1:"3 values from clotting assays the APC sensitivity status must be considered. When APC resistance and an apparent deficiency of PC or IS is found, a type I deficiency may be confirmed or ruled out using antigen assays, but at present, there seems to be no reliable way other than genetic analysis to address the question of dual defects with presumed type II deficiency, especially in the case of PS. Therefore, there is a clear need for analytical improvementS and the development of functional assays that are less sensitive to interference by APC resistance. The most common inherited risk factor for venous thrombosis is the resistance to activated protein C (APC). The main reason for this resistance is a point mutation in the gene of dotting factor V (F V). A nueleotide exchange leads to a different amino acid sequence in the protein and thus to the loss of one of the cleavage sides which is important for the inactivation of the procoagulatory form off V. The imbalance in the coagalation cascade leads to a strong risk for venous thrombosis, which is thought to be inoreaced by factor 7 to 10 for heterocygotes and about 80 for homocygote individuals. The prevalence in patients with a history of thrombosis is about 20%, among APCresistant patients as high as 80%. Prevalence-ofF V ,Leiden" is reported to be 2% of the normal population in Leiden/Netherlands and as high as 15% in a certain city in the south of Sweden. Due to the obviously different geographic distribution and the highly increased risk of venous thrombosis in carriers of the mutation investigated the distribution of this mutation in the normal population of different regions in Germany.

First attempts to assess the prevalence ofF V mutation by molecular methods (polymerase chain reaction followed by restriction fi'agment length polymorphism analysis) revealed that in the area of Wiirzburg 7.7% of blood donors carry the mutation, which is significantly different (Fischer T-Test, level of significance 0.05%) from published data observed in Freiburg (3.4%). Homburg/Saar and Harmover revealed 5.8% and 8.5%, respectively, and were not significantly different from Wfwzborg. Further investigations of blood somples fi:om other regions should give an overview of the geographic distribution of this mutation in Germany and might finally help to draw a map era regional risk of venous thrombosis. The regular APC resistance test is not applicable to plasma from Orally anticoagulated (OAC) or heparinized patients due to decreased levels of vitamin K-dependent clotting factors and to thrombin inhibition by antithrombin, respectively. The former limitation is overcome by prediluting samples 1-1-4 with FV-defident plasma.

For this purpose a special quality of FV-deficient plasma has been prepared, which also allows analysis of heparinized plasmas. This FV-deficient plasma has now been evaluated with the COATEST APC Resistance assay.

A complete APe ratio discrimination for the FV Q506 mutation is obtained for plasmas from OAC and heparin patients as well as from non-treated individuals. Ratios above 2.2 are obtained in normal genotype plasmas, whereas heterozygotes for the mutation yield ratios in the range 1.6-2.0. The basal APT times for 15 OACplasmas with widely spread INR-values were reestablished into the normal range.

.The stability of the reconstituted FV-deficient plasma was found to be' at least 3 hours at room tempetature, indicating its suitability for .routin.e u.~. F .u~ .e~mv~., the influence of preanalytical procedures is rmm~ single or double cenuifugation as well as freezing of samples do not alter the APC ratios to any significant extent.

Our results prove the usefulness of the modified COATEST APC Resistance kit method as a reliable, simple and rapid tool when screening for the presence of the FV Q506 mutation. Hereditary resistance against the anticoagulatory action of activated protein C (APC-resistance, APCR) which was first described by Dahlb~ick et al. (PNAS, 90:1004 , 1993 was identified as a possible new thrombophilic factor in a high percentage (17-60%) of young adults with thrombotic events. A single missense mutation (R506Q) due to a G/A transition (G1691A) in exon 10 of the factor V gene (Bertina et al., Nature, 369:64, 1994) is tightly linked with APCR and is regarded as the causative molecular defect. Identification of this mutation by PCR-based methods is easy to perform and avoids preanalytie and analytic errors in the eoagulometric assay for APCR. Since the impact of this mutation in children with thromboembolic disease has not been determined to date, we initiated a multi-center prevalence study on two pediatric populations, with and without thromboembolie events. We compared 125 pediatric patients with thrombosis, divided into 3 different age groups (0 to <0,5 years; >0,5 to <10 years; >10 to <18 years) with a normal population of 159 children. The mutation G1691A was found with an unexpected high prevalenee of 12% in our normal controls. However, the prevalence was significantly higher in the age groups: 0 to< 0,5 years (25%) and >10 to <18 years (30%). In patients between >0,5 to <10 years the overall prevalence was similar to the control (13%). However in patients of this age with spontaneous thrombosis APCR was also a significant risk factor (29%). Our results emphasize the impact of APCR for thrombogenesis in children. However, the significance is agedependent and does possibly reflect the different physiology of haemostasis in our three age groups. Activated Protein C (APC)-Resistanec is a newly reeognised risk factor for thrombosis. In at least 90% of the cases it is caused by a single point mutation in the factor V gene (G->A at nucleotide 1691), which predicts replacement of Arginin 506 with Ghitamin. One of the APC cleavage sites in factor Va is located C-terminal of Arginin 506, and mutated factor Va (Factor V Leiden) is resistant to APC-mediated inactivation. From epidemiologic studies it is known, that this abnormality can be found in about one third of patients with thrombosis. APC-Resistance is a major basis for venous thromboembolism and is prevalent in about 5.10% of the general caucasian population.

Recurrent spontaneous abortion (RSA) affects 1-5% of couples and represents a major concern for reproductive medicine. In spite of extensive endocrine, genetic, serologic and anatomic evaluation some 30-40% of RSA women remain unexplained. A frequent morphologic finding in placentae of aborted pregnancies is an increase of fibrin deposition within the intervitlous space.

Because of these findings we studied the prevalence of APC-Resistance in women with RSA (more than 3 miscarriages) of unknown origin. In 2 of 35 cases we found a pathologic APC-Resistance, both patients had a history of recurrent thrombosis and were heterozygous for Factor V Leiden. The prevalance of APC-Resistance is 5,7% and thus equals the prevalence in the general population.

Our data do not support the hypothesis that APC-Resistanee is a risk factor for recurrent spontaneous abortion. H~matologisches Zentrallabor der Universit~t, Inselspital, 3010 Bern Resistance to activated protein C (APC) due to the mutation 506 Arg --~ (]In of factor V (factor V Leiden mutation) is the most frequent hereditary thrombophilic defect known today, with a prevalence of 20 -50 % in patients with idiopathic venous thromboembolism and of about 3 -5 % in the general population. With an allele frequency of 2 % the expected number of homozygous individuals is about 4 in 10000. Homozygous and heterozygons individuals differ considerably with respect to the relative risk of thrombosis (80 -fold versus 7 -fold) as well as to the age of the first thrombotic event (31 versus 44 years). Deficiency of the vitamin K dependent protein S (P$), an important cofactor of APC, is another hereditary thrombophilia which is, however, much rarer than APC resistance with a prevalence of 5 to 8 % in patients with venous thromboembolism. Factor V Leiden mutation as well as PS deficiency are associated with impaired anticoagulatory activity of APC, which is most pronounced in case of the combination of the two defects. The combination of PS deficiency (with an assumed prevalence similar to that of PC deficiency) with heterozygous or homozygous APC resistance can be expected with a probability of 1: ~ 5000 or 1: ~ 500000, respectively. It is well known that PS levels decrease towards the low normal or even subnormal range during pregnancy. Moreovar, there is increasing evidence that the sensitivity of plasma to the antieoagulatory effect of APC decreases during pregnancy resulting in an acquired APC resistance. These pregnancy associated effects art obviously much more relevant in case of preexisting PS deficiency or APC resistance and should contribute to the elevated thrombotic risk during pregnancy in a subject with either of the two defects, and even more so for a woman who suffers from both defects. We describe a young woman with a combination of homozygens APC resistance ( APC ratio 1.10, normal range: 2.02 -3.73), pronounced PS deficiency (free PS 0.ll U/I, total PS 0.30 U/I, normal range: 0.55 -1.20 U/1 and 0.65 -lag U/I, respectively) and, moreover, impaired fibrinolysis (no change of euglobulin -lysis time after 10 rain venous occlusion) who developed deep vein thrombosis after cesarean section in her first pregnancy. Examination of her familiy showed heterozygous APC resistance in her asymptomatie father (APC -ratio 1.99) , combination of heterozygous APC resistance (APC -ratio 1.60) and PS deficiency (free PS 0.30 U/I, total PS 0.58 U/I) in her nsymptomatic mother and no defect in her sister. Considering the fact that the mother was still thrombosis free at the age of 49 one may assume that the thrombosis risk in the proposita was mainly influenced by the homozygnsity for APC resistance.

S. Ehrenforth, M. Adam, B. Zwinge, I. Scharrer University Hospital, Dept. of Angiology, Frankfurt a.M., Germany Introduction: APC resistance has been shown to be the most commonly inherited defect which constitutes a risk factor for venous thrombosis (VT). However, most of the present epidemiological studies concerning APC-R prevalence in thrombophilia were derived from results of tests conducted onplasmas collected under various conditions. This may influence the great differences reported on the prevalence of APC-R among these patients. For example, it has been shown that freezing of plasma specimens prior to analysis of APC-R causes a significant decrease in the assay results.The aim of our study was to evaluate the influence of eentrifugation conditions on the results obtained with the chromogenic APC-R assay. Patients and Methods: Blood was collected from 31 patients (t9 women, 12 men; FV gent.type: 13 R/R 506, 14 R/Q 506, 4 Q/Q 506) through veinpuncture into trisodmm ciwat (1:9). Platelet-rieh and platelet-poor plasma was obtained by immediately centrifugation at 4"C for 10, 20, 30, 40, 50, 60 rain at 1000, 2000, 3000, 4000, 5000 and 6000 rpm. Additional, PNP obtained from 14 healthy individuals (7 male, 7 female without hormonal trealraent) was prepared equally. APC-response was determined within one hour after centrifugation using the Coatest APC resistance kit from Chromogenix. Results: For both, PNP and sin/gle plasma samples, we observed continuous higher AF'C-ratios after increasing cenWifugation intensity. For example, an increase from 1000 to 6000 rpm resulted m an increased APCratio from 2.7 to 3.26 (20 min), from 2.88 to 3.326 (60 rain) respectively. Even though less distinctive, similar results were observed concerning the duration ol eentrifugation: when the duration was increased from 10 to 60 minutes we observed a continuous increase in APC-ratio, for example from 2.74 to 3.12 when using 2000 rpm and from 3.09 to 3.36 when using 4000 rpm. The decrease of the ratio after low eentrifugation is the eonse-9nence of the shortening of AFffT in the presence of APC, without a signhcant influence of basal Al:rl~ without APC. Conclusion: Our results demonstrate that centrifugation conditions are important to consider for the interpretation of APC-R results. Supporting our observations, recent studies from Sidelmann et al. have shown that an increase in plasma platelet concentration, low eentrifugation respectively, causes a signficant decrease in the APC-response. However, so far the mechanism responsible for the significant effect of both on APC-R assay results is unknown. Although technically simple, the biochemical cemplexitiy inherent in the chromogenic APC-R assay necessitates a standardized plasma handling procedure to secure a reproducible determination of APC-IL

COMPAPJSON OF DIFFERENT ASSAYS FOR DETERMINATION OF APC-RESISTANCE WITH THE GENO'FYPING FACTOR V (ARG -> GLU) G. Siegert*, S. Gehrisch*, E. Runge**. R. Naumann**, R. Kn6fler*** *Institute of Clinical Chemistry, **Clinic of Internal Medicine, *** Childrens Hospital

Resistance to APC diagnosed on the basis of prolongated clotting time in the aPTT assay" is now considered a major cause of thrombophilia. In the majority APC resistance is ~ted with a point mutation in factor V molecule (Arg506Glu), but both are not synonym. Protongated baseline aPTT is a limitation of the assay. Following the determination is not possible in risk groups of patients (factor)ill deficiency, Lupus Anticoagulan0 and in patients under anticoagulant therapy. In these causes a dilution of plasma in factor V deficient plasma is recommended. The Immunochrom assay is based on the inactivation of factor Villa by APC. The aim of the study was to compare different functional APC response assays with the result of the DNA analysis. APC response was tested in 100 healthy probands, 81 thro~ patients and 16 family members using the lmmtmochrem assay, the Contest (Chromogeaix) and the Contest with 1+ 4 dilution of the plasma in native factor V deficient plasma (Immune). The DNA analysis was performed as described by Bertina. One patiem was homozygoas for factor V mutstion~ a hetemzygous result was obtained in 4 members of the control group, in 28 patients and in 6 family members. In all cases with factor V mutation the ratio of the Immunochrom assay was lower than the laboratory own value, independent on anticoagulant therapy. Pathological ratios in this assay were also obtained in one member of a family" with high thrombotic incidence (DNA Arg/Arg) and in 17 patients under anticoagulant therapy ( two of this patients are one cloned twins). In the Contest a AI~ response was diagnosed in all cases with factor V mutation without anticoagulant therapy and in 40 % of heterozygous patients under anticoaglant therapy. Results of the test using the dilution in factor V deficient plasma showed a good agreement vAth the results of the DNA analysis but the method is obviously only sensitive for the factor V mutation. The reason for pathogical ratios in the lrnmunechrem assay in wildtype patients is unclear. The majority of this patients is treated with anticoagulants, a comparison with the Contest is not possible. Interestingly in one patient under heparin and low ratio in the Immunochrom assay' after reduction of hepann the ratio of the Coatest was also low. It seems necessary to investigate in which distance to the thrombotic events the APC resistance should be tested. Following pathological ratios in ftmctional APC assays must be discussed: high levels of factor VIII and or v WiUebrand antigen (acute phase reactien), other mutations in factor V and VIII. The factor V dilution assay should be replaced by the DNA analysis. Due to their differing compositions, the "sensitivities" of various APTF reagealts differ not only with respect to factor depletions, heparin and fibrin-fibrinogen degradation products, but also with regard to pathological inhibitors. For lupus anticoagulants this means that "lupus-sensitive" reagents can be delineated from "lupus-insensitive" reagents. With a "lupus-insensitive" AI~ reagent there is no or only slight prolongation of the APTT in the plasma under investigation, whereas with a "lupus-sensitive" reagent marked prolongation is observed. For the meaninof~l use of APTr reagents it is necessary to know the extent to which they are influenced by lupus anticoagulants. The following 14 APTI' reagents were tested: • PTT-Reageaz, P'rTa, PTTa liquid, PTT-LA, PTI'-LT (Boehringer/Stago) • Pathromtin, Pathromfin SL, Necthroratin (Behring) • Platelin S, PIatehn Excel LS (Organon Tekinka) • Actin-FS, Actin-FSL (Dade) • APTT silica lye, APTT silica liquid (Instntmentation Laboratory) The material for investigation consisted of 20 plasmas from patients with lupus anticoagulants. A confirmatory test (Lupus anticoagulant test, Immune) was positive for all of the patients. Measurements were made using the STA coagulation analyser (Boehringer/Stago). It can be seen from the results that in some instances very different prolongations were obtained in identical plasmas by using differing APTT reagents. Low susceptibility to lupus anticoagulants was shown by Actirt FS (Dade), PTT-Reagenz (Bcehrlnger) and Neothromfin (Behring). High susceptibility was shown by Platetin Excel LS (Organon Teknika), PTT-LA and PTI'-LT (Boehringer/Stago). Lupus anticoaguhant screening with the APTT reaction is promising when two APTr reagents differing as greatly as possible in their lupus anticoagulant sensitivity are used. The resistance to the anticoagulant response of activated protein C (APC) is a major cause of venous thrombosis. APCresistance is due to a single mutation in factor V gene, which predicts replacement of Arg 560 in the APC-cleavage site with Gln (Factor V Leiden mutation). In contrast to other known genetic risk factors for thrombosis, this Factor V 1691 G-A mutation has a high prevalence in the common population of Western Europe (average 4-5 %). We have determined the prevalence of the Factor V 1691 G-A mutation in a population of 700 probands of north-eastern part of Germany. The mutation was found in 7 %. (Heterozygoty were found in 48 subjects 1 person was homozygous.) The results are compared with our studies of populations from Argentine and Poland. Me analysed the factor V 1691 G-A mutation in 71 patients with thrombosis from Germany and Hungary. This mutation has been found in about 60 % of these patients. In contrast, the frequency of this mutation was strongly reduced in a group of 47 patients with thrombosis and pulmonary embolism of Argentine (3 heterozygotes in 47 patients; 6 %). The results of these different populations will be described and discussed. Past medical history: Venous thromboembolic events (rE) at 18, 19 and 2I years; intermittent oral anticoagulation (OAC) without TE's. Diagnosis of autoimmune disorder with elevated antinuelear-antibody-fiters and positive lupus-anticoagulant test. No other relevant illnesses; family history uneventful. Two weeks prior to the referral to us -acute febrile illness with nausea, diarrhea, abdominal pain; hospitalisation, treatment with iv antibiotics and anticoagulation with fraetionated heparin; development of extensive deep vein thrombosis (DVT) of the right leg; initiation of full-dose unfractionated heparin; decline of platelet count from 121 to a nadir of 21 G/l; referral to our department. On admission an extensive coagulation screen yielded the following results (n/normal, t/elevated, I/reduced, +/positive, -/negative): PT t, aPTT t, Tr n, factor II, V, VIII n, factor VII, IX, XI, XII /,, fibrinogan t, ATIII n, protein C, S *, activated protein C sensitivity ratio 1.92 ($), FV-Leidenmutation PCR -, fibrinolytic system n, TAT t, Ft÷2 t, lupus anticoagulant +, heparin induced platelet antibodies +; no diagnosis of a specific autoimmuna disorder could be made. An immunosuppressive therapy with corticosteroids and anticoagulation with recombinant hirudin were init'~at~; no p~ogr~zsion of the DVT oeeured and normalisation of the platelet count was observed.

During follow-up under OAC ) and low-dose corticosteroids, the patient was well, the pathologic coagulat;.on results, including lupusanticoagulant and activated protein C resistance, have returned to normal; no further TE's have been observed. In summary we present a case of a complex coagulation disorder as part of an autoimmune process, resulting in a clinically manifest prothrombotie dysbalance including lupus anticoagulant, acquired resistance against activated protein C and heparin induced thrombocytopenia (type II), entering complete remission under combined immunosuppressive and anticoagulant therapy.

In the last 30 years, a vast number of simplified analytical procedures have been developed for the diagnosis of haemostatic disorders. Today the detection method have evolved from the mechanical hooking method or ball coagulometry to optical systems, which additionally can utilise chromogenic substrates or immunological methods. In these systems the clotting time is derived from algorithms (e.g. threshold or maximum of the first or second integral).

We studied 50 healthy subjects, aged 21 to 65 years and 118 patients, aged 9 to 93 years using a new aPTT reagent (Pathromtin $L). The results were compared with those obtained with a routinely used reagent (Pathromtin). The reference range, factor-, heparin-and Lupus anticoagulant sensitivity were determined. Analysis was performed using the Behring Fibrintimer A (BFA) with optomechanical clot detection, the Behring Coagulation Timer (BCT) with op-"dcal clot detection by threshold and the DW test and DW confirm for Lupus anticoagulant diagnostic. Our results showed that the new Pathromtin SL reagent met the demands for a higher factor and Lupus anticoagulant sensitivity. It is highly suitable for monitoring heparin therapy and gave comparable results with the optical and the optomechanical analyser systems, hence reagent c~n be used for both systems. Restenosis following percutaneous transluminal angioplasty (PTA) continous to be a major clinical problem. Neoinfimal hyperplasia, being the major undedying cause, can not be sufficiently avoided. Vadous plasmatic coagulation and fibrinolytic factors, have been associated with artedal restenosis. Anticardiolipin antibodies (aCt_) have been established as dsk factors for venous or arterial thrombosis. Methods: In a cohort of 75 patients (53 men and 22 women, age 68±13 years) undergoing PTA of a peripheral artery we prospectively evaluated whether aCL could influence 6 months restenosis rate. Patients were clinically examined before, 3 and 6 months after PTA. Noninvasive grading of artedal stenosis was done by duplex scanning of jet peak velocities. Restenosis was arbitrarily defined as more than 50% occlusion of the lumen at the site of dilatation 6 months after successful intervention. Laboratory investigation at the same time included aCL and other known atherosklerosis risk markers, such as fibdnogen (Fbg), yon Willebrand factor (vWF), homocystein (Hcy), C-reactive protein (CRP). Thrombin generation markers, such as thrombin-antithrombin III complexes and prothrombin fragments 1+2, as well as thrombomodulin (FM) as an endothelial activation marker, were also measured. Results: 31/75 (41.3%) patients were considered to have developed restenosis after 6 months. 9/75 (12%) patients were found to have positive IgG-(19-35 GPL) and/or IgM-aCL (14-103 MPL) at all three measurements. 1/75 was negative before but seroconverted (IgM) 3 months after PTA. 2/10 (20%) aCL-positive and 29165 (44.6%) aCL-negative developed restenesis at 6 months (chi-square p-value=0.14). All above mentioned coagulation parameters did not differ between aCL-positive and -negative patients, measured before or 6 months after PTA. Some of them are shown below (values before PTA):

Fbg ( Basilar artery stenosis is a rare event in young children. Risk factors are head or neck trauma with consecutive dissection of the vertebral artery, cardiac diseases or hypercoagulability. Elevated lipoprotein (a) (Lp(a)) serum levels in adults can mediate atherosclerosis. In addition, Lp(a) might interfere with fibrinolysis. Here we report on a 3 year old boy , who presented with acute brain stem symptoms. History revealed neither trauma nor infectious disease. Conventional and MR angiography showed stenosis of basilar artery without ischemic lesions. Laboratory findings were normal in routine blood and CSF tests. Global coagulation parameters as well as procoagulant and anticoagulant factors were normal. Cardiac and autoimmune disease could be ruled out. Lp(a) serum levels were significantly elevated to 104 mg/dl (normal range <30 mg/dl). Analysis of other family members revealed a hereditary hyperlipoproteinemia (a) which might explain family history of an increased incidence of myocardial infarction and CVE in elderly family members. Clinically the patient recovered completely from brain stem symptoms after heparinization and subsequent oral anticoagulation with phenprocoumon. However, radiological signs of basilar artery stenosis were progredient. In a recently developed specific test, an elevated anti-phosphatidylserin antibody titer was detected one year after primary diagnosis. In conclusion, this is the first report on a child with stenosis of the basilar artery and elevated levels of Lp (a). It is unclear, whether APA contributed to the onset of basilar artery stenosis or developed secondary due to endothelial defects after thrombosis and anticoagulation. APA, however, might increase the risk of further thrombotic events in this patient. In 110 patients with thrombotic events respectively patients with systemic lupus erythematodes antioardiolipin antibodies (ACA) aund lupus anticoagulant (LA) were ~ea~ured. For ACA detecting we use the assays from elias for IgG-and Ig~}-antibodies. We use as sensitive methods for detecting LA in our laboratory the testkits from Diagnostlca Stago (Staclot LA with hexagonal array of phospholipids, ,PTT-LA a very sensitive PTTmethod and Staclot P~P-a platelet neutralization procedure) and the PTT from Organon Teknik~ (Platelin Excel LS with two incubation times, 1 and 10 minutes). i"~e results Of this tests were compared with three new or~e on German market: SpeckTin APOT (aktlvated plasma clotting time), SpeckTin APTT (APTT wlth purified soy extract) and 8peckTin LA (phospholipid preparation in concentrations between 10 and 500 ~g/ml); all WAK Chemie. Traditional aPTT reagents were developed for the sensitive detection of factor VIB an IX as a cause of hemorhage. High sensitivity against Lupus anticoagulants, which also prolong aPTT, was not required for this purpose, With increasing recognition of the importance of antiphospholipid antibodies as a risk factor for thrombembolism, more sensitive reagents were designed, which now reliable detect this condition. Using such reagents as a screening test in a general hospital makes it necessary to distinguish both conditions quickly. We here report an algorhythm, by which we use an inhibitor (Lupus anticoagulant) sensitive (STA APTT, Boehringer) and an inhibitor insensitive reagent (Actin FS, Dade) to distinguish anticoagulants and factor deficiencies as a cause of prolonged aPTT. Citrate plasma from 33 patients with various diseases showed an unexpectedly abnormal inhibitor sensitive aPTT (>40s). 13 plasmas with factor deficiencies remained abnormal with the insensitive aPTT reagent. A regular correction of their defect occured on mixing with normal plasma. By measurement of single coagulation factors five patients with contact factor XII deficiency were found. This condition is associated with thrombosis and very rarly with bleeding. Three patients with factor XI deficiency and two patient with factor IX deficiency were also identified. Antiplatelets, of any kind, permits a secondary prevention of myocard ischemic lesions. There is no general consensus regarding secondary prevention of cerebral ischemic lesions. Aspirin remains the most common substance, ticlopidlne also brings about prevention, but with important secondary effects. European Stroke Prevention Study i has demonstrated that the combination of antiplatelets, in particular aspirin/dipyridamole (Persantln), is also very active. To collect more information, ESPS 2 was organized and 6602 patients receiving either a placebo,either 50 mg aspirin,either 400 mg sustained release form of dipyridamole (Persantin (R)), or the combination aspirin/dipyrldamole, were recruited. It ended March 31st 1995 with the following conclusions: I-aspirin, 50 mg a day, brings about a significant secondary reduction of stroke (18.Z %), after a two year follow-up. Notwithstanding the low dose of aspirin, haemorrhages remain important. 2-dipyridamole, at 400 mg a day, brings about a significant reduction of stroke (i6.3~), similar to that of aspirin. One could thus substitute 50 mg aspirin by 400 mg dipyridamole. 3-the combination of 50 mg aspirin and 400 mg dipyridamole brings about a significantly greater reduction of stroke (37.0 ~). ESPS 2 revealed that a low dosage of aspirin is active, that dipyridamole alone is also active, but that the combination of both gives far better results. The study of the primary end-points,the study of the survival curves, the factorial statistical analysis and the pairwise comparison analysis, led to these conclusions. The conclusions drawn from ESP£ 2 underline that the combination aspirin/dipyridamole is a privileged choice for cerebral ischemia, The state of activation of circulating platelets in acute cerebral ischemia is controversial. Activation of platelets on single cell level can be assessed by determining the shape change or the expression of antigens such as p-selectin (CD62). Shape change is an early and rapidly reversible event in platelet activation whereas p-selectin is irreversibly expressed on the platelet surface upon stimulation. Methods: We investigated 20 untreated patients within one day after cerebral ischemia, 20 patients months after stroke treated with warfarin, and 21 age and sex matched control subjects without vascular risk factors. Venous blood was collected into a fixation solution blocking the metabolic processes in platelets within milliseconds. We determined the fraction of resting discoid platelets by phase contrast microscopy. The expression of p-selectin was measured by flowcytometry. Results: The fraction of platelets expressing p-selectin was higher in patients with acute cerebral ischemia (7.8_+4.7%) than in control subjects (1.9_+1.1%; p<0.001, u-test). Patients with stroke (n=15, 7.8+4.5%) and patients with transient ischemic attack (TIA; n=5, 7.6-+6.1%) had similar values. Patients months after stroke still had higher values (4.3+9,3 %, p<0.05) than control subjects. The rate of discoid platelets was not different between patients with acute ischemia (n=17, 85.6-+8.8 %), patients months after stroke (n=19, 85.7-+5.1%) and control subjects (n=21, 86.7_+5.8 %). Platelet count was not significantly different between groups. Conclusion: The elevated proportion of platelets expressing pselectin indicates strong platelet activation in acute cerebral ischemia and in a majority of patients months after stroke. Assessment of pselectin revealed a higher sensitivity for platelet activation after stroke or TIA than analysing the reversible shape change. Further studies have to clarify if monitoring of platelet activation by flowcytometry is helpful as a prognostic tool and to evaluate therapeutic strategies after stroke. Vascular smooth muscle cell (SMC) proliferation and migration into neointima are the hallmarks of atherogenesis. The complexity of these processes and their concerted action and interaction of molecules are yet to be fully elucidated, One crucial molecule seems to be the urokinase-type plasminogen activator receptor (uPAR) recently also assigned as CD87 antigen, uPAR serves a dual function: (1) it directs uPA proteolytic activity to a special location on the cell surface and (2) induces cellular signals leading to various phenotypic changes. We have investigated the signal-transducing capacity of uPAR in human SMCs and provide here a molecular explanation for uPARrelated cellular events. Activation of these cells with uPA (even with inactivated catalytic center) results in the induction of tyrosine phosphorylation, suggesting modulation of uPAR-associated protein tyrosine kinases (PTKs) upon ligand binding. We obtained patterns of tyrosine-phosphorylated proteins with molecular masses of ~ 55-65 and 35-40 kD. Using antibodies against different types of PTKs as well as immunoprecipitation-and immunoblotting techniques the PTKs involved in the uPAR-signalling complex were identified to be members of the Src-PTK family. The cotocalization of uPAR and PTKs at the cell surface of SMCs was further confirmed by confocal microscopy studies. We conclude that the uPAR-PTK complex is most likely involved in this signal transduction pathway that provides the coordinated action of extracellular proteolysis, adhesion, and cell activation, which is required for cell migration. This mechanism may be crucial for the progression of atherosclerotic plaques. Activation markers of haemostasis have been found elevated in relation to diabetic vascular lesions. Simultaneous pancreas-and kidney transplantation (PKT) in Type I Diabetes has been shown to improve diabetic complications and long term survival. We measured haemostatic vascular risk factors and activation markers in plasma of 18 patients after successful PKI', 17 patients after PKT and rejection of the pancreas graft and 5 patients after PKT and rejection of the renal graft. Blood samples were taken during routine ambulatory visits, patients were free of any ongoing acute disorder or transplant rejection and under continuous immunosuppressive medication. Despite individually adjusted insulin therapy HbA1 plasma levels increased after pancreas rejection (5,37 vs 7,I2, p<0.001). Platelet counts and plasma levels of fibrinogen, F1+2 fragment, TAT-, APP-complex and-Fibrin Monomer were found significantly elevated as compared to diabetic controls but not significantly different with respect to complete or partial successful PKT. One major reason of the increased activation state of haemostasis may be Cyclosporin treatment given to all patients, t-PA and PAl I plasma levels were within the normal range and significantly correlated to plasma triglyceddes (r.0.049; p<0.005). D-Dimer plasma levels were significantly lowered after pancreas rejection (772(375) vs 324(232) nglml; Mean(SEM) p<0.005), which might reflect impaired fibrin degradation related to increased glycosylation of fibrinolytic factors. In conclusion, despite the marked improvement of glucose and lipid metabolism, plasma markers of activation of coagulation and flbrinolysis are not decreased to normal after simultaneous pancreas and kidney transplantation. According to the investigations of Fowler et al. and Pepe et al. the probability of an ARDS occurring with one risk factor is 5-8%, and in the presence of several risk factors, 25%. Goris et al. and Johnson et al. determined the level of severity with the aid of a fixed scale: the injury severity score. All these investigations are however not to be interpreted as typical following coronary surgery. These investigations demonstrated that the kallikrein and factor XII systems are of great importance as intraoperative risk factors. Here the factor XII system plays a major role with direct or indirect activation of the kaUikrein-kinin system with the splitting products alpha-factor XIIa and Bfactor XHa respectively. All ARDS scores take the PMN-elastase into account. If the PMN-elastase values (1000 pg/l) are constantly high postoperatively then lung complications are to be expected. Patients developing an ARDS displayed significantly lower alpha2-macroglobulin values. Patients who developed a highly significantly raised kallikrein-like acdvity (>60 U/I) after the beginning of bypass and showed constantly high values during ECC are difficult to keep under control due to the blood pressure behaviour. The platelet PAl also shows a significant rise and intraoperatively runs analogous to platelet factor 4, only antiparailel, since it attacks the endothelium. We were able to show that PAI-1 is suitable as an indirect marker for a possibly developing restenosis. 85% of the patients investigated with lowered PAI-1 values in the postoperative phase did not develop a restenosis. However, with patients showing significantly rising PA[-1 values from the 1 st. to 3rd. postoperative day 50% of all the cases had a restenosis. A further risk factor in this respect are significantly raised fibrinogen levels which lay over 800% at the end of surgery. If these fibrinogen values do not fall from the 1st. postoperative day onwards a raised risk of thrombosis must be reckoned with in the absence of therapeutic intervention. The following parameters represented haemostaseological risk parameters with significant behaviour within the framework of this study: 1) regards the blood pressure behaviour, the kallikrein-like activity (>60 U/I); 2) with regards to the lung complications, aipha2-macroglobulin and PMN-elastase (>900 g/i); 3) and final/y as a possible marker for a developing restenosis PAI-1 and fibrinogen (>800%). Resulting from numerous clinical studies homocysteinemia is found to be an almost independent risk factor of atherosclerosis including thrombotic complications as well as of venous thromboembolism. Experimental investigations on the underlying mechanisms suggest endothelial cell damage accompartied by the development of an atherogenic and thrombogenic potential, increased platelet reactivity, oxidative modification of LDL, and enhanced affinity of Lp(a) for fibrin. To our knowledge no results are published on the influence of homoeysteine on leukoeytes although these cells are deeply involved in pathological events within the vasculature. Therefore, as a first approach different functional parameters of human polymorphonuclear leukozytes (PMNL) were followed under incubation with 60, 300, and 600 I.tM (final concentration) DL-homocysteine (HC) in isolated fractions or whole blood, respectively: l) Spontaneous mobility of PMNL, measured as migration distance into micropore filters in a modified Boyden-chamber, is found to be significantly enhanced by the two smaller HC concentrations. 2) Chemotaxis induced by 0.1 I.tM formylmethionylleueylphenylaianine (tMLP) shows no significant differences. 3)Monitoring of chemiluminescence signals (AutoLumat LB953, Berthold) is complicated as HC influences the luminol-mediated indicator reaction. Adjusting appropriate conditions the following results are obtained: Spontaneous chemiluminescence and that induced by zymosane, tMLP, and the Ca2+-ionophore A23187 are entranced by the two higher HC concentrations. There are, however, differences between the blood donors as a minority does not respond to HC in repeated measurements. With phorbol 12-myristate 13 acetate the signal is diminished by HC in all cases and with all concentrations. 4) Phagoeytosis induced by zymosane (microscopic evaluation) as well as by opsonized E. coil (cytoflowmetric evaluation) is significantly increased by the two higher HC concentrations. Conclusion: The activation of human PMNL is enhanced with respect to the majority of investigated stimuli by HC in concentrations reached under pathophysiological condititions. The effect of pysical exercise on hemostatic parameters was studied in 12 patients (male, mean age: 55 [range 36-68] yrs) with angiographically documented coronary artery disease (CAD) and in 11 controls (male, 53 [43-62] yrs) both participating in an 1 hour group exercise session for cardiac rehabilitation. In each group relevant arteriosclerotic lesions in carotid, abdominal and leg arteries were excluded by Doppler ultrasound examinations. Patients were all under 6-blocking agents and aspirin. Plasma levels of prothrombin fragment 1+2 (PTFI+2) and fibrinopeptide A (FPA) reflecting formation of thrombin and fibrin, respectively, were measured at rest and immediately after 1 hour of exercise consisting of jogging, light gymnastics and ball games. Training intensity in both groups was comparable as indicated by the mean heart rate during exercise corresponding in patients to 80+6% (mean-+SD) and in controls to 79-+5% of the maximal heart rate previously determined on a bicycle ergometer. Baseline values for PTF1 +2 were significantly lower in oatients (0.67-+0.15 nmol/I; mean-+SD) than in controls (1.01-+0.21; P<0.001 i. After exercise we found an increase of PTF1 +2 in controls to 1.14-+0.24 nmol/I (P<0.001) while in patients PTFI+2 remained unchanged (0.67-+ 0.14 after). Accordingly, exercise induced r se of FPA was more pronounced in controls (from 0.62-+0.24 to 1.60-+0.62ng/mt; P<0.001) than in patients (from 0.63-+0.26 to 1.20+0.46ng/ml; P<0.00t). We conclude that in terms of thrombin and fibrin generation exercise training does not exert detrimental effects on hemostasis in patients with CAD. Lower baseline values and lack of exercise induced increases of PTF1 +2 in patients with CAD might be attributed to medication with aspirin and/or B-blocking agents.

Periodontitis marginalis (Pm) is an inflammatory oral disease that is caused by gram-negative bacteria and that has a high incidence in the second half of the life. Clinical signs of Pm are gingival bleeding, periodontal pockets, alveolar bone destruction and loss of teeth. Recent epidemiologlcal studies have provided some evidence for an association between Pm and atherosclerosis. In the present paper we will summarise some of the results that we have obtained in studies on patients with Pm as well as on patients with hypercholesterolaemia (HC) and atherosclerosis. Pm was frequently found to be associated with HC (90 % in rapidly progressive Pm) and increased reactivity of peripheral blood neutrophils and platelets (e.g. generation of oxygen radicals and PAF-induced aggregation). Patients with HC and atherosclerosis had a higher frequency of severe Pm when compared with data on the community periodontal health. The severity of Pm was higher in patients with plasma cholesterol levels _> 6.5 mM when compared to those with plasma cholesterol < 6.5 mM. In patients with coronary atherosclerosis the severity of Pm was significantly correlated with plasma cholesterol level, systolic blood pressure and the number of diseased coronary arteries. These results provide further evidence for an association between Pm, HC and atherosclerosis. It can be speculated that HC is not only a risk factor for atheroscterosis but also a risk factor for Pm and acts by increasing the reactivity of neutrophils and platelets. On the other hand, Pm as a mild chronic inflammation could promote the development of atherosclerosis due to effects of endotoxins on vessel wall, blood cells and haemostatic factors. It has been also speculated that phagocyting leukocytes in the inflamed periodontal tissues could contribute to oxidative modification of LDL. So far, there is no evidence that atherosclerosis may contribute to the pathogenesis of Pro Protein Z (PZ) is a vitamin K dependant plasma protein synthesized in the liver. It promotes the association of thrombin with phosphorlipid surfaces. Recently it has been shown that a deficiency of PZ may lead to a bleeding tendency. In patients undergoing chronic hemodialysis, disorders of hemostasis are common. To examine if plasma levels of PZ are altered in patients with end stage renal disease we determined PZ in plasma of patients at the beginning of hemodialysis treatment. The results were compared with a group of 50 healthy controls. The difference of PZ levels in plasma of patients with end stage renal disease with the control group was not significant. Control group was 2900 + 487 ng/ml and in patient group was 3133 + -1394 ng/ml. One patient with marked bleeding tendency after hemodialysis PZ was 1387 ng/ml. We concluded that patients with bleeding disorders PZ determination may be helpful. The normal range of Actin FS was reinvestigated in a multicentric approach. A protocol was developed which requests from each center to assess the aPTT with one common and one variable lot of Actin FS in 50 samples of suspected normals. Inclusion and exclusion criteria based upon the results of clotting assays, liver enzymes and clinical data were defined. Results: A total of 1056 results was obtained. The majority of centers in this study used the Electra 1000 or 1600 C (MLA). Results for the Electra group (n = 6) showed a precision for the common lot of Actin FS with a common lot of a three level control from 1.5 % (Level 1) to 1.1% (level 3) with an excellent accuracy between the 6 centers. Clotting times with the variable lots of Actin FS were very similar. The results from normals, however, showed a somewhat higher dispersion using the common lot of Actin FS. 4 of 6 centers had almost identical mean values (range 26.6 to 27.2 sue) whereas one reported shorter and one longer clotting times (25.2 and 29.7 sec). Results with the variable lots gave almost identical results as the common one. A total of 556 results of all lots gave a normal range of 23.5 to 31.7 (5 -95 % percentiles) on Electra. Mean values on ACL (n = 200) were 26.3, on BCT, 27.65 sec, on AMGA coagulometric, 29.4 sec, on AMGA turbidimetric, 26.6 sec (n = 100 each). All centers used Sarstedt monovettes with 3.2 sodium citrate. Discussion: The results of this study demonstrate the lot to lot consistency of all lots of reagents included in this study since the common and variable lots showed very consistent results. Interestingly in the large group of Electra users the normal ranges showed some differences, though the controls in all centers were almost identical. This confirms the recommendation that a normal range as stated from the manufacturer should be used for orientation only and that each laboratory should assess its own range. Direct acting anticoagulant agents such as hirudin (r-H), argatroban (Arg), efegatran (Efe) and PEG-hirudin (PH), represent specific and potent inhibitors of thrombin. Blood samples collected in r-H (10 ~g/ml), Arg (50 ~tg/ml), Efe (25 ~tg/ml) and PH (10 ~tg/ml) do not clot for extended periods (>24 hours), thus allowing for the collection of plasma for analytical purposes. Unlike heparin, these agents do not require any plasma cofactor for their anticoagulant effect. In contrast to citrate, oxalate, EDTA and heparin, these antithrombin agents do not alter the electrolyte or protein composition of blood. Thus, blood collected in these agents may provide a physiologically intact (native) sample for clinical laboratory profiling. We have used all of these agents to prepare whole blood and plasma samples for various diuical laboratory measuroments. Plasma samples collected with these agents are obviously not suitable for global clotting tests (PT, APTT, thrombin time, fibrinogen); however, these agents are optimal anticoagulants for the collection of samples for the molecular markers of hemostatie activation, such as fibrinogen/fibrin related degradation products, prothrombin fragment, protease cleavage products, TFPI, TNF and other protein mediators. Electrolytes, blood gases, enzymes and protein profiling can also be satisfactorily measured on blood samples collected with these agents. Antithrombin anticoagelatad blood used fur hematologic analysis showed equivalent blood count and differential results as that obtained with EDTA blood. Unlike other anticoagulants, these agents do not interfere in the cell staining process. Washed blood cells can also be prepared using antithrombin aents supplemented buffers for morphologic and fuuctional studies. Thrombin inhibitors such as hirudin have also been used for flow cytometry and image analysis of blood cells and tissue exudates. Our observations suggest that these anticoagulants can be used as suitable anticoagulants for clinical laboratory blood sampling. These agents can also be used as a flush anticoagnlant fur most automated instruments as these exhibit superior anticoagulant properties to heparin. Furthermore, the hematologic parameters obtained in antithrombin anticeagnlated blood may be physiologically more relevant than those determined on blood collected in EDTA, citrate or heparin. Antithrombin Ul determination is one of the most popular method for in vitro diagnostic of number of different disorders. Human fhrombin a~nity purified on heparin-rnodified silica-based sorbents was used for level of antithrombine lU determination by Abilgaard method in blood of 40 patients with pregnancy pathology, acute leukemia, thrombocytopenia and anemia. It was founded, that antithrombin level is decreased to 50-60% of normal values in case of pregnancy pathology, to <50% -in case of acute leukemia and thrombocytopenia, to S0% -in case of anemia. Obtained results show the strong relationships between named disorders and patient antifhrombin III level. Therefore anfifhrombine III estimation may be used as simple and quick method for preliminary diagnosis of above named disorders. BM Coasys 110 is a complete automated analyzer system for coagulation tests. It is well suited for routine coagulation testing in random access in a medium throughput laboratory environment. Analytical performance and practicability were tested by a common evaluation program in five hospital laboratories.

Within run and day to day CV's were below 5 % in different samples (controls, patients) . Comparison in different therapeutic ranges confirms the declaration of the ISI-value for calculating INR-values. Normal values for coagulation tests with results in pdmary units were checked in 390 samples and confirmed. Due to the optical measuring principle of the BM Coasys 110 there was a little tendency to shorter times with the thrombin reagent.

In conclusion the performance of coagulation tests with the BM Coasys 110 was rated as well or better compared to existing systems in the laboratories with advantages due to short timed familiarizing and easy handling.

Flexibility and stability of the system permit optimal integration and innovation into the w0rkflow of the routine laboratory. The purified thrombin and antithrombin III (AT III) have a great interest in the clinical diagnostic and treatment practice, so their isolation methods are very important. Molecules of these proteins have some fragments replying for interaction with native glycosaminoglycan, heparin. This interaction is used for isolation and purification of thrombin and AT Ul from native materials, blood plasma or its fractional products. We have done comparative studying these proteins purification on heparin sorbenfs, which contain heparin immobilized on sificagel, modified by glycidooxipropyl, gamma-aminopropyl and tosyl chloride groups, or on cellulose: heparin-epoxy-silica (1), heparin-gammapropyl-silica (2), heparJn-tozylsilica (3), and heparin-cellulose (4). We founded that thrombin binds with all sorbents, while AT III doesn't binds with sorbents 2 and 3. There wasn't any difference between silica and cellulose sorbents in thrombin desorbfion by t M NaCI. AT III binds more stronger with heparin-ceUulose t[~,~n with silica sorbents but specific activity and purity degree were approximately the same on both kinds of sorbents. Thrombin specific activity and purity degree were approximately twice higher on sorbents 2 and 3 in comparison with sorbents ! and 4 (2250-3000 NIH units/mg versus 1260-t350 NIH unlts/mg). Therefore, sorbents 2 and 3 can be used for isolation and purification of thrombin and sorbents t and 4 can be used for isolation and puriiication of AT II1. We used these sorbents for large scale purification of named proteins. Purified thrombin was used for production of diagnostic kits for anfithrombine III, fibrinogen, fibrin/fibrinogen degradation products and thrombin time determination. After an aerobic or anaerobic physical exercise various alterations of the hemostatic system were detected. Numerous investigations of the hemostatic system exist of running and of bicycle ergometer exercise but not of swimming. Young volunteers (n=54; median age 25 years) were investigeted~ There was an aerobic exercise (achieved heart rate 130 --10/min, lactate < 4 mmol; n= 27) and an anaerobic exercise (achieved heart rate 150 ~ lO/min, lactate > 4 mmol/1; n= 27). In both groups there was a significant shortening of the PTT. Under anaerobic conditions hematocrit and quick significantly increased. Factor VIII activity rose significantly in both groups. Indicating plasmatic clotting activation there was a significant increase in molecular markers TAT and F1+2 only under anaerobic conditions (TAT from 2,5 to 5,4 pg/1; F1+2 from 0,87 to 0,93 nmol/1). Indicating activation of fibrinolysis t-PA activity increased significantly in the anaerobic group (from 0,1 to 0,4 IU/ml) but not in the other group. This findings indicate that there is e balance in the hemostatic system by activation of clotting as well as of fibrinolytic system in young volunteers during exercise by swimming dependend on the degree of exercise load.

Membranes as well as compact, porous disks are successfully used for fast analytical separations of biopolymers. As far as capacity, speed and performance of separation are concerned, the supports are as effective as other recently developed fast media for the separation of biopolymers °). So far, technical difficulties have prevented the proper scaling-up of the processes and the use of membranes and compact disks for preparative separations. In this report, the use of a compact tube made of poly(glycidyl methacrylate) for fast preparative separations of proteins is shown as a possible solution of these problems. The units have yielded excellent results, regarding performance and speed of separation as well as capacity. The application of compact tubes made of poly(glycidyl methacrylate) for the preparative isolation of the coagulation factors VIII and IX from human plasma shows that this method can even be used for the separation of very sensitive biopolymers. In terms of yield and purity of the isolated proteins, this method was comparable to preparative column chromatography. The period of time required for separation was five times shorter than with corresponding column chromatographical methods. Our measurements showed an excellent correlation of the two systems (r=0,99). The maximum amplitudes on the roTEG were on average 2.2% higher than on the HTEG, corresponding to a slightly lower reverse momentum of the measuring system in comparison to the HTEG. We report first results out of the evaluation of STA Compact (Boehringer Mannheim/Diagnostica STACK)). STA Compact is designed for automated analyses of routine and special coagulation (chronometfic, photometric [405 nm] and turbidimetric [540 run]) tests. In addition, it does measure ,,derived" fihrinogen. Tests as follows were evaluated: Prothrombin time (PT), partial thromboplastin time (aPTT), fibrinogen (Clauss method), thrombin time, AT III (chromogen), Hepato Quick, as well as the factors II, V, VII, X, and VIII. Results: Within run CVs of the clotting tests were below 2% (calculated on the basis of seconds) in most cases, day to day CVs below 4% (not measured for factors, yet). AT III yielded within run CVs below 3% in the decision range. Measuring ranges: AT III: 20-140 %; fibrinogen: 1.3-9.0 g/l (plasma -dilution 1/20), after rerun with other dilutions: from 0.3 g/1 (dilution: 1/5) to 18 g[L (dilution: 1/40). Method comparisons, using STA as reference, yielded slopes close to 1.00 and negligible intercepts. Throughput: With routine clotting tests about 100 tests/h, in a sample selective access mode. We conclude, that STA Compact allows precise measurement of routine and special coagulation tests. It is also a reliable system for photometric tests and well suited for intermediate workloads as well as STAT analyses. We did evaluate PTT LT, a new liquid, silica based PTT reagent. Special attention was given to reference interval and heparin sensitivity. The new reagent is well suited for the measurement of intrinsic clotting factors and is reported to have high sensitivity for lupus anticoagulants (higher sensitivity than STA aPTT [Boehringer Mannheim = BM]). It is stable for 4 days in the cooled compartment of the STA analyzer. Methods: All experiments were made on STA. For comparison, we used three other PTT reagents (a lab. routine, silica based aPTT, as well as STA aPTT and STA PTT Kaolin from BM). In addition, thrombin time (3 U/mL thrombin, STA Thrombin reagent) and heparin (chromogenic Xa test, Rotachrom Heparin) were measured. Results: Within mn imprecision (n=21) was below 0.7 % CV in the normal range and in two controls (mean values: 35 s, 76 s), and 1.4 % in a heparin plasma (mean: 81 s). Between day imprecision (d=10) was below 3% in two controls ( mean values: 34 s and 58 s). The upper limit of the reference range is 40 s (97.5 th perc., median: 32 s; 90 patients with normal coagulation status [routine aPTT, fib., PT], median age: 54 years); almost identical reference ranges were obtained with STA aPTT and the routine PTT reagent, while STA PTT Kaolin showed significantly lower values (97.5th perc.: 33 s, median 28 s). Method comparison study: good agreement using plasmas from patients without heparin: (y= 0A + 0.98 x, n= 198, range of(x) from 25 to 79 s, r = 0.97; x = STA aPTT). The median values from 54 patients under high dose heparin were: routine PTT: 81 s, STA aPTT: 75 s, PTT -LT: 82 s0 STA PTT Kaolin 54 s, thrombin time 37 s and heparin 0.5 IU/mL In conclusion, results of the new reagent compare well to our routine PTT and to STA aPTT system reagent. It allows sensitive monitoring of high dose heparin therapy and is well suited for detecting abnormalities of the intrinsic clotting factor pathway. is a standard technique since many years. The interpretation of the thrombelastograms has been widely based on phenomenologic observations, while there is a lack of exact information concerning the coagulation mechanisms leading to the TEG amplitude (A'rE~). The Aa'Ec is a measure for the mechanical stiffness of the clot and depends on: A) Fibrin formation and adequate polymerisatiun of a 3-dlmensional network: measurements with nonrecalcified citrated blood activated with ADP or epinephrine (both n=10) did not show any clot formation in the TEG This relies on the need for a mechanical coupling between the TEG pin and cup over a distance of 1 mm, which is accomplished by the fibrin network Therefore, TEG can only be performed under thrombin formation and thus under thrombin-activation of the platelets in the sample. Factors, which inhibit platelet aggregation but don't limit thrombin-activation of platelets, cannot be monitored by TEG. B) The attachment of the dot on the surface of the TEG pin and cup. According to recent literature we suggest that the attachment of the clot in the TEG relies exclusively on fibrinogen/fibrin adsorption to the surfaces of the pin and cup. Interruption of this attachment can result in lower amplitudes or the so-called ,,stairway" phenomenon. We could show a complete interruption of the clot attachment by dipping the pin for one second in 30% albumine solution (n=10). C) The fibrinogen concentration (Fg) and platelet count (PC) of the sample. In 50 volunteers we found only a poor correlation of the maximum amplitude (MA) with Fg alone (r=0.40) or PC respectively (r=0.50), while there was a very good nonlinear correlation to the product of Fg and PC. We suggest that the fibrin network forms the main structure of the clot while the thrombocytes enhance its stiffness in a concentration-dependent manner. This effect of the ptatelets can be completely reversed by GplIbfllla antagonists. D) Adequate coagulation activation: In nonactivated TEG even small amounts of inhibitors can lead to a significant reduction of the ATEG. Conclusion: Alterations in TEG measurements can be judged more properly when the underlying mechanisms are understood. The consideration of the limitations of the method allows a more specific interpretation of the results. As a response on a customer request we did investigate the sample stability of blood samples for the aPTT. The study was set up in a way that simulated the conditions of a large private laboratory in which the samples arrive several hours after blood collection. Blood was drawn from 10 donors into 3.2 % sodium citrate and mixed well before it was divided into several aliquots Which were kept at room temperature. The aliquots were centrifuged after ~ 0,5, 11, 23 and 29 h after venopuncture and the plasma was analyzed immediately with 3 different reagents on Electra 1000. Results: There was a clear difference in the change of the aPTTover time with these reagents. Also F VIII (determined with a chromogenic assay with complete and standardized activation) change considerably. 2 Reagent A: ellagic acid, plant phospholipid, reagent B: sulfatide/kaolin, phopholipids, reagent C: ¢llagi¢ acid, plant and rabbit brain phospholipids The increase of aPTT was apparently not a function of the decrease of FVIII because the in vitro F VIII sensitivity of reagent B. was inferior to reagent A though reagent B showed more prolongation of the aPTT than reagent A. Reagent C, however, showed only minor changes in the aPTT. Discussion: These data show that the sample stabifity of the aPTT is reagent dependent and that it is not simply a function off VIII sensitivity. Other factors such as the buffer system but also the sensitivitiy towards other factors than F VIII seem to contribute.

A COMPARISON OF THE TECHNICAL PRINCIPLE OF THE roTEG COAGULATION ANALYSER AND CONVENTIONAL THROMBELASTOGRAPHIC SYSTEMS An. Calatzis, P. Fritzsche. AL Calatzis, +M. Kling, +R. Hipp, A. Stemberger Institute for Experimental Surgery and +Institute of Anesthesiology Yechnische Universit~t M0nchen Thrombelastography (TEG) was introduced by Hartert in 1948 as a method for continous registration of the coagulation process. In 1995 we presented the roTEG Coagulation Analyser, using a newly developed technical method. In TEG systems according to I/artert the sample (blood or plasma) is placed in a cup which is alternately rotated to the right and left by 4,75 °. A cylindrical pin, which is suspended freely on a torsion wire, is lowered into the blood. When coagulation starts, the clot begins to transfer the rotation of the cup to the pin against the reverse momentum of the torsion wire. The angle of the pin is electromagnetically detected, transformed to the TEG amplitude and continously recorded. In the roTEG the pin is attached to a short axis, which is guided by a ball beating. Thus all possible movement is limited to rpotation (r_oTEG). The cup is stationary, and the pin is rotated alternately by 5 ° to the right and left by a feather system. When a clot is formed, it attaches to the surfaces of the pin and cup and starts preventing their relative movements against the reverse momentum of the feather. Here the reduction of the rotation of the pin, which is detected optically, is tranformed to the TEG amplitude. As can be shown by theoretical analysis and by control measurements, the roTEG provides the same measuring capabilities as conventional TEG systems. The main advantage is the solid guiding of the measuring system, which makes the roTEG easily transportable and less susceptible to shock or vibration during measurement. Yhrombelastography (TEG) is a standard monitoring procedure for evaluation of coagulation. Usually only nonactivated native blood TEG measurements (NaTEG) are performed, which leads to a) a long time interval until coagulation and fibrinolysis parameters are available b) very high susceptibility of the measurement to inhibitors like heparin, which disturbes the judgement of other components of coagulation, c) unspecific results. Our aim was to develop a coagulation monitoring system based on TEG providing fast and specific information on the different components of coagulation. Methods: The following measurements are performed in paralel using disposable pins/cups (Haemoscope): a) Extrinsic activated TEG (ExTEG): 3551al whole blood (WB) + 5~tl Innovin (recombinant thromboplastin reagent, Dade). b) Intrinsic activated TEG (InTEG): 3551al WB + 5~tl Kaolin (suspension 5g/l, Behring). c) Aprotinin TEG (ApTEG): ExTEG + 20 KIE Aprotinin (Trasylol, Bayer). d) Heparinase TEG (HepTEG) as decribed in (1). Results: ExTEG and InTEG provide information on the extrinsic/intrinsic system within 5-10 min and information on the platelet/fibrinogen status within 10-20 min. Because of the addition of potent activators InTEG and ExTEG can be performed when inhibitors like Heparin are present in the circulation. Fibrinolysis effects can be seen on ExTEG and InTEG and by comparison of ExTEG and ApTEG (ApTEG: invitro-fibrinolysis inhibition by Aprotinin). If fibrinolysis is detected by ExTEG or InTEG and aprotinin-susceptibility is verified by ApTEG, aprotinin therapy will be initiated. Heparin effects are revealed by HepTEG. Discussion: By the comparison of parallel TEG measurements which have been differently activated, specific and fast information on the different aspects of the clinical coagulation status is provided. The presented tests can be easily performed bedside and only a small specimen of whole blood is needed (0,4-1,8 ml). Introduction: A severely prolonged aPTT (333s; normal: 40~Os) was observed during preoperative screening for a planned splenectomy in a 71-year-old man with an 8 year history of osteomyelofibrosis. Fellewing neer-normal~atien (71s) of the aP'CI" after 10 rain preincubation in a kaolin based aPTT assay, PK deficiency was suspected and studies were performed to further investigate the nature of the PK deficiency as well as the mechanism underlying the normalization of the prolonged aPTT by increasing the preincubation time. Methods: The aPl-r assay was peal'armed using kaolin/inesithin. High molecular weight kininogen clotting activitiy (HK:C), FXII:C end PK:C were measured by an aPTT based assay using Neothromtin ® (Behnng) and 1 rain (PK:C) or 4 min (HK:C, FXlI:C) preincubation. PK amidolytic activity (PK:Am) was assayed using Cosset PK ~ (Chromogenix) and PK antigen (PK:Ag) by quantitative immunoblotting. FXll and HK proteolysis dunng activation of plasma by kaolin (10mg/ml at 37=C) or DS (12.5~tglml at 4=C) was demonstrated by immunotilotting assays of FXII and HK following SDS-PAGE.

Assay PK:C PK:Am PK:A~I FXfi: The propositus had PK:C<5%, PK:Am=5% and PI~Ag<2.5% as compared to normal pooled human p(asma (NHP). His son and two daughters had PK:C-50% and normal aPTT values, incubation of the propositus' plasma with DS did not result in FXII or HK cleavage within 180 rain, whereas Jn NHP detectable F×II and HK proteolysis occurred after 5 rain and complete proteolysis was observed after -120 rain. In contrast, kaolin activation of propositus' plasma led to slow activation of FXII after 10 rain, presumably by autoactivation, and to FXlla-induced HK proteolysis. Near-normalization of the propositus' aPTT by prolongation of the preincubation time paralleled FXII autoactivation as evidenced by immunobletting.

We describe a propositus with severely prolonged aPTT due to hereditary, CRM negative PK deficiency suffering from OMF. Activation with a particulate suspension of kaolin led to slow FXII autoactivation and HK proteolysis, whereas DS in solution did not induce FXII or HK cleavage. FXII autoactivation seems to be responsible for the normalization of the prolonged aPTT in PK deficiency after prolonged preincubation times. In our study we compared a conventional bag with silicone tubing (A) for blood donation with 2 new ones (]3 from Biotrans and C from Baxter) with a newly developed Y-shaped adapter. This adapter is integrated into the tubing and therefore provides the advantage for drawing blood samples in a closed system. The 3 systems were identical in amount and content of anticoagulant, i. e. 63 ml of CPD per bag resulting in approximately 14% of the final whole blood volume. The purpose of the study was to determine whether the different tubings can influence the quality of plasma products conceming the blood coagulation system. In plasma samples we measured several factors of the procoagnlatory and fibrinolytic systems. Intralndividual control eitrated (.135 M) blood samples were initially drawn from the contralateral cubital vein from the same male donor (34 in each group). In all bag samples we found small but significantly higher levels of the global test parameters aP'IT and TI" compared to controls, indicating a higher amount of anticoagulant. PT, however, revealed no differences, thus suggesting that factor activities were not altered (statistics according to Mann-Whimey). Increase of procoagulatory activity measured as TAT complexes showed elevated levels in bags A and C whereas prothrombin fragments Fl+2 decreased only in A. Conceming the fibrinolytic system, plasminogen a~tivators and PAI-1 values were diminished in all three systems 03 < A < C) compared to controls. D-dimers were lowest in A followed by slightly higher values in C, controls and B. Fibrin monomers did not reveal any significant differences: A < C < controls < B. In summary, the quality, of the 3 different blood sampling devices was comparable to the intraindividual controls as to factor activities measured by global tests. The activation of the procoagulatory and fibrinotytic systems was slightly but in most cases significantly higher in the two new devices than in the conventional one. All values, however, obtained from the plasma samples did not exceed the normal range of healthy blood donors. Therefore we concluded that the two new closed blood drawing systems are favorable for blood donating procedures. In 20 patients with acute myocardial infarction (AMI) and thromholytie therapy (13 patients with rt-PA, 6 patients with streptokinase and one with heparln) with CK, myoglobin and EKG criterions the patients were divided in two groups (reperfusion/no fellow two hours after starting the thrombolytic therapy) . Blood samples were taken before, 30 rain, I h, 2 h, 4 h, 8 h,12 h after lysis and than every day till day 10.

Because of the central role of factor XII in activation of coagulation, fibrinolysis, kallikreln-kinin-system and complement cascade we investlgate the role of factor XIla initiated by AMI and the relation of factor XIIa to the thrombolytie agent and reoeclusion rate. For the investigatlens we take the kits from SHIELD DIAGNOSTICS (XIIa), Behring Diagnostica (C~-Inactivator, pl~nogen, ~-antip]~n~n, PAP), Chromogefiilx AB (prekallikrein) and Di~nostica Stage (Vile).

The results: There is an increase of factor XIIa immediately after starting the fihrinolysis (max. 30 rain after starting); the increase 5/I independently of the thrombolytie agent. Parallel to factor XIIa raises factor VIIa without significant changes of C1-1naotor and prekalllkrein. That means: activation of XIIa and fibrinolytic pathway leads to relatively mild c.hanges in kallikrein system, hut to significant activation of extrinsic system by VIla-tissue factor. In some patients is an additional rise in the system XIIa -VIIa, when the fibrinolytic system is already in the normal range. There will need further investigations to define the risk of reocclusion as a result of activation of faktor VIIa by faktor >LI Ia. Autoimmune thrombocytopenic purpura (AITP) is a frequent complication of chronic lymphocytic leukemia (CLL] which developes on different stages of the disease and needs special treatment measure. Mechanism of autoimmune disorders in CLL remains uncleared. We investigated immunologic phenotype of blood lymphoid cells in 22 patients suffering from CLL with AITP. In these patients we did not observe disorders in expression of B-lineage markers as compared with CLL patients without immune complications (13 patients). But in the 1st group of the patients the greater number of B-celts expressed markers of activation. According to Ig heavy chain expression, the lymphocytes in most cases of CLL complicated by AITP had more mature phenotype. In all patients with k phenofype of CLL lymphocytes we found immune disorders. The development of AITP was accompanied by lowered level of T cells and changed dis'flibution of their immunoreguiatory subsets: diminished number of CDZ~cells and increased one of CD~'÷lymphocytes. The results of our investigations undirectly proved that malignant B-cells in CLL are involved in production of autoantibodies against blood cells. Dysbalance in T-cell system with functional disturbances of immunoregulation are significant in development of autoimmune complications in CLL A24 In women with severe FVII deficency (<10%) hypermenorrhagia may cause life threatening blood loss. Therefore, hysterectomy at a young age is reported frequently in the literature.

A 12 year old girl without history for a bleeding disorder was transfered with hypermenorrhagia. The initial laboratory data revealed an abnormal Quick-test of 30% due to FVll of 9,0%, normal platelet count and hemoglobin level of 7,2 g/dl. Antifibrinolytic therapy (tranexamic acid 4x15mg/kg BW/d) and lynestrenol substitution were started to reduce the hemorrrhage. Despite treatment the daily blood loss increased to a maximum of 290ml. Therefore, substitution therapy with recombinant FVIla (rFVIla) (NovoNordisk) was started at a dose of 15 Ilg/kg BW q 6 h. Subsequently blood loss decreased to 30ml/d, but even with an increasing dose of rFVlla up to 35 I~g/kg BWq 4h (FVil activity max. 7400% 10 min after injection) and additional hormonal support with a LH-FSH-anatgonist some hemorrhage remained. A short .course of methergin was stopped due to severe pain. Ultrasound of the uterus revealed a hypertrophic endometrium causing the persistent bleeding. It decreased slowly over several weeks and hemorrhage stopped completely after 40 d. The total rFVlla dose administered was 118 rag. No side effects were observed. No transfusions of blood products were necessary. Currently, menstrual cycle is suppressed by estriosuccinate. Conclusion: Due to close cooperation with a specialised gynecologist, hypermenorrhagia was controlled and in this woman with severe FVll deficiency hysterectomy was avoided. In three male members aged between 27 and 52 years of a family suffering from inherited bleeding disorders the diagnosis of protein Z deficiency was established. Plasma protein Z evaluated by ELISA (Asserachrom Protein Z, Diagnostica Stago, France) ranged between 200 and 300 ng/ml. The patients mostly suffered from moderate bleeding complications like prolonged bleeding secondary to trauma or invasive measures and also spontaneous hematuria. Previous laboratory investigations revealed variable platelet function deficiencies and transitory boderline decrease of von-Willebrand factor. Spontaneous bleedings were rarely recognized, however, they occured more frequently when analgetics were taken. Bleeding complications showed good response to hemostyptic measures and antifibrinolytic therapy. The use of PCC containing a high level of protein Z in these patients is restrained to severe bleeding disorders or major surgery. Defibrotide is a mammalian polydeoxyribonucleotide derived anti-ischemic and antithrombotic drug (Crinos S.p.A., V"flla Guardia, Italy). While the drug is known to produce polytherapeutic effects owing to its multicomponent nature, the exact mechanisms of its anti-ischemic effects remain unknown at this time. Since defibrotide is found to be effective in ischemic disorders such as PAOD, VOD related occlusive disorders and related rnicroangiopathic conditions, we studied the effect of this drug on the contraction of dog and pig arterial strip/rings obtained from various sites. In vitro supplementation ofdefibrotide to the organ bath containing control dog and pig arterial rings did not modulate the serotonin and thromboxane (generated) contraction, however, tissues obtained from dogs treated with 10 mg/kg defibrotide IV exhibited a profound desensitization to the agonist induced contractile process. The time course of these effects was found to be much larger than the plasma half-life of defibrotide. This presentation will provide additional data on the effect of defibrotide on the contraction of vascular smooth muscles as a possible explanation for the anti-ischemic effects of defibrotide.

A. Wehmeier, A. Popescu, W. Schneider Klinik for H,~matologie, Onkologie und klinische Immunologie der Heinrich-Heine-Universit&t D0sseldorf

In chronic myeloid leukemia (CML), evolution of blast crisis is the limiting factor of survival. However, as in other chronic myeloproliferative disorders, bleeding and thrombotic complications are a major source of morbidity but their incidence has rarely been analysed in larger patient groups. We retrospectively evaluated 182 patients with CML during chronic phase (170 cases), accelerated disease (58 cases), and blast cdsis (72 cases), and determined the incidence of thrombohemorrhagic complications in relation to the stage of the disease. In chronic phase, 28 patients had bleeding complications (8.4%/patient year) and 15 patients thrombotic episodes (4%/patient year). The incidence of bleeding increased significantly in accelerated disease (18 patients, 51.2%/patient year) and blast crisis (37 patients, 347%/patient year), and many patients had repeated complications. Contrary to our expectations, the incidence of thrombotic complications also increased to 10.2%/patient year in accelerated phase and 39.8% /patient year in blast crisis, tn chronic phase, 3 patients died because of bleeding events. In accelerated phase, 5 patients died due to bleeding and 1 patient due to thrombotic complications. In blast crisis, bleeding was associated with 21 deaths, and pulmonary embolism with 2 deaths. Analysis of the cause of thrombohemorrhagic complications revealed that in chronic phase, bleeding was often associated with uncontrolled busutfan therapy, whereas in blast crisis, severe bleeding occurred mainly when platelet counts were low and peripheral blasts increased. However, there was no obvious explanation for thrombotic complications. We conclude that bleeding and thrombotic complications are a major source of morbidity and mortality also in CML, and that the incidence of such complications increase in advanced stages of the disease. Klinik for Innere Medizin °, Klinikum Schwerin Patients suffering from primary or secondary amyloidosis may occasionally acquire a coagulation disorder characterised by isolated factor X deficiency. We report on a 60-years-old man who presented with lower gastrointestinal bleeding and prolonged prothrombin time (Quick 50 %). Amyloidosis was suspected and proven using biopsy of the rectum and histological analysis. In addition, a monoclonal gammopathy of undetermined significance was diagnosed by immunofixation (light chain, type X). Detailed investigation of the prolonged prothrombin time led to the discovery of a pronounced factor X deficiency (residual activity 4 %). Inhibitors of coagulation factors could not be demonstrated. The treatment of the patient consisted of red blood cell transfusion and infusion of prothrombin complex concentrates. Due to the extremely rapid clearance of infused factor X, no increase of its activity was observed. Chemotherapy of the monoclonal gammopathy was initiated (melphalan/ prednisone). Over the following six months the frequency of major bleeding episodes gradually decreased. However, subclinical occult bleeding continued. The factor X activity was repeatedly found between 10 and 12 %. We support the suggestion from literature data that clinically relevant bleeding episodes are likely to occur in patients with amyloidosis-associated factor X deficiency if the residual activity is below 10 %. Sepsis and septic shock is a disease entity which is characterized by inflammatory reactions (SIRS), coagulation abnormalities (DIC), organ failure (MOF) and severe hemodynamic alteration frequently leading to death in a shock. The aim of our studies was to investigate the efficacy of antithrombin III (Kybernin ®) on ~he outcome of septic shock in a pig endotoxemic model. Pigs, in this model respond to LPS with elevated TNFlevels, decreased leukocytes and platelets counts, increased TAT and fibrin monomer levels, hypotension and in increase of the pulmonary arterial pressure (PAP), indicating impaired lung function. A total number of 13 male castrated juvenile domestic pigs (25 -30 kg) were anaesthetized, ventilated mechanically and infused with Saimonella abortus equi lipopolysaccharide (S. equ-LPS) over three hours (0.5 ~g/kg * h). A Swan-Ganz-Catheter was inserted into the pulmonary artery to measure the PAP. Animals were allocated to two groups,, the treatment group (n = 7) received antithrombin III (AT III) according to the following regimen: 250 U/kg (t = 60 -30, i. v. infusion), 125 U/kg (i. v. bolus, t = 0) and 250 U/kg (t = 180 -240 rain, i. v. infusion). The placebo group ( n = 6) received the appropriate amount of human serum albumin: 50 -25 -50 mg/kg (same schedule as with AT III). Main objective was defined as the mortality rate at six hours a_~er S. equ-LPS infusion. Whereas in the placebo group 4 out of 6 animal died (mortality rate: 66 %) all AT III-treated pigs survived the observation period of 6 hours (p < 0.05, X2-test). The AT III group was shown to have a lower PAP than the control group, especially the second peak of hypertension was abolished by AT III. It is therefore concluded that AT III should be a useful tool for the treatment of severe sepsis and septic shock. In a nationwide monthly survey all childrens hospitals in Germany (ESPED) were asked to clinical and therapeutical informations about children suffering from PMI. During July 94 till June 95 299 children were registered. From these, 87 had either ecchymoses and/or necroses related to an increased mordibity and mortality (20%), whereas 212 showed no bleeding signs except for petechiae. Of these children one died. The therapeutic interventions concerning hemostasis are listed according to the defined two risk ~oups. From the patients with ecchymoses or necroses, 13/87 received combination therapy (compared to 5/212 with petechiae or no bleeding sign) of AT III, heparin and/or plasma. Only t child received protein C concentrate. The data show that children with low risk did in part receive higher doses of heparin and/or AT III concentrate than did high risk patients, whereas plasma therapy was adjusted to severity of eoagnlopathy. Furthermore, the wide range of given therapeutics allows no information about the different medications. Therefore, controlled studies with respect to the different therapeutic interventions in children with high risk PMI is desirable.

A FULLY AUTOMATED PROCEDURE FOR THE REPTILASE TIME ASSAY Y. Schmitt (1) and H.J. Kolde (2) (1) Institute for Laboratory Medicine, St~dtisches Klinikum, Darmstadt, FRG, (2) Dade Diagnostics, Unterschlei6heim

The reptilase time assay is a relatively simple technique for the detection of fibrinogen degradation products and fibrinogen deficiency or abnormality. The procedure is performed with citrated plasma and batroxobin reagent, a snake venom enzyme from Bothrops atrox. This enzyme cleaves fibrinogen by releasing fibdno peptide A only but not fibdno peptide B. In contrast to the physiological enzyme thrombin that is readily neutralized by antithrombin III and hepadn batroxobin is not inactivated by physiological inhibitors. At present this assay is mainly performed manually or on mechanical instruments. We have adapted this assay to the Electra 1000 fully automated coagulation analyzer (Medical Laboratory Automation, Pleasantville, N.Y.) using the thrombin clotting time procedure in the instrument software with batroxobin reagent (Dade Dia-

The clot formation is registrated turbidimetrically and the dotting time is pdnted. The within run precision (n= 10) of this procedure was tested with two plasmas from the daily routine and was between 2.8 and 3.4 %.

In 25 normal samples we found clotting times from 10.5 to 12.8 sec. In 30 samples with liver disease (confirmed by pseudochlinesterase < 2000 U/ML) or on thrombolysis therapy with streptokinase or urokinase the fully automated assay on the Electra was compared to the semiautomatic method using a KC 10 coagulometer (Amelung, Lemgo, Germany) based on a rolling metal ball pdnciple and magnetic endpoint detection. The two assays agreed very well with a correlation coefficient of r = 0,948 and a regression line according to Passing and Bablok of y = 1.0 x + 1.7. These data show that the reptilase time can be performed with good precision and with good correlation to the manual technique on mechanical instruments on the Electra 1000. Introduction: Disseminated Intravasal Coagulation (DIC), due to a massive activation of the coagulation system, is frequently observed in intensive care patients suffering from severe underlying diseases. Laboratory diagnosis of DIC is based on different coagulation tests, but unfortunately the routine haemostaseological parameters react with latency in the course of acute DIC Objective: In four cases from a cohort of 43 patients with severe sepsis and DIC we analysed special haemostaseological parameters (TAT, F1-T2, D-Dimers, Human-Leucocyte-Elastese (FILE), Catepsin G and Heparin Cofactor II (HC II)) and correlated them with a MOF-score in order to test their predictability on the prognosis of these patients. Results: All patients were substituted with AT III concentrate. L,1 the investigated patients median time of treatment with AT III concentrate was 8 (6-9) days and median time of DIC-duration was 6 (4-8) days. None of the presented patients died during observation period. All analysed parameters, except D-Dimers, showed a sufficient correlation with the evaluated MOF-score (TAT: r= 0,78; F1-F2: r= 0,84; HLE: r= 0,71; Catepsin G: r=-0,75; HC II: 1"=-0,88). The D-Dimers did not correlate with the MOF-score, which is probably due to the delayed reactive hyperfibrinolysis in the course of DIC. Furthermore, the decrease of the TAT-complexes, F1-F2, HLE and Catepsin G levels were followed by an increase of AT HI and HC II activity. Conclusion: In general the analysed activation markers and coagulation parameters are sufficiently to describe the ongoing process of the DIC. The hyperfibrinolytic activity of DIC is sufficiently represented by the D-Dimer test, but is of defered reactivity in the course of DIC. Unfortunately these parameters are not established in the routine monitoring of DIC on intensive care units and therefore further studies are needed to investigate the practicability and reliability in the daily routine monitoring. We have previously reported that Notoginsenoside R1 (NG-R1) has an effect on counteracting lipopolysaccharide (LPS) induced upregulation of plasminogen activator inhibitor-1 and tissue factor expression in cultured human umbilical vein endothelial ceils in vitro and in mice in vivo [Fibrinolysis 1994;8:(suppl 1)119]. In this study we investigated the effect of NG-R1 on prevention of LPS induced lethal toxicity in mice. Because mice are relatively resistant to LPS when applied as a single agent, we sensitized them by simultaneous treatment with D-galactosamlne. The 80% lethality induced by LPS (1.5 mg/mouse) plus D-galactosamine (8 mg/mouse) in C3HS-Ie mice was reduced to 16% by simultaneous administration of NG-R1 (1.5 mg/mouse) with LPS/galactosamine (P<0.05 by X 2 test). NG-R1 also significantly delayed LPS/galactosamine induced lethal toxicity from 12 hours to 30 hours with all animals surviving beyond 30 hours. Because lethality induced by LPS involves the synergistic effect of multiple effector molecules such as tumor necrosis factor (TNF)-ct, interleukin (IL)-I, interferon 3' etc., we also investigated the effect of NG-R1 on LPS induced TNF-ct production from leukocytes in cultured human whole blood cells (HWBCs) ex vivo. The production of TNF--ct induced by LPS (1 ng/mL for 24 hours) in the supernatant of HWBCs was inhibited by 46% and 22% respectively, when the cells were incubated 1 ng/mL or 10 ng/mL LPS together with 100 I~g/mL NG-R1, respectively (TNF-ct concentration, 1 ng/mL LPS treated cells: 297+192 pg/mL, I ng/mL LPS plus 100 l.tg/mL NG-RI treated cells: 162+137 pg/mL, P<0.01; 10 ng/mL LPS treated cells: 3094_+487 pg/mL, 10 ng/mL LPS plus 100 pg/mL NG-R1 treated cells 2423+713 pg/mL, /'=-0.02). The present results suggest that NG-R1 can prevent the onset of LPS toxicity as well as the LPS induction of cytokines. Therefor NG-RI may be effective in preventing the effects of septic shock in Gram-negative infections. To elucidate the mechanisms by which coagulation is initiated in septic patients in vivo, coagulation measurements were prospectively evaluated in patients with severe chemotherapyinduced neutropenia. This group of patients was chosen because of their high risk of developing severe septic complications, thus allowing serial prospective coagulation testing prior to and during evolving sepsis or septic shock. 62 patients with febrile infectious events were accrued to the study. Of these, 13 patients progressed to severe sepsis and an additional 13 patients to septic shock. At onset of fever, Factor (F) Vlla activity, F VII antigen and antithrombin III (AT III) activity decreased from normal baseline revels and were significantly lower in the group of patients who progressed to septic shock compared to those that developed severe sepsis (medians: 0.3 versus 1.4 ng/ml, 21 versus 86 U/dl and 45 versus 95%; P < 0.001 ). The decrease of these variables in septic shock was accompanied by an increase in a marker of thrombin generation like prothrombin fragment 1 + 2 (medians: 3.6 versus 1.4 riM; P=O.05). These differences were sustained throughout the septic episode (P < 0.0001 ). F Vlla and AT Ill levels of <0.8 ng/ml and <70%, respectively, at onset of fever predicted a lethal outcome with a sensitivity of 100 and 85%, and a specificity of 75 and 85%, respectively. In contrast, FXIla-alpha antigen levels were not different between both groups at onset of fever and were only marginally higher further during the course of septic shock (P=O.001). Thus, septic shock in neutropenia is associated with significant coagulation activation, presumably driven by the tissue factor pathway rather than the contact system. Furthermore, in septicemia both F Vlla and AT III measurements are sensitive markers of an unfavourable prognosis.

Hemostatic parameters in sepsis patients treated with anti-TNFct monoclonal antibodies C. Salat 1, P. Boekstegers 2, E. Holler 1,3, B. Reinhardt I, R. Pihusch 1, K. Werdan 2, M. Kaul 4, T. Beinert 2, E. Hiller 1 Med. Klinik III 1 und I 2, Klinikum Grosshadern der Ludwig-Maximilians-Universitat MOnchen, H~imatologikum der GSF 3, Knoll AG Ludwigshafen 4

Tumor necrosis factor et (TNFc~) is a central mediator in the pathogenesis of sepsis and septic shock. As administration of anti-TNFct monoclonal antibodies was able to protect animals from an otherwise lethal endotoxin challenge clinical studies were initiated in patients with sepis. TNFct exerts a procoagulant effect, e.g. by enhancing PAI-I and activating thrombin as indicated by an increase in TAT and PF 1/2 levels. Therefore it may be involved in disseminated intravascular coagulation in sepsis. We determined TAT, PF 1/2, D-dimers, tPA, uPA, PAI-I and vWF levels in 30 patients with sepsis or septic shock. 14 patients received the anti-TNFa monoclonal antibody MAK 195F (Knoll AG, Ludwigshafen), whereas 16 patients served as controls. We found a significantly lower level ofuPA in anti-TNFc~ treated patients. Since the difference existed before onset of treatment it can not be attributed to TNFot antagonisation. All other parameters investigated did not differ significantly between the two groups throughout the study period. Failure to detect modulation of hemostasis by anti-TNF~ might be explained by delayed initiation of treatment in clinical sepsis. In animal experiments it has been observed that the antibody prevented lethal endotoxin effects when given prophylactically or 30 minutes after endotoxin challenge, but not when it was administered 2.5 hours later. In addition, beneficial clinical and hemostatic effects of TNFet antagonisation might be observed only in subgroups of patients with hyperinflammatory sepsis. Larger studies addressing this point are under way. Protease receptors for thrombin and trypsin have been described for different cell lines. We investigated the ability of trypsin to activate human umbilical vein endothelial cells (HUVEC). Cell activation was measured by the increase of intracellular free Ca 2* (Caff) with help of microscope fiuorometry (FURA-2) and by the von Willebrand factor release measured by a sandwich ELISA. Incubation of HUVEC with thrombin (1U/ml) or trypsin (10nM) showed a 2-10 fold increase of C~ff. A subsequent homologous stimulation after 80 s lead to a 2-5 fold lower concentration of Ca~ 2÷ compared to the first stimulation. Therefore cells have been desensitised by the first stimulation. Inhibition of the proteolytical activity of trypsin by soybean trypsin inhibitor was followed by failure of trypsin inducing an increase of Ca~ 2÷ concentration. In cross stimulation experiments with thrombin and trypsin, we could demonstrate, that cells first stimulated with thrombin showed a second maximal response by subsequent stimulation with trypsin. The same effect was measured with first stimulus trypsin and second stimulus thrombin. Trypsin and thrombin induced a release of von Willebrand factor (2-5 fold in comparison to unstimulated cells). We found a vWf release dependent on the concentration of trypsin similar to thrombin. An electrophoretic analysis of the released von Willebrand factor showed a different multimeric composition of vWf between trypsin and thrombin stimulation. These results indicate, that there might be a protease receptor on HUVEC for trypsin being different from the thrombin receptor. Clinical and laboratory findings of coagulopathy were investigated by an 1-year-survey to 320 children's hospitals. 291 meningococcal infections were evaluable. Severe disease (characterized by need for mechanical ventilation, dialysis and/or catecholamines) was seen in 42 of these children; 29 of those survived and 13 died. Clinical signs of severe coagulopathy were seen in 83 children: Ecchymoses (n = 73) and skin necrosis (n = 36) were associated with increased mortality (16% and 20%, resp., compared to 4.5% overall mortality). Five of 29 surviving children with skin necroses required surgical interventions (skin transplantation and/or amputations). Petechiae were frequent (n = 156) and as isolated finding not related to severe disease or fatal outcome (6% mortaliy). Platelet counts at admission were lower in non-survivors (10th-90th percentile: 30 -450.000/gl, median: 139.000/I.tl) than in survivors (10th-90th percentile: 140 -480.000/I.tl, median: 242.000/gl). AT III values showed no difference between survivors and non-survivors. Protein C was available in few patients (n =14): in this subgroup, protein C was lowered in patients with limited disease (10th-90th percentile: 20 -105%, median: 48%) as well as severe disease (10th-90th percentile: 30 -75%, median: 60%). In conclusion, the findings "ecehymoses" and "skin necroses" were related to fatal outcome and therefore included in a prognostic score for severity of meningncoccal disease.

The influence of irradiation on PAI-I and vWF levels in human umbilical vein endothelial cell cultures K. Fragiadaki, C. Salat, R. Pihusch, B. Reinhardt, M Penovici, E. Hiller Med. Klinik III, Klinikum Grosshadern der Ludwig-Maximilians-Universitat MOnchen An elevation of PAI-1 in bone marrow transplant recipients developing veno-occlusive disease (VOD) of the liver has been described earlier. Endothelial cell damage due to the preparative myeloablative radioehemotherapy is supposed to be an important step in the pathogenesis of the disease, which is characterized by an obstruction of small intrahepatic venules. In order to investigate a possible role of irradiation we studied the influence of several doses (0, 5, 15, 30 gy) on PAI-1 and vWF levels in the supematant of human umbilical vein endothelial cell cultures (HUVEC). PAI-1 antigen and vWF were determined by enzyme immunoassays. Whereas PAI-1 and vWF levels remained unchanged alter irradiation with 5 Gy and in control cultures, a rise was observed one day after irradiation with 15 Gy (mean day 0"-)day +1) in PAI-1 (100,0% --)171,2 %) and vWF (100%--)159,7%) levels. The increase was more pronounced and reached levels of statistical significance after a dose of 30 Cry (PAI-1 100%--) 278,7% and vWF 100%--)168%). Both PAI-1 and vWF levels decreased on day 2 after irradiation with 15 and 30 gy.

Our results indicate that irradiation induces an increase of PAl-1 and vWF in endothelial cells. Nevertheless, this effect was observed only in doses above those ones used during conditioning when patients receive 3x4 gy. Additional factors seem to be of significance. Cytokines like TNFo~ enhance PAI-1 and vWF in endothelial cell cultures and are known to be elevated in BMT-associated complications. It can be speculated that irradiation in concert with these factors may contribute to the development of veno-occlusive disease. Disseminated intravascular coagulation is characterized by high consumption of coagulation factors, systemic elevation of fibrinolysis by tPA and concomitant elevation of PAI-I secreted from inflamed endothelial cells. In an attempt to investigate the contribution of inflammatory cytokines, endothelial cells lines of microvascular origin were stimulated in vitro and PAl-1 antigen was measured 2h, 4h and 24h after stimulation. In contrast to results published from experiments performed with macrovascular human umbilical vein cells (HUVE), our results obtained with 3 different microvascular endothelia isolated from skin, solid tumor tissue and bone marrow revealed that inflammatory cytokines reduced PAl-1 antigen levels. In addition to TNF-a (25ng/ml) and LPS (10pg/ml), we found that IL-10 (100 U/ml) and GM-CSF (100 U/rot) also reduced PAI-I levels within the first 2h of incubation (from 120ng/ml to 80-110 ng/mll and the effect was even more pronounced after 4h and 24h (from 380 ng/ml to 250 ng/ml). IL-1 (10 U/ml) and LPS (10 pg/l) also reduced constitutive levels of PAl-1 but the effect occured later than 4h after addition of the stimulator. The strongest synergistic effect was demonstrated with GM-CSF plus IL-1 resulting in PAl-1 suppression of 50% after 2h and 30% after 24h. In contrast, G-CSF (300 U/ml) induced the immediate (120 to 140 ng/ml after 2h and 380 to 420 ng/ml after 24h) upregulation of PAl-1 antigen. Stimulation of PAt-1 levels was also observed with TGF-I~ (10 pg/ml), however not earlier than 18h of incubation. Interestingly, both stimulatory cytokines, ie. G-CSF and TGF-13, alone were able to counteract the decrease of PAt-1 antigen by TNF-a but only a combination of G-CSF plus TGF-g neutralized the effect by IL-1. Results indicate that inflammatory cytokines regulate PAl-1 fibrinolysis in a synergistic and antagonistic fashion. We established the culture of human brain microvascular endothelial cells (HBMEC) in order to investigate the pathophysiology of hu~man cerebral malada, which is still associated with a high mortality rate. It is widely accepted that among the reasons for the fatal outcome of cerebral malaria, the interaction of endothelial cells with cytokines and paras lites with subsequent changes in haemostaseological parameters is involved. The human microvascular endothelium may therefore play a deci §ive role in the pathophysiology of cerebral malaria. Ery throcytes containing later stages of P. falciparum specifically bind to capillary EC in vivo (sequestration). TNF-cq IL-1 and IL-6 are considerably elevated in severe malaria. Coagulation factors such as tissue factor and von Willebrand factor are affected by malada suggesting the involvement of the HBMEC in cerebral malada. So far, research on the involvement of the HBMEC has been performed on EC cultured from human umblilical veins (HUVEC). The relevance of this model may be questioned on t, ,he grounds that the capillary endothelium probably plays a greater role than the endothelium of the large vessels. Besides, some propertie.$ of the endothelioum seem to vary, upon the organ of origi/n. For the~ reasons, our laboratory has established the HBMEC as a model to study the pathophysiology of human cerebral malaria. To demonstrate the relevance of this model in the context of malaria, HBMEC were challenged with sera from different patients with severe P. falciparum malaria and with serum from a healthy donor. We can demonstrate that in cells challenged with malaria patient sera ICAM-1 and Substance P were upregulated. On the other hand cells challenged with serum from a healthy donor expressed neither ICAM-1 nor Substance P. These results strongly suggest the relevance of this model for vessel involvement in malaria. Both, histamine and serotonin have been described as potent stimulators of yon Willebrand factor (vWf) release from human umbilical vein endothelial cells (HUVEC). We performed experiments to differentiate the receptors for histamine and serotonin induced vWf release. Absolutely unexpected we don't found any significant vWf release after the addition of serotonin to HUVEC or human artery endothelial cells (HUAEC) in concentrations from 0.1 IJM to 50 pM. In the case of histamine (0.1 pM -50 pM) we measured a vWf release 2-5 fold compared to unstimulated cells. This release was in the same order of magnitude as the release induced with 11U thrombin. To verify these results we measured the effect of histamine and serotonin on the intracellular Ca 2÷ concentration (Ca~ 2÷) in HUVEC and HUAEC. Cells were labelled with FURA-2 and the change in fluorescence after agonist addition was measured with a microscope fluorometer. Using the same agonist concentrations as above we found an 5-10 fold increase of Caj 2. with histamine or thrombin but no effect by addition of serotonin. This results indicate a similar activation of human endothelial cells by histamine and thrombin and that serotonin don't stimulate endothelial vWf release or increase of Cay. Activation and/or dysfunction of the endothelium can be triggered by cytokines (e.g. interleukin-2, tumor necrosis factor-alpha) or bacterial substances (e.g. endotoxins) and may contribute to shock and multi organ failure. PAl-l and TM were assessed as parameters of activated endothelium following BSCT in three to four days intervals from start of conditioning therapy through day +35. Data were compared to the occurrence of sepsis, veno-occlusive disease (VOD), capillary leakage syndrome (CLS) and graftversus-host-disease (GvHD). Patients with neither complication served as controls.

No *days after stem cell tranplantation PAI-1 and TM were increased in all patients with sepsis, CLS~ VOD and/or GvHD. PAI-1 peaked at days 14 to 18 and the increase was highest in sepsis and lowest in CLS. The increase in TM values was somewhat delayed (day +24) and was highest in VOD and CLS and lowest in GvHD. PAI-1 and TM are sensitive markers of endothelial activation in sepsis, VOD, CLS, and/or GvHD, but they do not allow a differention between these complications. 

Endothelin (ET) is the most potent vasoconstrictor. It is known that ET plasma concentration is correlated with a poor prognosis in patients with non ischemic cardiomyopathy (CM). The contribution of the heart to the production of ET is still unknown.

To investigate the pathogenetic mechanism in patients without coronary artery disease (CAD), we examined 13 patients with hypertension ( . Pulmonary capillary wedge pressure (PCWP) was measured in all patients. ET and its precursor big-endothelin (BET) were determined at rest and after pharmacological stimulation with dipyridamole (0.5 mg/kg body weight), that increases coronary blood flow by factor 2 -4 on a non endothelial pathway. Cardiac coronary ET and BET concentrations were determined from the arterial blood samples, obtained from the aorta, and simultaneously from the coronary sinus (venous blood). Blood samples were collected into ice chilled Vacutainer tubes and stored after centrifugation at -70 *C. ET and BET were analysed after extraction by a SePal< C 18 cartridge by radio immuno assay technique (Immundiagnostik). It is concluded that ET is increased with elevated filling pressures of the heart in patients with CM. It is not produced in considerable quantity by the heart neither at rest nor at increased blood flow. There4ore the lung has to be considered as the major organ for the production of ET and BET in patients without CAD. To characterize the incompatibility of blood with foreign surfaces valide in vitro methods especially in testing of platelet function are neceessary. It seems to be effective to use test systems which can also be helpful lateron in the clinic when foreign surfaces (e.g. venous catheters) are used and evaluated in so called phase-4-studies. We studied the influence of 21 reference polymers under standardized and controlled flow conditions on platelets in citrated blood specimen of healthy blood donors.The following tests were performed pre and post platelet-pol)aner contact: decrease of platelet count, platelet aggregation (Wu-Gmtemeyer index), analysis of platelet spreading capacity on standardized plastic surfaces by using a visual microscopic evaluation according to Breddin and Bfirck (1963) and an interactive computer-aided system (IBAS, Kontron GmbH, Manchen, FRG) by digitalizing the morphological picture of the platelet slides and area detection with a resolution of 512x512 pixels. Results: Platelet counts showed significant differences pre and post polymer contact, the Wu-Grotemeyer index demonstrated platelet activation only by blood contact with large volumes of polymeric material whereas both visual and computer-assisted evaluation of platelet spreading ability revealed a marked shift in the different classes of platelets: platelet activation results in a decrease of large structural elements and an increase of elements with spider threads. (Pre contact (n=1000): 27:~-6 large forms of platelets, 700~-39 small forms and 275:L-41 spider forms; post contact (n=1000): 6-+-5 large forms, 510a:56 small forms and 484±58 platelets with spider threads). In some series there were significant differences between visual and computer-aided evaluation in the detection of small and spider forms. However, the relative increase of these nonspread spider forms could be stated with beth methods (Wilcoxon test). We therefore conclude, that platelet morphometry with both methods is a sensitive and reliable ex vivo method to evaluate platelet interactions with artificial surfaces and can also be used lateron in phase-4-studies in patients. However, the IBAS-system requires further maprovement in hard-and so,ware to reduce the high expenditure of this method. Despite for the most part standardised methods such as hypothermia, cardioplegia the perioperative myocardial infartion rate is still high at approx. 6%. In cardiovascular surgery it is well known that various cardioplegic solutions are employed for myocardial protection during the ischemic phase. In order to evaluate the possible influence of these solutions we selected two of the most commonly used cardioplegic solutions for investigation in a randomised double-blind study: HTK (group 1) and St. Thomas (group 2). After randomisation each group consisted of 20 patients who had to undergo aortocoronary bypass surgery. Aim of the investigation was to establish possible varying cellular changes during the reperfusion phase or in the early operative phase in order to be better able to apply reinforcing clinical measures.

In the context of this study the classical enzyme-diagnostical methods CK,CK-MB and LDH as most useful, however not as convincing. Still, we have in the meanwhile been able to show that the cardiac muscle Troponin T proves a particularly sensitive parameter regards differentiated ischemic damage to the myocardium. ~his we were able to confLrm in extensive preliminary trials. Cardiac Troponin T was registered with a one-step lmmunoassay using two highly specific monoclonal antibodies directly via two different epitopes of cardiac Troponin T. Simultaneously the corresponding pre-and postoperative ECG was registered. Further, within this context we investigated parameters that indicate cellular damage, such as platelet factor 4 (PF4), t-PA, Interleukin-6 and PMN-elastase. In the reperfusion phase in group 2 there is a significant rise in Tmponin T while in group 1 these values remain practically unchanged up to the 1st. postoperative day. Of special importance is interleucin 6 since according to most recent studies the release of this substance leads to platelet activation via the arachidonic acid metabolism. This pathway must, further, be regarded within the context of free radical formation. On the 1st. postoperative day the 11 6 values in group 2 are significantly higher. The effects of membrane damage is also observed via PF4 and the PMN-elastase to be different in both groups.

On the basis of this study we arrive at the conclusion that the HTKcardioplegia is essentially less damaging than that of the St. Thomas solution. (2) R. Hetzer (2) (1) Department of Hematology and Oncology, Vimhow Klinikum, Humboldt University, Berlin, Germany (2) We investigated the influence of two different VAD systems on these hemostatic changes.

VADs were implanted in 18 patients [11 bi-VAD (Berlin Heart), 7 left VAD (Novacor N 100)] with end-stage heart disease who were awaiting heart transplantation.

The following hemostatic parameters were measured during the first 51 days of bddging or until heart transplantation: thrombin-antithrembin III (TAT) complexes, prekallikrein, factor (F) Xll, plasminogen, or2 -antiplasmin, and I?,thremboglobulin. RESULTS: During the first week of bridging, significantly higher TAT levels were observed in Novacor patients compared to Berlin Heart patients. Prekallikrein activity levels were significantly lower in the Berlin Heart patients in the early bridging period. All other parameters were comparable in both groups throughout the entire observation period.

Differences in hemostatic parameters became apparent only in the early bridging period with more enhanced pmthrombin activation in the Novacor group and more prominent contact activation in the Berlin heart group. Avoidance of the transmission of viral infections and saving in the use of blood products encouraged the use of apparatwe intraoperative autetransfusion techniques. Patients and methods: ARer randomization apparative intraoperative autotransfusion was performed in 5x7 patients during elective hip surgery using I-Iaemonetics Cell saver Ill, Haemonetics Cell saver V, Electromedics Elmd, HaemoLite 3 and Fresenius Continuous AutoTransfnsion System (CATS). At defined tmaes we detenmned a lab panel (clinical chemistry, lipids, proteolytic capacity, hemolysis, coagulation panel) at 9 determination points in the reservoir, the retransfused blood and in the patient. Results: No significant differences concerning proteolytic capacity, prothrombin time, platelets, lipids, electrolytes. Increased hemolysis (p<0.01) in the HCS III group vs. the other groups (lO rain. after application of the retransfnsed blood). Low heparin concentrations of retransfused blood in the HCS III group( 0.32+-0.3 U/ml) vs. high concentrations in the CATS group (0.47 +-0.3;p--0.01). Parameters of thrombin generation were elevated in the HCS III group vs. the other groups (p=0.02). Conclusions: The use of 5 different apparative autotransfnsion systems dunng elective hip surgery results in dysturbances of hemocompatibility. The activation of the coagulation system during the collection and filtering is partly influenced by the elimination kinetics and the dose regime of heparin. However intraoperative autotransfusion must be roan~ged very carefully and possibly adverse effects of perioperadve heparin peak levels have to be considered. Little information is available on the management of patients with factor VIII deficiency who require cardiac surgery. We report the case of a 54 year old man with factor VIII deficiency and combined severe aortic stenosis and incompetence and mitral incompetence who underwent a double valve replacement at our institution. He had a history of several bleeding episodes following minor surgery. Previous factor VIII levels were between 8 and 26%. Using standard cardiopulmonary bypass, a double valve replacement with a 23 and 29 mm bileaflet prosthesis in aortic and mitral position, respectively, was performed. A high dose aprotinin regime was used (5.5 x 10 a IU). Three doses of factor VIII concentrate were given in the perioperative period, totalUng 7000 1U until the 1st postoperative day. Repeated measurements of the factor VIII level were performed. The postoperative chest tube drainage was 350 rot. Until the 4th postoperative day an additional dose of 3000 IU of factor VIII was given to maintain a level of at least 30%. The obligatory anticoagulation was achieved initially with heparin i.v. in therapeutic dosage. Due to a persistent 3rd degree AV block a permanent pacemaker was inserted with additional 2000 IU of factor VIII. On the 17th postoperative day Warfarin was commenced aiming for an INR of 3.0 -3.5. The patient was discharged home thereaRer. He was trained to monitor his INR with a Coagu Chek device. No bleeding episode occurred during the first 3 months follow up. Open heart surgery can be performed safely in patients with factor VIII deficiency with the use of factor VIII concentrates and monitoring of factor VIII levels. Coating of biomaterials was developed using synthetic polymers with incorporated anticoagulants. Stents were coated with a thin layer consisting of a polylactide polymer containing PEG-Hirudin and a stable prostacyclin analogue. These materials were tested with a ,,human shunt model" using nonant/coagulated blood of healthy volunteers. Within minutes uncoated stents were covered by fibrin and aggregated platelets, which could be seen macroscopically and by scanning electron microscopy; coated stents were free from coaguiation plugs. This observations were supported by analysis of coagulatiuon activation markers. Unlike coated stents, uncoated stents revealed high levels (>detection limit) of TAT complexes and prothrombin fragments (F1-2). In a series of experiments stents were tested in sheep. In 16 sheep stents (coated/uncoated Patmaz-Schatz stents) were ptaced by conventional techniques in the left anterior descending artery. Anticoagulant therapy consisted of a heparin bolus and intravenously given aspirin before stent implantation. No ant/coagulation was given thereafter. Existing data show hyperplasia in the area of uncoated stents which was reduced around coated stents (this study will be finished in January 1996). This coating technique with incorporated anticoagulants reduces thrombogenicity during the early and late phase of biomaterial implantation. Studies concerning catheters, vascular prosthesis and oxygenators are in progress. The mechanical circulatory support (MCS) is a therapy for patients (pts) with endstage cardiac insufficiency. During MCS thrombeembolic events, due to the surface thrombogenicity of the implanted device, are feared complications. Activated blood platelcts play a major role in this context. Therefore, patient's platelet morphology was investigated. During the period of MCS, using the Novacor left ventricular assist system N100, blood samples of 8 pts were observed by means of scanning electron microscopy (SEM). Blood was collected preoperatively and after implantation daily during the first week as well as weekly for the first 3 months. Samples were drawn via an 18gauge cannula into caeodylic-acid buffered glutaraldehyde and platelets were prepared for morphological investigations. Platelet alterations were classified as non activated, activated and aggregated, based on "shape change" morphology. Additionally, the common blood coagulation parameters were evaluated. Preoperatively, 15.0 + 4.6 % of activated platelets were found. Within the first postoperative week, the mean level of activated platelets raised to 32.8 + 8.0 % (p<0.05). Comparing short-(<30 days) vs. long-term (>30 days) MCS, a significant difference of activated ptatelets (overall mean values) could be seen (24.3 +_ 3.3 % vs. 34.8 _+ 3.4 %, p=0.004). During MCS a correlation between hemolysis and platelet aggregates, as well as the values of activated dotting time and activated platelets were observed. Also, specific platelet deformations and damages appeared during MCS, which could not be found preoperatively. All pts with MCS showed alterations of their platelet morphology induced by the activation of the implanted synthetic material. With regard to the postoperative antithrombotic therapy, these observations should be taken into consideration. During extracorporeal circulation (ECC) the blood and its compenents are exposed to artificial surfaces and inflammatory respenses are activated, especially the complement, coagulation, fibrinolytic and kallikrein systems. Furthermore leukocyte activation occurs and platelet function is impaired. These humoral and cellular systemic responses are known as the "pustperfusion syndrome" with clinical symptomes like lenkocytosis, increased capillary perraeability, accumulation of interstitial fluid and organ dysfunction. The impertance and even perhaps the existence of the damaging effects of CPB have been widely debated in the literature over the past 30 years. Many efforts have been made to reduce traumatizing factors, e.g. the use of membrane instead of bubble oxygenators.

Recently, heparin-coated equipmen~ and tubings have been proposed to avoid excessive contact activation during CPB, The here presented study was designed to assess changes in coagulation and flbrinolytie activity in 20 patients undergoing CPB. In this regard we investigated coagulation parameters like fibrinogen, antithrombin, pmthrombin-fragments Fl+2, thrombin-anthhmmhin complex, tissue-factor, fibrin-monomeres and parameters of the fibrinolytic system like tissue-plasminogen-activator, plasminantiplasmin-complex, d-dimers and plasminogen-activator inhibitor before, during and after CPB. The activation of the complement cascade was followed by measuring the concentration of C5a, C4 and C3c.

The results demonstrate distinct alterations in above mentioned parameters. In spite of a high dose hepariulzation (ACT>450s) combined with an antifibrinolytic tw, atment an activation of the coagulation system was observed immediately after the onset of CPB followed by an activation of the fibrinolytic system. Therefore further efforts should be done to develop new anticoagulatory regiments and improve the biocompatibility of materials used for CPB. During cardiopulmonary bypass blood is exposed to nonphysiologic conditions. The contact with artificial surfaces and mechanical stress result in a periopemtive response which includes activation of the complement, coagulation, fibrinolytic and kallikrein system, activation of nentrophils with degranulation and pmtease enzyme release, oxygen radical production and the synthesis of various proinflammatory cytokines. This so-called "pest-pump intlammatory response" has been linked to respiratory distress syndrome, renal failure and neurologic injmy.

Our goal was to investigate the time course of eytokine levels and the activation of leukozytes and platelets and to quantitate leucocyte subpepulatioas in 20 patients undergoing CPB. At different time points, pre, during and pest CPB, we determined the levels of interleukin (IL) 113, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor ¢z (TNF-a) and interferon "1' (IFN'--/) using ELISA-techulques. Lymphozyte subpepulations were characterized by flow cytometry and specific monoclonal antibodies against CD3 (pan T-cell marker), CD4 (surface antigen on T-helper cells), CD19 (surface antigen on B-cells), monocytes were determined by CD14 and platelets by CD41 (act. GPIlb/llla) and CD42b (GP Ib). Single cell activation was analyzed using markers against CD25 (IL-2 receptor), CD126 (IL-6 receptor), HLA-DR (MHC class II), CD71 (transferrin receptor) and CD69 (activation inducer molecule), platelet activation was monitored with an antibody against CD62 (GMP-140).

Preliminary results revealed distinct increases in R,-6, IL-8, and IL-IO following CPB whereas TNF-a and IFN--/levels were not significantly influenced. Fttnhermore, activation of particular cell populations was observed. Finally, our investigations should contribute to a better understanding of the complex humeral and cellular respenses induced by CPB and thus might help to develop new strategies to circumvent the negative impacts of CPB. Optimal adjustment of anticoagulation in machine plasmapheresis is important for the quality of the prepared fresh frozen plasma (FFP) as well as for the safety of the donation. In the present study the suitability of prothrombin fragment ( Ft+2 ) in the assessment of anticoagulation during plasmapheresis was investigated.

Matarlal and Methods: 75 plasmapheresis procedures were performed on 25 donors (10 ~, 15o" ) using 3 different plasmapheresis machines (A 200, Baxter; MCS 3p, Haemonetics; PPH 900, Electromedics/Medtronic). Acid Citrate Dextrose formula A (ACD-A) in a ratio to whole blood of 8 : 92 was used for anticoagulation. The concentration of FI+2 in the donor's blood was measured before and after plasmapheresis and in the prepared FFP. The actual ACD-A volume used was also registered. Results: There was a significant rise of the Ft+2 -concentration in the donors blood after plasmapheresis with each of the three automatons: A 200:1.32 vs 1.14, p < 0.05; MCS 3p: 1.26 vs 0.98, p < 0.05; PPH 900:1.20 vs 1.05, p < 0.05. The FFP prepared with each machine showed the following F~+2concentrations: 0.91± 0.18, 1.0:2 ± 0.17 and 0.93 ± 0.11 respectively. The difference between the groups was not significant. The elevation of the Ft+2 -concentration in the donor's blood showed a negative correlation with the volume of the ACD-A used. During 6 of the 75 procedures technical problems occurred (inadequate venous acces, occlusion of the citrate tube, reduced whole blood flow). After these procedures there was a marked elevation of F~+2 in the donors blood (2.74 ± 0.53), accompanied by an elevated F~+2 -concentration in the prepared FFP's. Conclusion: These data show that Ft+2 is a suitable parameter for the assessment of anticoagulation during plasmapheresis. Several epidemiologic studies demonstrated that fibrinogen is an independent cardiovascular risk factor and should be considered for screening programs. Prothrombin time derived fibrinogen (DF) measurement combines the advantage of an established highly reproducible automated method with no additional reagents, except for calibration. Several studies showed that the DF values correspond well with the Clanss method except in cases such as thrombolytic therapy in which the DF results are higher. However, no results exist whether in patients with coronary heart disease with fibrinogen as a risk factor the DF values are also comparable to the established Clausss method. The aim of our study was to compare DF values to Clauss method results in cardiac patients, especially in patients before and after coronary bypass grafting (CAB(]). Measurements of DF were performed on an ACL 3000 (IL) using the PT-Fibrinogen-HS reagent. Fibrinogen Clanss method was done on the ACL using Fibrinogen C reagent (IL) and on a KC4 (Amelung) with Fibrinogen a reagent (Boehringer Maanheim). For calibration we used the Calibration plasma half volume (It.) with the fihrinogen concentration proposed by the manufacturer. Plasma samples were obtained from 24 patients at admission before CABG and postoperatively up to 1 week, and from 23 healthy persons (staff). Within assay imprecisious using normal and abnormal controls (IL) were comparable with both methods showing cvs between 1.99 and 4.22 %. In normal healthy persons the medians of the DF and the Clanss method run on the ACL were very similar (296 vs 302 rag/all), whereas KC4 values were about 10% lower (268 md/dl). In CABG patients at admission we found the same differences as in normals with the Clanss method (ACL: 363 vs KC4: 337rag/all), however the DF values were siginficantly higher (median 418mg/dl). If we took a cutoff value of 320 mg/dl, as suggested by the results from the Northwick Park Heart Study, we would categorize into the high risk group 21 out of 24 patients using the DF method, 20 with the Clanss-ACL method and 16 with the Clanss-KC4 method, i.e. nearly 30% more patients were classified in the high risk group using the DF method. Postoperative samples showed the expected increases due to the acute phase response with the same magnitude of differences. Because of its rapidity and reproducibility the DF method is well suited for routine measurements, however, standardization remains an urgent task in order to avoid misinterpretation of results. For fibdnogen measurements in clinical laboratories, the two most widely used methods are the clotting time method according to CLAUSS (CFIB) and the sn called "derived" fibrinogen method (DFIB) implemented in optical coagulometera with the fibrinogen concentration being derived flora the optical density of the fibrin clot in a standard prothrnmbin time (PT) assay. It is well known that under certain circumstances, e.g. in the presence of fibrin(ogen) degradation products (FDP), there is a discrepancy between the two methods with higher values for DFIB than for CFIB. Yet the opposite discrepancy, i.e. fibrinogen values derived from the optical density of the clot grossly lower than values from dotting time assays, seems to be very rare and is poorly understood so far. The patient (male, 26 years) had ingested the esterase inhibitor parathion (E605) in an attempt o f suizide and was treated with high doses of atmpin. He had no clinical signs or history or family history of bleeding or thrombotic disorders. Except for a very low pseudocholinesterase activity, all laboratory results were normal ineinding PT, aFFT, thrombin time, and factor XiII. PT and aPTT did nnt differ between an optical COagulometer (Electra 1000C, MLA) and a mechanical one (KC..4, Amelung). There was no evidence of disorders known to interfere with hemostasis like paraproteinemia or dyslipldemia. However, in all 7 blood samples received for dotting tests during a period of 7 days the macroscopic appearance of the fibrin clot was quite unusual (only slightly turbid/almost transparent) and there was a striking discrepancy between a very low or low DFIB on the Electra (PT reagent: Thromboplastin IS, Dade) and a normal or high CFIB (KC4; thrombin reagent, Dade). On admission, values were 57 mgMl (derived) vs. 275 mgldl (CLAUSS). CFIB rose to S42 mg]dl with DFIB at 155 mg/dl in the last sample on day 7. ~ al! samples DFIB was about 20 % (lS-23) of CF[B. When the patient's plasma was added m normal pooled plasma it caused, in a dose-dependent manner, values lower than predicted for DFIB and values slightly higher than predicted for CFIB. In the absence of data from additional (e.g. immunologic) methods the following principal possibilities (and combinations) have to be considered: 1) normal fibrinogen concentration and clot formation rate, but abnormal optical properties of the clot (CFIB correct, DFIB falsely tow); 2) normal optical properties of the clot, but accelerated clot formation and very low fibrinogen concentration (DFIB correct, CFIB falsely high). In either case, the molecular basis could be: A) a genetic or acquired molecular abnnrmality of fibrin/fibfinogen; B) an interfering substance. Direct effects of the loxic agent parathion and/or the antidot drug atropin are not likely to be the cause since other patients, often with more severe parathion inmxicatian requiring higher doses of atmpin, showed normal optical density of the clot. We hope to perform a more in depth investigation of this abnormality in the future, including various methods, reagents, and instruments for fibrinogen measurement, a survey of the patient "s family, and studies of the molecular nature of the phenomenon. Increased fibrinogen is known to be an independent predictor of subseqtmnt acut~ coronary syndromes. However. a multitude of methods for fibrinogen determination is available. There is a lack of standardisation among fibrinogen assays. In a family cohort study (patients'with combined hyperlipidaemia and f or hypemricaemia) fibrinogen was determined in plasma samples from 340 family members using a functional and an immunochemical assay. The fimctional assay according to Clauss was performed on the analyser CA 5000 using the test fibrinogen a from Boehringer. The immmmephelometric assay was performed on ~e Behring Nephelometer System using the reagent and standard from Behring. A good similarity between both assays was obtained at low and high flbrinogen levels as well as in samples with increased C-reactive protein (CRP). Values obtained by both assays correlated similar with total cholesterol, LDL--cbelesterol and apolipeprntein B. The ratio functional fibrinagen / immlmochemial fibrinogen showed no dependence on cholesterol, t-PA, v WiUebrand factor and CRP. Release of two fibrinopeptides A from fibrinogen generates DesAA-fibrin monomer, which rapidly aggregates, forming fibrin complexes. Fibrin monomers can be detected in plasma samples after chemical desaggregation of fibrin complexes using thiocyanate by monoclonal antibody binding to the alpha-chain Neo-N-termini generated by fibrinopeptide release. Although postulated, an intermediate of fibrin formation, carrying one fibrinopeptide A and one fibrin alpha-chain Neo-N-terminus has so far escaped analytical procedures. We have employed a monoctonal antibody specific for fibrin alpha-chain neo-N-terminus, MAb 2B5, attached to magnetic microparticles, for isolation of fibrin-related material from plasma samples of patients with elevated soluble fibrin. The material was desorbed by SDS-urea buffer and subjected to SDS-PAGE and immunoblotting. Immunostaining with panspecific anti-fibrinogen and anti-FDP-E antisera showed a range of bands corresponding to fibrin monomers, and fibrin derivatives containing the fibrin E-domain. lmmunostaining with monoclonal anti-fibrinopeptide A antibody resulted in a doublet band corresponding in size to fibrin monomer. Similar results were obtained with polyclonal antisera against fibrinopeptide A. For a more quantitative approach, desA-fibrin monomer was detected by an ELISA procedure using MAb 2B5 as capture and monoclonal anti-fibrinopeptide A antibody as tag. A sample with extremely high level of desAA-fibrin monomer, determined by ELISA (Enzymun®-Test FM) was used for calibration, since reference material is not available. A correlation of r=O.g4 was found between DesAA-fibrin monomer and relative DesA-fibrin monomer levels. Detection of DesA-fibrin monomer required sample pretreatment with thiocyanate for desaggregadon of fibrin complexes. From these preliminary data it appears that DesA-fibrin monomer accounts for a fairly constant proportion of soluble fibrin and is a polymerizing species. Fibrinogen has been shown to be a major cardiovascular risk factor. Especially for epidemiological studies, exact quantitation of fibrinogen in clinical plasma samples is of great imporance. Fibrinogen levels are generally measured by clotting assay according to Clauss, or by determination of derived fibrinogen values upon photometric measurement of prothrombin time (derFbg). The clotting assay has been shown to be influenced by high levels of soluble fibrin derivatives. The PT-derived fibrinogen levels appear rather convenient in clinical routine, since no additional reagents are needed. We have compared the Clauss assay and derFbg with a turbidimetric fibrinogen assay using snake venom protease for fibrinopeptide release, performed in photometric autoanalyzers. D-direct antigen was measured in parallel using TINAqaant D-dimer LPIA. Results were correlated with total fibrinopeptide A release by thrombin, measured by ELISA. A total of 484 samples were included, of which 29 samples (6 %) were recorded as above measurung range by derFbg. These samples encompassed a range of 5.90-10.40 g/l and 5.21-12.37 g/l in Clauss, and turbidimetric assay, respectively. The range of values measured by derFbg assay was 0.72-9.14 g/I, corresponding to 0.26-11.00 gll and 0.24-10.48 g/1 in the Clauss and turbidimetric assay, respectively. The correlation of derFbg with the Clauss assay was re0.91, correlation with turbidimetric assay was r=0.92 for the values actually detected. The correlation between Clauss and turbidimetric assay was r=0.93 for all values. There was no dependency of test results or inter-test variation upon D-direct. Correlation graphs displayed a decreased test response of Clauss assay in the high concentration range, resulting in an underestimation of fibrinogen concentration. The derFbg assay, in contrast, showed normal range values in samples from patients with fibrinotytic treatment and low fibrinogen levels in the other assays. Correlation with fibrinopeptide A release was r=0.88 for Clauss assay, r=0.89 for turbidimetric assay, and r=0.82 for derFbg. For clinical routine, derFbg appears to be applicable for all samples between 1.00 and 5.00 g/l with exclusion of samples from patients with fibrinolytic treatment or endogeneous hyperfibrinolysis. Other samples may be analyzed by clotting assay or turbidimetric assay, although the latter appears to be more suited for measurement Of high range samples. for inhibition of PK is 0.067 pmol/L The antifibrinolytic activity of the inhibitors was determined by measuring the lysis of radiolabelled human plasma clots• The compounds which inhibit plasmin and PK influence remarkably the streptokinase-induced clot lysis but not lysis induced by UK and tPA. Surprisingly, inhibitors of UK and tPA do not influence clot lysis induced by UK or tPA. The structure-activity relationships for inhibition of ptasmin, UK, tPA and PK could help in the design of more potent inhibitors of fibrinotytic enzymes. UK inhibitors are of interest for the development of anti-invasiveness drugs, while plasmin/PK inhibitors could be prototypes of a "synthetic aprotinin". In the ECAT Angina Pectoris Study t-PA antigen was an indepcndem risk factor of subsequent acute coronary syndromes. PAt indicates the risk bat depends on other known risk factors. It should be tested in 183 members of a family cohort study (patients with combined hyperlipidaemia and / or hyperuricaemia), if the active PAl antigen or the whole PAI antigen showed a stronger relation to t-PA and metabolic variables. The active PAll antigen was determined using ELISA Actibind PAt-1 (Technoclone / lmmuno) , the whole PAI-I antigen was measured using the F_LISA PAt-1 (Technoclone I Immuno). t-PA activity was determined with the Coaset t-PA from Chromogenix, the TintElize tPA from Biopool was the used test for determination of t-PA antigen. The active PAt antigen showed a stronger correlation to t-PA activity and t-PA antigen than the whole PAl antigen. Circulating t-PA activity was influenced predominantly by the active PAl antigen. Both PAt antigens were correlated in similar manner with metabolic variables, lipoproteins and B/VII. Table: Correlations of active and whole PAl antigen (** p < 0,001) active PAl antigen whole PAl antigen active PAt antigen 1,000 0,851 ** whole PAl antigen 0,851 ** 1,000 t-PA activity -0,594 ** -0,492 ** bPA antigen 0,604 ** 0,497 ** body mass index 0,502 ** 0,462 ** triglycerides 0,452 ** 0,441 ** total cholesterol 0,252 ** 0,255 ** LDL-cholesterol 0,263 ** 0,264 ** HDL-cholesterol -0,357 ** -0,355 ** apolipoprotein B 0,428 ** 0,402 ** apolipoprotein A I -0,233 ** -0,211 ** The lower relationship of the whole PAt antigen to t-PA is obviously caused by patient samples with high levels of whole PAt antigen in contrast to normal values of active PAt as well'as of t-PA. Possibly, a high ratio of whole PAI antigen / active PAt antigen is caused by a raise of latent PAL the main form of PAt in the platelets. The clinical importance of an increased ratio whole PAl antigen / active PAl antigen remains under investigation. The cyclic antibiotics-polypeptides Bacifracin A, Bacilliquin from Boci/lu~, licheniformis and Gramicidin S from Bocil/us brevis, var. (3. B., were used for investigation. We studied their influence on the fibrinoly~c and coagulation activity in vitro• Me~hods. To solution of human plasmin (thrombin). containing 0.2 mg of protein (1 NIH unff)/ml, the analyses' solution of antibiotics (0.1-8,0 mg) was added. Then we defined the tlbrinolytlc activity of the mixes using azofibrin lysis, and fhrombin activity was determined according to the speed of fibrin clots formation from fibrinogen solution.

Results. In following table are submifled the results received in our laboratory {we also offer results of antibiotics influence on urokinase activity):

Ki, mM

Ki --the constant of inhibition. N. D. ~ in studied lirnils the inhibitor's activity was not observed. ---the inhibitor's activity was not define. I. --the inhibitor% activity was observed but Ki not determined. +, +% +++ --effect of inhibffion (in rela*iive indexes). Conc/us/on.~ The results received by us testify to the necessity of cautious approach to the use of antibiofics-polypeptides for various sorts of therapy in view of their possible influence on fibrinolytic and coagulation actlvlfy, of the organism. These results were used for preparation in our laboratory of biospeciflc sorbents containing C-ramicidin A, Bacil}iquirt and Gramicidin S.as ligands, They can reversibly bind thrombin, plasmin {plosminogen) and urokinase directly from crude exkacts. The enzymes are selectively eluted without substantial losses of specific activity in e yield of 60-90%. There is a great body of rather contradictory informations dealing with fibrinolysis in liver.. cirrhosis, which can be accelerated, normal or reduced, depending on the type of cirrhosis and investigation techniques (clot-lysis, fibrinolytic component measurements). Our previous finding was, that in vitro plasma-clot lysis, induced by exogeneously added tPA or streptokinase proved to be reduced, and this had a good correlation with severity of the disease and the elevation of plasmatic yon Willebrand factor levels. In vitro clo~/-[ lysis tests, induced by tPA were performed in 41 patients with alcoholic liver cirrhosis, utilising a microplate light-scattering assessment method.

The tests were repeated using the same plasma samples in each patients with a microplate which was covered by cultured endothelial-cell monolayer (umbilical vein, HUVEC}. Clot lysis speed proved to be 1.5-2 times slower with HUVEC milieu in the control group, while in the cirrhotic patients this inhibition was stronger and resulted in 5-fold reduction of lysis speed. Our results suggest, that cirrhotic plasma is able to accelerate the release of fibrinolytic inhibitors from cultured endothelial cells, which phenomenon may also contribute to the complex alterations of in vivo fibrinolysis in cirrhotic patients. Deep vein thrombosis (DVT) is a systemic disease with prolonged clinical manifectation. Anticoagulation therapy in DVT is not completely effective. Thrombolytic therapy may give rise to a systemic lytic state, The fibrinospesific agents (scu-PA and t-PA) have short half-lives in the circulation. We investigated the potency of the acylated plasminogen streptokinase activator complex (GBPg-SK) to deep vein clot dissolution as compared to well known SK and APSAC both in v~tro and in vivo in the model of venous thrombosis in artherio-venous shunt in rats. It was shown in in vitro study that fibrinolytic activity of plasminogen activators mainly depends on their stability in plasma. Stability studies carried out by incubating SK and Pg-SK activator complexes in plasma with euglobulin precipitation . Total fibrinolytic activity was measured by the fibrin plate method. GBPg-SK possessed the greatest stability in human plasma than APSAC or SK because of its prolonged inactivation period (the deacylation half-life for GBPg-SK was 230 :E 21 rain in contrast with 73 -~ 6 min for APSAC). The stability degree of two acylated thrombolytics (GBPg-SK and APSAC) was in order to inverse proportion of their first order rate deacylation constants (2.9 • 10 -4 and 6.0 • 10-s sec-1 respectively). The fibrinolytic potency of SK, APSAC and GBPg-SK was measured by 1251-labeled fibrin clot lysis in plasma and in vivo by lysis of the preliminary formed 1251-labeled fibrin clot inserted into the jugular vein.

FibrinolytJc activity of acylated plasminogen activators gradually increased in time. Under SK administration, the clot lysis came to the end by 2 hours while APSAC and GBPg-SK haven't lost their activity for 5 -6 hours. GBPg-SK possessed significantly more prolonged fibrinolytic activity than APSAC, The acyl-enzymes did not significantly influence on plasminogen,,.~2-anfiplasmin and fibrinogen levels in plasma according to their activity specific to fibrin-bound plasminogen. In opposite, SK produced a significant depletion of plasminogen, ~-2antiplasmin and fibdnogen levels in plasma. It seems, on the basis on in vitro and an animal experimentation, than APSAC with its moderately fast deacylation rate is more suitable for rapid thrombolytic effect, but GBPg-SK with its slow deacylation rate is suitable for deep vein thrombosis, when the rapid thrombolysis is less critical. It's well known that the complete lysis of thrombi usually isn't observed at the thrombolytic therapy. At present study we have attempted to quantify the possible mechanism of fibrinolysis inhibition during the thrombolysis. 125I-Labelled partially cross-linked fibrin clots of different volume (0.1-0.35 ml) were immersed in Tris-HCl buffer (3 ml) containing plasmin (5-100 nM) at 37°C. The lysis rate was detected by counting of soluble fibrin degradation products (FDP). At all the eases lysis slowed down and stopped in 3 hs though clots dissolved up to only 60-85%. No irreversible inlaibition of plasmin caused by denaturation occur as was judged by the measurement of fibrinolytic activity at the diluted samples. However the increase of FDP concentration in surrounding buffer led to the reversible inhibition of fibrinolytic activity of plasmin up to 5% of baseline. The SDS-PAGE analysis under non-reduced conditions shown the acoumulation of high-molecular weight FDP at the surrounding buffer. The inhibition phenomenon could be connected with the specific binding of plasrnin with soluble FDP having exposed lysine residues and the subsequent removal of enzyme from fibrin surface. Unexpectedly since the heterogeneous character of occurred reactions tile change of the clots surface area during lysis didn't affect the fibrinolysis kinetics in all the concentration intervals. To estimate the kinetic parameters the kinetic curves were linear in the coordinates [P1/t (l/t*ln(ISl°{(lSlo.lPi)). The obtained parameters were following: keat=l.36 min-l,KM=l.33 ixM,Kp=0.12 ~tM. The clinical trials have shown that FDP concentrations at the thrombolytic therapy of deep venous thrombosis and acute myocardial infarction usually was approximately in the range 0.05-0.2 ~tM. Therefore the described phenomenon of fibrinolysis inhibition by formed FDP may take place during thrombolytic therapy.

Al. Calatzis, An. Calatzis, +M. Klmg, +L. Mielke, +R. Hipp, A. Stemberger Institute for Experimental Surgery and +Institute of Anesthesiology Technische Universit~.t MOnchen Thrombelastography (TEG) is an established method for the detection of fibrinolysis. Fibfinolysis is usually determined when the TEG amplitude decreases by more than 15% atter the maximum amplitude is reached. This takes a considerable amount of time (more than 30 minutes). Our approach bases on the understanding of fibrinolysis as a process which runs in paraUe[ to coagulation and is not exclusively subsidiary to it. The effect of fibrinolysis on the growing clot in the TEG is shown by the comparison of two parallely performed TEG measurements: ExTEG: TEG measurement with standardised activation of the extrinsic system. ApTEG: ExTEG with in-vitro-fibrinolysis inhibition via aprotinin.

ExTEG-Reagent (Ex): 1:2 dilution of Innovin (recombinant thromboplastin reagent, Dade) with aqua dest. ApTEG-Reagent (Ap): 5 parts Innovin, 2 parts Trasy[ol (Aprotinin, Bayer, I0.000 KIE/ml), 3 parts aqua dest. Test procedure: l0 p1 Ex or Ap + 300 ~l citrated blood (CB) + 50 lal CaCl2-solution 0,15 M. The only difference of the two reagents is the addition of 20 KIE Aprotinin in the ApTEG, leading to an in-vitro fibrinolysis inhibition. The usage of disposable pins and cups (Haemoscope, Illinois, USA/E.M.S., Vienna) is recommended for ensuring standardised conditions for both measurements. Results and discussion: When there is a better clot formation in the ApTEG (corresponding to a lower so-cafled K-value) than in the ExTEG, fibdnolysis can be suspected. This technique requires only commercially available reagents and is easy to perform on conventional TEG systems. Due to the standardised coagulation activation with a thromboplastin reagent, fibrinolysis can be detected also when inhibitors like heparin are present in the circulation. According to our experience using this technique during liver transplantation, clinical relevant fibrinolysis can be detected as described in less than l0 minutes. Many thromboembolic (massive pulmonary embolism, proximal deepvein thrombosis, etc.) and coronary diseases (infarction, acute phase, etc.) require fibrinolytic therapy to early recanalizafion. The application of the well-known or new thrombolytic agents needs the use of specific, simple and reproducible methods for the determination of fibdnolyfic activity. We suggest new methods for measuring the blood plasma concentrations of plasmin, plasminogen, antiplasmins, and urine urokinase activity. These methods involve the employment of chromogenic substrafe azofibrin (human fibrin, covalently labeled with p-diazobenzenesulfonic acid). Method~. 0.2 ml of studied solution was added to 0,8 ml of azofibrin suspension in certain buffer (5-10 mg/ml) and the mixture incubated at 37 oc for 10-60 rain. After the end of incubation the mixture was filtered, the volume of solution brought up to 4 ml by 0.02 M NaOH and the optical density was determined at 440 nm. Resuffs. Azofibrin can be used for quantitative determination of proteinases activity in search of new fibrinolytic means. For comparison the results of our studies fibrinolytic activity of some proteinases with the use of azofibrin are presented:

Activity. with an increase of PAl and LDL-and a decrease of HDL-cholesterol concentrations k is concluded that the increased cardiovascular risk in diabetes meilitus was partly caused by a down regulation of the fibrinolytic system, increase of erythrocyte aggregation and plasma viscosity. Also disturbances of lipid metabolism an abnormal WHR seems to be of an additional atherogenous factor in DM. Plasma concentrations of thrombin-anfithrombin-III (TAT), alpha-2antiplasmin-plasmin (APP) complexes and DDimer were investigated in 50 patients treated with thrombolytic therapy for acute myocardial infarction (AMI) either with streptokinase (n=24), urokinase (n=16) or recombinant t-PA (rt-PA, n=10). All patients received an intravenous heparin bolus of 5,000 IU on admission, which was followed at once by an infusion of 1,000 IU/hr for the next three days titrated to maintain the partial thromboplastin time at twice control value. TAT, PAP and DDimer were measured by enzyme immunoassay on admission, 1, 2, 4, 6, 8, 12, 24 hours and on day 3 and 7 after admission. Groups did not differ significantly in regard to age, sex, delay and infarct location. On admission, no marker differed significantly between groups. Thereafter, TAT levels increased significantly exclusively in rtPA treated group. From 2 to 6 hours after admission, TAT were significantly higher in rtPA treated patients than in streptokinase and urokinase treated group (p<0.02). However, during continous heparin infusion, which was started immediately after stop of thrombolytic therapy, in each group TAT concentrations decreased below admission values. APP were significantly higher only 1 hour after admission in the rt-PA group (p=0.03). DDimer did not differ signifieanfly between groups. Our results demonstrate, that rtPA induces a hypercoagulable state, which may contribute to reocclusion after successful reopening of the infarctrelated coronary artery. The significant TAT decrease during continous heparin infusion supports the concomitant use of thrombin inhibitors as adjunctive therapy with thrombolytlc treatment for AMI. Thus, in acute myocardial infarction patients, thrombin generation is markedly influenced by the thrombolytic agent used and concomitant heparin therapy. Endothelium derived relaxing factor-NO (EDRF-NO) plays a major role in regulation of vascular tonicity and also exerts platelet inhibitory action~ However, due to the chemical nature of EDRF-NO few is known about its production and activity as a general index or marker of vascular function in human diseases. One way to achieve this can be measurement of nitrate/nitrite excretion in the urine, which seems to reflect vascular EDRF-NO production. In this report a self-developed ELISA method is described, which was used for this perpose. Nitrate/nitrite urinary exretion proved to significantly decreased in insulin dependent and in non-insulin dependent diabetes mellitus as well After a comparison of the excretion values to other markers of angiopathy (yon Willebrand factoD soluble thrombomodulin, beta -thromboglobulin) it seems to be acceptable, that urinary nitrate/nitrite excretion can be a useful indicate of diabetic vascular disorders. Two major concerns still accompany the application of Prothrombin Complex Concentrates (PCC). Viral safety has to be guaranteed and therefore several measures for virus inactivation or elimination are taken during the manufacturing process. The inherent risk of thrombo-embolic side effects has to be considered. To minimize these risks and to achieve good clinical efficiency the quality criteria for PCC's are under pending discussion. It is generally accepted that a modem PCC-preparation should contain all of the four coagulation factors in a well balanced proportion and that it should also contain protein C and protein S. Additionally, the concentration of activated coagulation factors should be kept at a minimum. A present PCC-produedon process mainly consists of a QAE-Sephadex extraction of cryopeer plasma followed by a solvent/detergent virus inactivation step. Further purification is achieved by subsequent chromatography on DEAE-Sephamse. The aim of this study was to improve product quality by avoiding F VIIactivation without implementing major changes to the production process. At the same time, a second virus eliminating step was added to the production process. It could be shown that speeding up the chromatographical process by switching the DEAE-Sepharose-chromatography from a classical axial column to a radial chromatography resulted in a significant reduction of F VIIa-genemtion. Mainly the reduction of contact time, resulting from the highest possible flow rates, leads to the wanted effect. The relation between F VII/F VIIa was 10 : 1 or more. In order to investigate the feasibility of virus filtration the eluate of the DEAE-Sepharose column was filtered through a virus removing Ultipor VFfilter. The analysis of the solution before and after fillration showed that the filtration had no influence on coagulation factors activity, protein content, proteolytic activity etc. Preliminary studies showed significant virus reduction values. In the past few years the problem of expediency of the treatment aimed at developing immunological tolerance in hemophil;a patients by way of complete removal of inhibitor with high doses of factor VIII has been discussed in literature. We observed 121 patients with hemophilia. Inhibitors to factor VIII:C were revealed in 32.7 % of patients with hemophilia A and fo factor IX --in 1.6 % of patients with hemophilia B. The level of an inhibitor was not higher than 87 Befhesda U/ml, that is those patients were not regarded as "high responders". A high incidence of inhibifors in young patients [from 7 to 26 years of age, 51.9 %) compared with older patients (from 27 to 40 years of age, 11.2 %) testifies to the probability of inhibitors development during treatment with modern concentrated preparation of factor VIII, IX. Inhibitor development in patients (40.5 %] in the course of antihemophilic concentrates transfusions is an evidence of alloimmunization of patients with proteins. The investigations show that in the course of transfusion therapy patients develop secondary immunodeficiency due to chronic antigenic stimulation of immune system with high doses of allogenic proteins. Against the background of immunodeficiency patients with hemophilia develop complications of immune character: infections complications --53.9 %, aufoimmune processes --44.9 %, secondary tumours --1.2 %. Plasmapheresis is the most rational method of removing inhibitor in patients with low level of inhibitor ("low responders", < 10 BU/ ml) and in patients with mean response. Thus it should be noted that the treatment of patients aimed at developing immunological tolerance is not only expensive and economically unprofitable but also not indifferent fo the organism. In a recent multicenter study 73 previously untreated patientens (PUPs) with severe hemophilia A were treated with a recombinant factor VIII concentrate (rfVIII, Recombinate©). During fVIII treatment 21 (29%) developed inhibitors, 6 high titer (>5 Bethesda units (BU)/ml), 4 low titer (<5 BU/ml) and 11 transient inhibitors. Plasma samples from before treatment and during treatment but before inhibitor occurrence were available in 12 inhibitor patients. These plasma samples were analyzed by a highly sensitive immunoprecipitation (IP) assay for the presence of anti-tViiI antibodies. In 9 (66%) a significant increase of anti-fV]]I antibodies was seen indicating the development of a clinical relevant inhibitor titer. This immune response occurred after 2 to 17 (median 5) exposure days (ED). In the same period only 3 out of 15 inhibitor patients showed a decreased in vivo recovery. In 16 PUPs who developed no inhibitors plasma samples from the entire treatment period were available. An immune response to rfVIII treatment was seen in 7 PUPs after 2 to 43 ED (median 24 ED). The immune response was later and less pronounced in comparison to inhibitor PUPs before inhibitor occurrence. With the IP method the detection of an early immune response is possible which might be predictive for a later inhibitor development. The inclusion of the lip method should be considered for future multicenter PUP studies. In the past anaphylactie reactions to plasma and plasma components have been a common complication of replacement therapy in patients with hemophilia A and B. We report on 3 severe bleeding episodes in 2 patients with hemophilia A and B, respectively. Both patients had a history of life threatening anaphylactic reactions after exposure to different plasma derived clotting factor concentrations including intermediate purity factor VIII-and factor IX-concentrate, respectively. High purity factor concentrates were tolerated well without any allergic side effects.

A 67 years old patient with a moderate form of hemophilia A (F VIII 4 %) had a history of severe immediate reactions with skin manifestations and bronchospasm after exposure to fresh frozen plasma, ctyoprecipitate and 3 different plasma derived factor VIII-concentrates of intermediate purity. In all episodes pretreatment with corticosteroids and antihistamines was unsuccessfull in avoiding severe bronchospasm. Replacement therapy with two different recombinant factor VIII concentrates was tolerated well without any side effects. A 12 years old haemophiita B patient developed hypersensitivity reactions to prophylactic factor IX substitution, which could be overcome by using a factor IX .concentrate with improved purity. A recent recurrence of hypersensitMty under this treatment was finally overcome by the use of highly purified (monoclonal antibodies) factor IX concentrate. We conclude from these findings that high purity of factor concentrates, possibly due to the absence of soluble HLA-antigens, are advantageous in patients disposed to allergic reactions. Introduction: Antibody formation against factor (F) VIII remains one of the most severe complications of repeatedly transfused patients with haemophilia A. As reported previously in our study about the incidence of FVIII inhibitors, we have observed a high incidence of FVIII inhibitors among our haemophilia A patients. It is still not clear why certain haemophiliacs develop antibodies and others do not. A number of previous studies suggest that there is a genetic predisposition for the FVIII inhibitor development. Thus, the purpose of our study was to examine, if there is a correlation between FVIII antibody-formation and genetically determined histoeompatibility antigen (HLA) patterns in our haemophiliacs. Patients and methods: HLA-class I (A, B, C) and HLA-class II (DR, DQ) typing was carried out for 51 respectively 44 multi-transfused paediatric haemophilia A patients (FVIII:C activity < 5%), including 22 who had developed an antibody to FVIII: 19 were high responders (> 5 BU), 3 were low responders (< 5 BU). HLA-typing has been performed by a standurcl two-stage microlymphoc~.ftotoxicity procedure (DRK Frankfurt) using antisera with defiend HLA-specifity (Biotest Diagnostica). Results: We found an under-representation of HLA-A2 in FVIII inhibitor patients when compared with the subgroup without inhibitor. In regard to the HLA-B and HLA-C antigen frequencies there are no apparent differences between the groups. Among the class II antigens there were higher frequencies of DR1, DRw52 and DQwl in the non-inhibitor group. However, the reduction in HLA-A1, HLA-Cw5, HLA-DQw3 respectively HLA-DR4 frequency for inhibitor patients as reported previously could not be confirmed in our study. Conclusion: So far it remains unclear if there is a significant association of a certain HLA allels with the development of FVIII antibodies. Recombinant factor SQ (r-Viii SQ, Pharmacia) is a B-domain-deleted recombinant factor VIII. It is formulated without albumin (HSA). The product has been shown to have in vitro and in vivo biochemical characteristics similar to a plasma derived full-length protein (p-Viii).

The international clinical trial programme was initiated in March 1993. Pharmacokinetic studies have shown that the B-deleted r-VIII SQ should be given according to the same dosage principles as a full length p-VIII. At present, the product is being tested in previously treated patients (PTPs) and untreated patients (PUPs) with severe haemophilia A (VIII:C < 2 %), both during long-term treatment (on demand therapy or prophylaxis) as well as during surgery.

The long-term study in previously treated patients in Germany was started in January 1994. Thirteen patients have been included in 8 centers. All patients are still on treatment with r-VIII SQ, most of them receiving prophylactic treatment.

Global treatment efficacy has in general been considered excellent or good. No serious clinical adverse events related to the study product have been reported, nor have any inhibiting antibodies to factor VIII or antibodies to mouse-lgG or CHO-cell components developed in the patients. Further results such as data on efficacy, half-life, recovery and safety will be presented in detail at the meeting. Nowadays it is not sufficient to regard hemophilia only as hemorrhagic diafhesis of coagulation genesis, caused by deficiency or molecular anomalies of coagulation factor, without taking into account the immunity state. On examination of 125 patients (pts) (hemophilia A --110 pts, hemophilia B --11 pts, Willebrandt's disease u 4 pfs) the development of immune complications was revealed in 34.4 %. Chronic persistent hepatitis (3.2 %), chronic active hepatitis (3.2 %), herpes simplex (1.2 %), chlamidiosis (1.2 %), bacterial infection (7.2 %} were regarded as infectious complications. Bacterial infections have a routine course due to preserved phagocytic function of neufrophils. And viral infections, whose ability to resistance is connected with T -cell link immunity, take on a chronic persistent course, Mechanism of the development of autoimmune processes (autoimmune thrombocytopenic purpura --2.4 % of pts, immunocomplex disease --4.9 % of pts, the appearance of immune inhibifors --34.4 % of pts} is connected with the impairment of immunological surveillance over B -cells aufoimmune clones as a result of dysbalance in the system of T -lymphocyfes immunoregulatory subpopulations. Lymphadenopathy and splenomegaly (4.9%) develop due fo benign proliferation of lymphoid tissue as a result of impairment of regulatory function of T -lymphocytes system, or they may be an evidence of virus infection. We observed one episode of acute leukemia. Immune complications in hemophilia patients develop against the background of secondary immunodeficiency caused by chronic antigenic stimulation of patients' immune system with high doses of allogenic proteins, which plasma preparations contain. In immune complications hemophilia patients develop hemorrhages, whose pathogenesis is quite different from that caused by coagulation factor, so it should be taken into account in the course of treatment.

Control of hemophilia therapy classically was based on four parameters: life span expectancy of patients, orthopedic status (normal zero), Pettersson score and social integration. ORen, however, these parameters described an irreversible status with permanent damage particularly of the joints, especially when patients were grown-up. In order to establish risk-adapted therapy protocols to prevent hemophllic osteoarthropathies, quality control programs have to he set-up that allow for early adjustment of dosage and substitution frequency. Here bleeding frequency is one the main parameters, being a clear hint for the possible development of a target joint.

Since 1988 we have established a computer database (Haemopat) that contains data from all patients treated in our center. Tables and graphs allow for early detection of increased bleeding tendency in a given joint, and accordingly for adjustment of therapy. The results of 8 years of measuring reasons of joint damage and not documenting the orthopathies as such will be demonstrated. Parallelly a new program (Haemopat Win 1.0) will he introduced allowing for easier handling of data and their evaluation. This program will be used as of December 1995. In combination with a substitution calender to be filled in by all patients, in which factor concentrates, lot numbers, dosage, and date of administration will he constantly recorded, this program will extend our existing database in order to follow closely clinical and orthopedic parameters of each patient, and consequently acts as strict control of therapy quality. Additionally, it provides sufficient data to fulfil any documentation needs, requested by medical authorities.

The program will be available for all those interested free of charge. 2) Kinderklinik der Westf. Wilhelms Univ. Mttuster 3-6) Biotest Pharma GmbH, Dreieich Haemoctin® SDH; the FVIII SDH (SDH = solvent detergent and dry heat = 100 °C, 30 rain) from Biotest Pharma is a high purity (specific activity ~ 100) FVIII concentrate manufactured from large human plasma pools. Virus validation studies have shown virus inactivation/reduction (log 10) during the manufacturing process for lipid coated vints~ such as: H]V-1 > 16.2; PSR > 16.8; VSV > 14,5; BVDV > 15.7; HCV > 4.5* and non enveloped vimsas such as: Parvo** = 2.7; Reo > 5.3*** and HAV > 13.9. More than 50 hemophilia A patients (PTPs = previously treated patients), baseline FVIII activity < 1%, were included in an international drug monitoring study to follow their FVIII inhibitur status. The hemophilia Centers included were three Centers from Hungaria (Helm Pal Children Hospital and the National Inst. of Haematology, Budapest and Regional Blood Transfusion Center, Debrecen) and four Centers from Germany (two from Berlin, one FraukfurffMain and one MOnster). Patients were enrolled in the drug monitoring beginning Aug. 1993. At the entry none of the patients had a detectable inhibitor. At the end of Sept. 1995 there were no side effects or adverse events in connection with the use of Haemuetin®. Before the Haemoctin drug monitoring study, the patients were treated with cryoprecipitate, or purified FVIII products. Inhibitor testing was done on patients plasma samples using the Bethesda method. Repeated FVIII recovery determination at one time (between 12 to 24 hrs) after Haemoctin® application demonstrated the expected recovery and normal half life time. None of the hemophilia A patients, treated with Haemuetin® SDH developed a clinical relevant inhibitor. At the beginning of the stud)', the clinical efficacy of Haemuetin® was studied in 16 hemophilia A patients and shown to give an in vivo recovery of 71 + 15 % by one stage assay and 77 + 17 % by a chromogenic assay. T ½ values were 13 + 2.8 and 12.7 + 3.2 hrs respectively. The study for the clinical efficacy of Haemoctin® SDH was repeated in a group of 6 patients approximately two years later. Although CD4 lymphocyte counts are known as reasonable predictors of prognosis in HIV infection, the CD4 count is not in all cases an infallible indicator of prognosis. Therefore several serological markers are used to predict disease outcome, including beta-2 microglobulin (132m), immunoglobulin A (IgA), lymphocyte counts (lymph) and others. In this study we followed a cohort of 23 haemophiliacs (19 with haemophilia A, 4 with haemophilia B) and 2 patients with severe von Willebrands disease over a period of 28 months (mean, range: 22-34). Testing for l~2m, IgG, IgA, IgM, CD4 and CD8 cell counts (abs. and relat.), CD4/CD8 ratio, and absolute resp. relative leucocyte and lymphocyte counts was performed at least 3 times a year. At the same time clinical examinations and review of history were undertaken. Mean of laboratory tests for every quarter of a year and significant changes during time of observation were calculated and correlated with clinical data.

1-4 5-8 9-12 13-16 17-20 21-24 CD41 440+956 344+924 278+925 302.-1:240 273.+.220 166+125 CD8 ~ 1165+474 1171+523 1236+1187 1017+439 930±412 873+478 1~2m z 2.0+0.6 3.0+1.0 3.0+1.2 3.0+1.1 3.5±1.0 3.5±1.3 lymph ~ 1.98+0.6 1.83+0.6 1.72+0.7 1.67±0.6 1.46±0.6 1.31±0.5 means/pL ± standard deviation means mg/L ± standard deviation During time ef observation we found significant changes of CD4 (abs. and relat.), abs. CD8 counts, CD4/CD8 ratio, f~2m, leucocytes and lymphocytes. The abs. CD4 and CD8 counts correlated clearly with lymphocytes und leucocytes counts but not with 1~2m. The prognostic value of the tested parameters is discussed by calculation of correlations with clinical data, anti-retroviral treatment and treatment of haemophilia. The availability of high purity factor concentrates has recently encouraged clinicians to use perioperative continuous infusion of FVIII or FIX to prevent or reduce bleeding in patients with haemophilia. In conlIast to repeated highdose bolus injections, the continuous infusion trealment regime maintains constant coagulation factor activity at a level necessary for hemostasis, reducing the total cost of treatment by about 20% and preventing possible side effects of bolus doses. The new application mode, however, requires stable products which tolerate slow passage through an infusion device.

Our objective was to test in vitro the FVIII concentrate IMMUNATE (STIM plus) and the FIX concentrate IMMI.YNINE (STIM plus) at room temperature, under conditions of long-term contact with polypropylene tubing in an infusion pump. Infusion rates were chosen to mimic clinical situation. The control samples were not infused through the pump but were otherwise treated identically. Test samples were drawn before and at 1, 4, 8, 24 and 48 hours after the onset of each infusion run. FVIII (one-stage, two-stage and chromogenic assay) and FIX (one-stage) activity were measured using IMMUNO reagents. Presence of activated factors were measured by NAPT'I', while Flla, FXa, plasmin and pre-kallikrein activator were detected with specific chromogenic substrates. The data showed equivalent results between test and control samples with no loss of FVIII or FIX activity. The potencies of both IMMUNATE (STIM plus) and IMMUNINE (STIM plus) remained within 100 + 20% of labeUed values within 48 hours after onset of infusion.

In conclusion, IMMUNATE (STIM plus) and IMMUNINE (ST1M plus) are suitable for contiuous infusion when using automatic infusion device within applied test criteria. In htanans, circulating half-lives of asparaginase enzymes from E. coli and Erwinia chrysanthemi vary within a wide range. Moreover, half-lives differ not only among different E. coli strains but also among commercial E. coli preparations. To investigate the possible influence of two different sources of E. coil asparagmase (ASN) preparations on the fibfinolytic system of leukemic children a prospective randomized study was performed correlating ASN pharmacokiuetics (ASN activity, asparagine depletion) with fibrinolytic parameters (plasminogen (plas), o.2-antiphismin (ct2AP), tissue-type plasminogen activator (t-PA), tissue type plasminogen activator inhibitor 1 (PAl 1), D -I)imer (I)-D)). Together with prednisono, vincristine and an anthracycline 20 children received I0000 IU-/m 2 ASN Medae R (originally purchased: Kyowa Hakko, Kyogo Japan) and 20 children 10000 IU/m 2 Crasintin R (Bayer, Leverkusen, Germany). Blood samples for pharmacokinetic and coagulation analysis were drawn before the first ASN administration and every third day whilst on medication. The results are shown in the 0.05 ASN activity shows a negative correlation (Spearman: rho/p) to plas (-,637/0.0003) and ct2AP (-, 751/0.0001). A positive correlation was found between ASN activity and D -Dimer formation (0.475/0.01). T-PA and PAl 1 showed no relationship to ASN activity. All children showed complete aspamgiue depletion at a detection limit of 0.1 uM during the course of ASN admiatstration. Two thrombotic events occurred in the Kyowa group, One of the distinctions between the two E. coli ASN preparations administered ill this stndy is the absence of cystine in the Kyowa ASN, which also has a lower isoelectric point and a longer half-life than the Bayer type A ASN. With respect to this observations this may lead to longer inhibition of protein synthesis, which then may be the cause of a bigher rate of side effects. Along with studies on ASN pharmacokinoties dose recommendations need to be tailored to the specific ASN preparation employed to ensure optimal antineoplastic efficacy while minimizing the hazard of complications.

DIFFERENT TYPES OF COAGULOPATHY IN HEPATIC VENO-OCCLUSIVE DISEASE (VOD) AND CAPILLARY LEAKAGE SYN-DROME (CLS) AFTER BONE MARROW TRANSPLANTATION W. Ntimberger, S. Eckhof-Donovan, St. Burdaeh and U. G6bel Department for Pediatric Hematology and Oncology, Heinrich Heine University Medical Center, Diisseldoff, Germany It is generally accepted, that CLS, coagulation activation and refractoriness to platelet transfusions are part of the syndrome of hepatic VOD. We assessed patients with either VOD or CLS or both VOD and CLS, in order to analyze the influence of either syndrome on different aspects of hemostasis. VOD was diagnosed according to Jones et al. [Transplantation 44 (1987) 778]. Diagnosis of CLS was >_3% increase of body weight in the past 24 hours and non-responsiveness to furosemide [Niirnberger et al., Ann Hematol 17 (1993) 67] . Patients with VOD, CLS or both were compared to control patients without either diagnosis. Eight patients suffered from both VOD and CLS, 5 patients only from VOD, and 8 only from CLS. 61 patients had neither syndrome and served as control population. Activation of the coagulation system was assessed by increase of TAT-complexes and/or increased consumption of AT IIL The hemostasis patterns were as follows:

No. Introduction: Lung cancer goes along with coagulation activation and increased thromboembolic risk. Acute phase reaction in cancer patients leads to elevated levels of C4b-binding protein (C4b-BP) followed by a shift from free to C4b-BP-bound protein S. We tried to find out whether there is a correlation between alterations of C4b-BP, protein C protein S system and interleukin 6 (IL-6), which is one of the most potent inducers of hepatic acute phase reaction. Patients: I. 25 patients with lung cancer; 2. Control group: 11 patients in complete remission after lung cancer. Methods:

Clotting methods: protein C and S activity; ELISA tests: protein C antigen, TAT-complexes, prothrombin fragments F I+2, IL-6. Electroimmuno-diffusion (Laurell): free and total protein S, C4b-BP. Results: TAT-complexes and F I+2 were elevated in cancer patients. C4b-BP levels were slighthly increased (129±19 % of n.), protein S activity was 89±33 % of n. (control group: 108±23 % of n.). IL-6 in lung cancer patients was 37.2252.9 pg/l (control: 27.2±8.2 pg/l). Conclusion: One source of the hypercoagulable state in lung cancer patients is decreased protein S activity due to elevated C4b-BP levels. This is probably caused by hepatic acute phase reaction which is triggered by increased IL-6 levels. These plasma levels correlate with levels of the tumor marker CA 125 and with the stage of the disease but correlations with patient outcome (disease recurrence and overall survival) have not previously been shown. Plasma levels of D -Dimer and CA 125 (determined by sandwich ELISA assays} were measured prior to treatment in 36 women with FIGO Stage t to III ovarian cancer and correlated with tumor stage, relapse and overall survival over a mean follow -up period of 28 months (range 16 to 40 months). Levels in 71 healthy women and 27 patients with benign ovarian disease served as controls. The occurrence of deep vein thrombosis in the cancer patients was also determined by impedance plethysmography that, when positive , was confirmed by contrast venography. Preoperative D -Dimer and CA i25 levels in ovarian cancer patients were statistically signfficantly higher than in controls. Preoperative cut off values were calculated for the prediction of cancer relapse and survival for both measurements. D -Dimer levels above a cut off level of 2060 ng/ml were statistically significantly associated with the rate of relapse but CA 125 levels were not. Deep venous thrombosis occurred in 33 % of cases but there was no difference between properafive levels of D -Dimer in patients who subsequently did versus did not develop deep vein thrombosis. High levels of D -Dimer are associated with more advanced disease and with poor prognosis in patients with ovarian cancer. The high levels of D -Dimer are a biologic feature of the malignancy itself that may be attributable, at least in part, to increased conversion of fibrinogen to fibrin in the tumor bed with subsequent degradation of fibrin by the fibrinolytic mechanism. Thus D -Dimer levels may serve as a marker for overall tumor burden as well as "disease activity". A high incidence of deep vein thrombosis exists in the course of the disease in ovarian cancer patients but preoperative levels of D -Dimer are not predictive of this occurence.

yon Tempelhoff Georg -Friedrich, Michael Dietrich, Dirk Schneider, Lothar Heilmann. Dept. Obstet. Gynecol. City Hospital of Ruesselsheim. -Germany. An increase of Plasminogen Activator Inhibitor activity (PAI act.) in the plasma of cancer patients has been recently discribed. We have longitudinally investigated PAI act. in 136 patients with primary breast cancer and compared the results with the outcome of malignancy. Patients with untreated primary breast cancer and without proof of metastasis (T 1-4 N0-2 M0) were eligible for this study. In all patients coagulation tests including fibrinogen {method according to Clauss), d -dimer (ELISA} and PAl act. (uPA dependent inhibition test) were performed prior to primary operation, 6 months thereafter and at the time of cancer relapse. Seventy -two healthy women and 43 patients with benign breast disease served as controls. During a mean follow -up of 32 + 16 months 34 patients (25 %) developed cancer recurrence and 13 (9.6 %) patients died. In all cancer patients preoperative levels of fibrinogen and PAI act. were significantly higher compared to healthy women and to patients with benign breast disease. Preoperatively only PAl act. was significantly higher in patients with vs. without cancer recurrence (4.52 _+ 1.67 U/ml vs. 3.4 1 + 1.55 U/ml; p = 0.002). In patients with later recurrence PAI a~t. significantly dropped 6 months after operation (p = 0.02) and was again significantly increased at the time of cancer recurrence (4.90 _+ 2.89; p = 0.001). A preoperative cut off value (calculated via Cox model) of PAI act. above 3.52 U/ml was significantly associated with the rate of relapse (tog rank: p = 0.0005) and in 70 % of patients who died of cancer preoperative PAI act. were also above this cut off. Impaired fibrinolysis in patients with breast cancer is significantly associated with the outcome of cancer. A monoclonal heparin antibody (mab) has been raised against native heparin using a heparin-bovine serum albumin conjugate prepared by reductive amination. For further analyses tyramine, which was covalently bound to low molecular mass heparin by endP0int attachment (Malsch R et al: Anal Biochem 1994; 217: 255-264) , was labeled with 125-iodine at the aryl residue. The tracer antibody complex was immunoprecipitated by goat anti-mouse immunoglobuline IgG. The mab recognized specifically intact heparin and heparin fractions. The lower detection limit of heparin preparations was 100 ng/ml. No cross reactivity of the mab occurred with other glycosaminoglycans such as heparan sulfate, dermatan sulfate, chondroitin sulfate A and C. Oversulfated heparin showed lower affinity to the antibody Hl.18 than 2-0-and 6-0-desulfated beparin. The method established for the purification of the mab was ammonium sulfate precipitation with followed dialysis. SDS-PAGE and high pressure capillary electrophoresis prooved the high purity of the received antibody. The biological activity of mab was tested by the chromogenic assay $2222 and remained stabile while purified. In conclusion, the present abstract describes an purified IgG 1 monoclonal antibody directed against heparin and heparin fractions, which can be used for biological measurements. The concentration of heparin and dermatan sulfate in biological fluids is usually measured using radiolabeling. For this purpose aromatic compounds are usually used to insert radioactive iodine labeling at the saccharide backbone of the glycosaminoglycan. We developed methods for the specific labeling of hepann and dermatan sulfate at the terminal residue. Tyramine was bound by reductive amination to the 2,5 anhydromannitosyl end of heparin, produced by nitrous acid degradation and confirmed by 13C.NM R spectroscopy. (Anal Biochem 217: 254-264, 1994) This method was also used to produce a low molecular mass dermatan sulfate (LMMD)derivative after partial deacatylation. In order to choose the proper method for evaluating the specific anticoagulant activity in the row of chitosan polysulphate (CP) samples with different degrees of pol3~merization and sulphation we applied to Pharmaeapea Article (A~) when assessing the ability of direct anticoagulants to depress the coagulability of recalcificated sheep blood (using the 3rd International heparin standard), and to measuring such acti¢ity as per pharmacokinetic model (A2). The model admits the "kinetics of CP elimination be linear in ease of intravenous injection to rabbits, as it is observed in heparin: Ct=Co exp(-I~ x t), where Ct is CP concentration at the time moment t; Co is CP concentration at the moment of injection; I~ is the elimination constant. Besides, it is assumed that there is a linear approximation of the anticoagulant effect on the dose, which finally makes it possible to calculate the specific actidty A2 : T=KT Ct + Tin, where T is the time of clot formation at different tlme intervals after of CP injection; T~, is the time of clot formation prior to CP injection. T value was assessed in two tests: in blood coagulation time (BCT) and in activated partial thromboplastin time (APTT). No correlation was observed between A1 and A2. At the same time the values of Ifm and the period of semieliminatinn (Tvz) with the use of the original method that were obtained with the help of the quantitative determination of CP in rabbit's blood taken at different time intervals after injection, showed a close correlation (1"=0,94 p<0,05) between the same parameters, obtained with the help of the of the pharmacokinetic model in BCT test. Thus, experimentally it was proved that the assumption of the linear elimination and the effect-dose dependence was true, which is necessary for A2 calculation. We recommend to use intravenous injection of the samples to animals with further assessment of the results according to the pliarmacokinetic model to calculate the specific anticoagulant activity in the row of chemically related potential direct anticoagulants. In this investigation we compared the biological activity of a Low-Molecular-Heparm (LMW-heparin, Mono Embolcx®) after intravenous, subcutaneous and oral application in rats. Sprague-Dawly rats were anaesthetized by Ketamine/Diazepam and the blood samples were taken from the retro orbital sinuus. 150 aXa U/kg body weight of the LMW-Heparin were injected intravenously and subcutaneously to 10 rats each. Between 3 minutes and 10 hours after injection serial blood samples were taken. 200 mg/kg (20.000 aXa U/kg) body weight of the LMW-heparin were applicated orally using a stomach tube. Blood samples were taken between 1 and 24 hours after oral application. The antifactor Xa and antithrombin activities of the plasma samples were measured, using ehromogenic assays and the substances S 2222 and S 2238 (Kabi Vitmm).

After i.v. injection the maximum aXa and alia activities were 2.8 aXa U/ml and 0.8 aIIa U/nil respectively. After s.c. application the antifactor Xa activity of the LMW-heparin showed a maximum of 0.5 aXa U/ml atter 120 minuteS. The antithrombin activity exhibited an eatiier maximum activity of 0.2 alia U/nil 60 minutes after injection. After the oral application no increase of the aXa or alia activities was measured. The LMW-heparin has a high antifaetor Xa and antithrombin activity after i.v. and s.c. injection. After oral application no activity of the LMW-Heparin was measurable. These results implicate that fractionated heparin is not absorbed after oral application or is inactivated in the gastrointestinal tract. To improve the activity after oral application modified hepatins have to be synthesized. In an in vitro study the effect of various heparin derivatives (calciparin, fraxiparin, CY 222, CY 231, astenose, hexasaccharide, SSH 14) on thrombin-and ADP-induced platelet aggregation as well as on ADPmediated platelet activation in whole blood was investigated. All heparin derivatives caused a concentration-dependent inhibition of thrombin-induced aggregation of washed platelets. Calciparin and astenose were found to be the most effective compounds with IC5o values of 0.67 and 1.2 p, mol/l, resp.; higher concentrations (5-30 times) were required for the other compounds. Furthermore, the heparin derivatives were studied with regard to their potentiating effect on ADP-induced platelet aggregation. In a concenWation range from 1 to 10 U/nil calciparin, fraxiparin, CY 222 and astenose led to a potentiation of the ADPinduced aggregation whereas CY 231, hexasaccharide and SSH 14 did not show this effect. The increase in aggregation was associated with an increase in thromboxane A2 lbrmation. In addition, the effect of calciparin, fraxiparin, CY 222 and astenose on ADP-induced platelet activation in whole blood was investigated by flow cytometric analysis using monoelonal antibodies to platelet surface receptors OPIIIa (CD-61) and P-selectin (CD-62). At concentrations that caused a maximum potentiation of ADP-induced platelet aggregation these substances led to a strong increase of ADP-mediated activation of platelets in whole blood. The effect was most pronounced when the blood was anticoagulated with calciparin and astenose, resp. In conclusion, the results suggest that the aggregation-promoting effect of heparin derivatives included in this study is dependent on the molecular weight and the degree of sulfation and is in part due to the generation of thromboxane. Heparins are negatively charged polysaccharides and bind protamine forming a stable complex. Here we report on the properties of microbeads (4.5 pro) coated by protamine. Protamine chloride (0.16 IJM) was covalently bound to 0.5 mg paramagnetic tosyl..activated microbeads M-450 (Dynal). The covalent binding of protamine was from 1.0 to 13,0 mg/g beads. Protamine-Dynabeads were produced in a phosphate buffer at different pH (7,0; 7,5; 8,0 and 8,5). The Protamine-Dynabeads produced pH 7.5 showed the best properties for flow cytometry analysis. In saline solution they bound LMM-heparin-tyramine-fitc (LMMH-Tyr-Fitc) dose dependently from 0.001 to 2 U/ml, whereas in plasma and blood they bound LMMH-Tyr-Fitc from 0.05 to 2 U/ml. Dependent on the binding protocol, the microbeads also bind proteins unspecifically, i.e bovine serum albumine and protamine to a lower extent.The adsorbed proteins, however do not bind LMMH-Tyr-Fitc dose dependently. The saturation of the proteins on the beads was determined as their relative fluorescence intensity (rfi). In saline solution the saturation was measured at 380 rfi, in human plasma at 325 rfi and in whole blood at 252 rfi. Using flow cytometry erythrocyctes, lymphocytes, monocytes and granulocytes were not bound to Protamine Dynabeads. These data demonstrate that Protamine-Dynabeads can be used to measure the concentration of LMMH-Tyr-Fitc in saline solution, plasma and blood because they do not bind to human blood cells. The present study was designed to investigate the anticoagulant action of inhaled low molecular weight (LMW)-heparin in healthy volunteers. 3,000 IU (group t), 9,000 IU (group 2), 27,000 IU (group 3) or 54,000 IU (group 4) LMW-heparin were given to 20 healthy volunteers each at 4 weeks intervals. In group 1 tissue iactor pathway inhibitor (TFPI) antigen and activity, 82222 chromogenic factor Xa assay, heptest, APTT and thrombin clotting time (TOT) remained unchanged during the 10 days observation period. In group 2 TFPI antigen and activity, APTT, TCT and the $2222 method remained uneffected. Heptest coagulation times were 18.7 + 2.0 before, 26.1 + 5.2 sec. 6 hrs and to 20.5 + 1.9 sec. 24 hrs after inhalation. In group 3 TFPI antigen increased from 74.1 + 13.9 to 80.5 + 14.2 ng/ml 3 hrs after inhalation. TFPI activity remained unchanged. $2222 method increased from 0.01 to 0.08 + 0.06 IU/ml 6 hrs after inhalation. Heptest coagulation values were prolonged up to 42 _+ 7.6 s ec after 6 hrs and returned to normal within 72 hrs after inhalation. APTT and TCT remained unchanged. After inhalation of 54,000 IU LMW-heparin, the following changes were observed: TFPI antigen increased to 103 +_. 17.9 ng/ml and normalized within 24 hrs. -I'FPI activity increased to 1.14 _+ 0.23 U 3 hrs after inhalation and was normal after 24 hrs. Antifactor Xa activity, as measured by S2222 method, increased to 0.343 + 0.196 U/ml after 6 hrs and was normal after 72 hrs. Heptest coagulation values increased to 77.5 + 11.8 sec 6 hrs after inhalation and normalized after 144 hrs. APTT and TCT did not change throughout the observation period. The data demonstrate a resorption of LMW-heparin by intrapulmonary route in man. No side effects were observed. Recently we developed a tritium-labelled arachidonic acid ([3H]AA) release test with high sensitivity to membrane-toxic agents. The assay performed in U937 cells is intended to evaluate ehemicals, drugs and biomatefials with regard to their eytomembrane toxicity [KlOeking et at. (1994) , Toxicology in vitro 8, 775-777]. Local irritation reactions are described in patients receiving therapeurieat dosages of LMW heparin. This fact prompted us to examine the following LMW hepafins and heparinoids for their membrane toxicity in U937 cells: reviparin-sodium, enoxaparine-sodium, mueopolysccharide polysulphate (MPS), pentosan polysulfate sodium (PPS), polysulfated bis-lactobionic acid amide derivatives LW10082 (aprosulate) and LW10086. For this purpose, [31--1]AA labelled U937 ceils were incubated with different concentrations of LMW heparins and heparinoids at 37°C for 1 hour. Compared with untreated cells, the [~H]AA release of cells treated with 5 mg of the drugs was two times higher with reviparin sodium, three rimes higher with bis-lactobionic acid amide LW10086, five times higher with pentosan polysulfate, 20 times higher with ertoxaparine-sodlum, but it was equal to the control with mucopolysaccharide polysulphate. The rate of araehidonic acid release in response to a test chemical may therefore be used to assess the membrane-toxic effect of this substance and to predict its the inflammatory potential in the skin. Semi-synthetic glyensaminoglycans (GAGs) with antithrombotic properties can be prepared from the E. Coli K5 polysaecharide by coupled chemical and enzymatic methods. The molecular weight of these semi-synthetic GAGs can be adjusted to obtain products mimicking the molecular profile of a low molecular weight hepatm. In order to compare the biochemical and pharmacologic properties of a semi-synthetic GAG (SR 80486A, Sanofi/Choay) with a commereiany available low molecular weight heparin, Fraxiparine (Sanofi, Paris, France), valid biooheanical and pharmacologic methods were used. The molecular profile of this agent as determined by HPLC exhibited a comparable distribution profile (Mr=5.7 kDa) in comparison to Fraxiparine (Ma=5.1 kDa) . The anticoagulant properties of SR 80486A were comparable to Fraxiparine in the APTT and Heptest. However, in the USP assay, this agent showed slightly weaker activity. SR 80486A also exhibi~d comparable affinity to ATffl and HCII. In comparison to Fraxiparine, it produced a much weaker response In the HIT screening system. In~ viv0 studies, SR 80486A preduecd strong dose-dependent antithrombotic actions in both the IV and SC studies in the rabbit jugular vein stasis thrombosis model (ED50=I 5-60 gg/kg). Additionally, it also produced antithrombotic aefiorts in a rat jugular vein clamping model. The hemorrhagic effects of this agent were comparable to those of Fraxipafine as measured in a rabbit ear blood loss model. Intravenous administration of SR 80486A also revealed a comparable pharmaeokinetie behavior to Fraxiparine. No abnomaiitias of the clinical chemistry (change in liver enzymes) and hematology profile (thrombocytopenia and lencecytosis, etc.) were noted In primates. At a dosage of I and 2.5 mg/kg IV, this agent also caused a release of functional TFPI which was comparable to the observed responses of other low molecular weight heparins. These studies suggest that SR 80486A is capable of producing similar pharmacologic effects as other low molecular weight heparms, however, additional optimization studies are required for demonslrating product equivalence. Limited information on the comparative pharmacoldnetics of low molecular weight heparin (LMWH) is available on the data obtained from APTT, Heptest, anti-Xa and antMIa assays. Since these drugs are currently used for therapeutic indications using relatively high dosages and intravenous administration. APTT, Heptest and antMIa test may be valuable in the assessment of their effects. In order to investigate the relative pharmacokinetics of LMWH using APT'I', Heptest, anti-Xa and anti-IIa methods, Certoparin (Sandoz, Basel, Switzerland) was administered to individual groups of healthy male volunteers (52-70 kg) via intravenous (30 mg) and subcutaneous (50 nag) routes in a crossover study. Blood samples were drawn at 0, 5, 15, 30, 60, 90, 180, 360, 540 and 720 minutes. Using a baseline pool plasma obtained from the same volunteers, calibration curves for each of the individual tests were constructed to extrapolate circulating levels of Certoparin. A non-compartmental model using trapezoidal technique was used to obtain pharmacokinetic parameters such as t 1/2, Vd, and CLsys. In the intravenous studies, the t 1/2 was found to be dosedependent for APTT, Heptest, anti-Xa and antM]a. The AUC, however, was significantly different for each test and was dose-dependent following the order: APTT<Heptest<Anti-Xa<anti-IIa. In the subcutaneous studies, 1the t 1/2 was both test and dose-dependent. The AUC was also dose-dependent and followed the order: anti-Xa>Heptest>APTT>antMIa. The CLsys of the antMa was much faster in comparison to the other tests. The CLsys of the APTT and Heptest was independent of dose. However, anti-Xa CLsys by this route was lower than other tests. The apparent Vd followed the order APTT>antMIa>Heptest>anti-Xa. The bioavailability of the Certoparin as measured by various tests ranged from 81-119%. These studies suggest that beside providing pharmacokinetic data, APTT, Heptest and anti-IIa assays may provide useful data on thier safety and efficacy at high dosages. The immunological type of heparin associated thrombocytopenla (HAT II) is a severe complication of heparin treatment and is associated with arterial and venous thrombosis. Only patients with absolute thrombocytopenia have prompted suspicion of HAT in clinical practice. We report on a 44 year old male, who developed thromboembolic episodes after coronary angiography like reinfarction and thrombotic episodes of A. brachialis.

Fibrinolytic therapy combined with i.v. unffactionated heparin treatment was the therapy of choice and was followed by severe fua~er thromboembolic adverse effects. Besides an impaired fibrinolytic response and elevated antiphospholipid anitbodies, we diagnosed HAT type II in HIPA and ELISA (Stago-Boehringer, Marmheim). This special patient had platelet counts within a normal range, when developing the thromboembolic episodes. It appears that the normal platelet count during the thromboembolic episodes reflect a relative thrombocytopenia. From a clinical point of view we recommend the use of a lab panel to exclude HAT type II in patients with thromboembolic episodes under therapy with fractionated or unfractionated hepafin. Platelet counts within a normal range are no absolute exclusion criterion for HAT II. Low molecular weight heparins (LMWHs) are now commonly used for the prophylaxis of post-surgical thromboembolic complications. In this indication, LMWHs are administered as a single or twice a day subcutaneous regimen. Usually these agents are administered at 30-40 mg total dose which is equal to 3000-4000 anti-Xa (AXa) IU. Newer methods such as ehromogenic substrate based AXa methods and the heptest clotting time can be used to determine the effects of LMWHs during the initial phases of prophylactic therapy. This may be useful in the elderly and weight compromised patients where a fixed dosage may not be optimal and may produce bleeding effects. Similarly in the overweight patients, a fixed dose may not be efficacious. Thus, monitoring of LMWHs in these patients may be useful in the optimization of their therapy. LMWHs are also used in the treatment of deep vein thrombosis using both intravenous and subcutaneous protocols. High dosages of up to 200 mg SC/day and infusions of up to 30 AXa IU/kg/hr have been administered. In these conditions, the monitoring of the circulating LMWH levels may be useful in optimizing the dosage. We have modified the ACA heparin (Do_Pont Merck, Wilmington, DE) method to measure the LMWH levels in the plasma of patients treated with both the prophylactic and therapeutic dosage. Owing to the required turnaround time, simple operation and reliable results, this method was found to be of value in the monitoring of these agents. This presentation provides an overview of the clinical application of various LMWHs with particular reference to the need of monitoring for their effects to optimize the clinical outcome. A double-blind, multicentric, controlled trial was performed in order to compare the antithrombotic efficacy and safety of single daily doses of 5000 IE anti-Xa of low molecular weight heparin (LMWH) Sandoz (Certoparin) and 5000 IE unfractionated heparin (UFH) tid.

In 288 patients undergoing elective total hip replacement blood samples were drawn before the first subcutaneous injection of LMWH or UFH resp., two hours after administration on the first and 7th postop, day and on the last day of prophylaxis (day 10-14), Anti-Xaactivity was measured by chromogenic substrate assay, Heptest and aPTT by clotting assays and tissue factor pathway inhibitor (TFPI) and heparin-Pf4-antibodies by ELISA techiques. As expected, the anti-Xa-activity and the Heptest values were significantly higher in the LMWH-group at all time points after administration of the drugs; the mean values of Heptest were 35 sec in the UFH-and 75 sec in the LMWH-group respectively, The aPTT was not different in both groups. At the end of prophylaxis positive antibodies to heparin-Pf4complexes were detected ~n both groups; this however was not correlated with clinical thrombocytopenia. A detailed correlation between patients with deep vein thrombosis (DVT) and positive antibodies has still to be done (all patients were screened for asymptomatic DVT between day 10-14 by bilateral phlebography. TFPI was markedly increased in the LMWH-and only slightly elevated in the UFH-group; the differences are statistically significant.

Summarizing it can be concluded that antibodies to heparin-Pf4complexes may occur without clinical symptoms of hepafin-induced thrombocytopenia type II and that TFPI may play a sigificant role for the antithrombotic efficacy of UFH and LMWH. Unfractienated heparin represents one of the most severe and frequent causes of drug-induced thrombooytopenia. Heparin-indueed thrombocytopeala (HIT) occurring early in therapy is often mild and serf-limited, appearing to be caused by a direct aggregant effect of heparin on platelets (HIT type I). HIT type II, however, is immune-related an may result in absolute thrombocytopenia (platelet count <I50/~L) or a relative decrease in platelet count (?_50% reduction from baseline values). Unlike most other immune thrombecytopenias in which bleeding complications often predominate, HIT type II may results in severe arterial and/or venous thromboses. The management of patients with HIT type II and thrombosis is often difficult. Heparin must be stopped immediately. Antithrombotic, fibfinolytic, or antiplatelet agents have been used in these situations, as have anticoagulants other than unfraetionated hepafirL Plasmapheresis, used in other immune-related disorders, has also been occasionally reported. At our institution this intervention appears to provide significant elininal benefit to HIT patients. Several recent reports have noted that the heparin-dependent antibodies in HIT type II are directed against a complex of heparin bound to platelet factor 4 (PF4) and an ELISA-based test system for hepafin-PF4 eomplexinduced antibodies (HPIA; Stago) has been develuped~ We report our experience in 3 patients with HIT type II in whom plasmapheresis was instituted and the antibody levels in blood were measured pre-and post-treatment to identify wkether circulating antibody titers decreased thus providing some explanation for the clinical benefit. The patients developed profound absolute thrombocytopenia (range, 3-21 K/~tL) and positive heparin-induced platetet aggregation studies following treamlent with uofractionated heparin. Thromh0cytopenia induced by hepariu (HAT-Syndrome) typically appears several days after initiation of heparin therapy. This phenomenon is caused by antibodies of the lgG type which are induced by a heparin-PF 4-complex and which also activate platelets. In a recent study (N Engl J Med 1995; 332:1330-5) the HAT-Syndrom was diagnosed in 2.7 % (9 of 332) patients who had received unfractionated heparin (UFH) and in none of those 333 patients who had been treated with low-molecular-weight heparin (LMWH). 8 out of the 9 patients treated with UFH presented with at least one thrombotic event (7 times a venous, once an arterial event). The incidence of heparin-dependent IgG antibodies (7.8 %) was higher in the UFH treated group than in patients treated with LMWH (2.2 %). Our patient was a male caucasian white, 62 years of age, otherwise in good clinical condition with no severe diseases in his PMH. Both parents, however, had suffered several thrombotic episodes. The patient was admitted to the hospital in September "95 with clinical signs of a deep vein thrombosis (DVT) of the right leg. The diagnosis was phiebographically confirmed. After having given informed consent the patient was treated with 5 cycles of r-TPA (loco-regional application) and 3 cycles of UHSK as part of a clinical trial (UHSK vs. rTPA, Boehringer/M). Lysis was successful with complete recanalisation of the veins. After completion of lysis the patient was further anticoagulated with coumarin overlapped by heparin (UFH). As a complication the patient suffered from a retroperitoneal and an upper GI4ract bleeding from esophagitis grade II to HI while being on coumarin prophylaxis. Therefore the patient was put on LMWH and then discharged with this prophylaxis. After less than two weeks the patient presented again to the hospital with a relapse of DVT in the same leg. Phiebographically nearly all deep veins were occluded to a higher degree than before, i. e. also with concomitant pelvic vein affection and with hea~ 3, swelling of the whole leg. Blood count showed a marked decrease of platelets to 27/nl. The platelet aggregation test demonstrated a HAT-syndrome which was confirmed by the HIPA-assay. The heparinoid Orgaran® was not reactive in the assay. Having seen a previous bleeding episode in this patient, we did not see an indication for another b'sis therapy. Therefore we decided to put the patient on an intermediate dose of coumarin overlapped by hiradin (HAT study, Behring) instead of Orgaran®, since hirudin allows a much more precise dose adjustment. After 10 days the patient was discharged with no further complications. Risk factors for DVT have not been detected until now. To our knowledge this patient is one of the first cases of HAT-syndrome induced by LMVeH. Under routine conditions aPTT is the most frequently used test method for the surveillance of patients undergoing hepann therapy. In literature a 1,5 fold to 2,5 fold prolongation of the initial value is recommended for individual therapy a~IXatian. In contrast to the control of the enmarin therapy by means of prothrombin time measurement the individual hupafin sensitivity of the ~ reagents of this standard value hardly receives notice. Commercially marketed ~ reagents differ from one another method and concentration of the activator as well as their phosphelipids. When choosing a reagent the user must only take into consideration the sensitivity against its factors, heparin and lupus but also the adalXability of the reagent towards the given measurement system ( software variability towards incubation time, sodimentalion problems in reagents with high kaolin concentration). Reagents offering a high heparin sensitivity already are showing a prolongation of the coagulation time at low heparin dosage (0.1 -0.3 U/ml). In the therapeutic range (0.4 -0.8 U/ml) very often no linear relationship between prolongation of the aPTr and heparin concentration level is found_ Therefore very often coagulation times above the routine measuring range (180 s) can be observed in this range. Moreover, various medical measuring devices differ in their ability to recogniso the retarded entry of coagulation. Coagulation limes with different aPq'T reagents in correlation with the heparin concentration level can be found in the adjoined table. 

Significantiy prolonged coagulation ti.~es of aPTT lead to a reduction of the hel~ dose in clinical practice. Due to the discrepancy between heparin concentmon level and the prolongation of the aFrT in reagents with a non-linear heparin sensitivity a reduction of heparin dosage leads to an inmt~cient anticeagulalion and the risk of further thrombosis. Therefore the producers should be asked to include precise directions on the heparin sensitivity of the aPTT reagent in the therapeutic range in order to enable an optimal surveillance of the heparin therapy. A coordination between clinic and laboratory should be compulsory for the choosing of the aPTT reagent. Introduction: HAT is a rare but sometimes fatal complication of therapy with unfractionated (UFH) and low molecular weight beparins (LMWH). We report on our experience with the clinical coume and management in 12 patients (pts). In all cases a severe decline of platelet count and in 11 cases a positive HIPA test (= heparin-induced platelet antibodies) were documented.

Patients, clinical course and outcome: 8 female and 4 male patients (median age 68 years, range 22-78) were analysed in this retrospective study. 9 pts were referred from outside hospitals. All pts had been treated with UFH (5 pts additionally with LMWH) for prophylaxis (n = 9. in 2 pts pedoperatively) or treatment (n = 3) of venous thromboembolism (V'TE). In 11 pts at least one VTE occured during heparin therapy, in all cases prior to HAT-diagnosis (VTE: n = 13; putmonary embolism: n = 6; artenal embolism n = 1; thrombosis of sinus transversus n = 1). HAT was suspected 16.5 days (median; range 12 -48) after first heparin application. Hepadn therapy was discontinued immediately, At that time the median plateiet count was 35 G/I (range 10 -117). 9 pts were treated with Orgaran R, followed by recombinant hirudin (r-hirudin, Behdngwerke AG: clinical study) in 4 cases; 3 pts were treated exclusively with r-hirudin. Plat~let count normalized in 9 pts within 3 days (median; range 1-10). One patient in the Orgaran" group died on recurrent pulmonary embolism. 11 pts recovered without any further thromboembolic complication. Conclusion: In our small series of 10ts HAT was complicated with VTE in 11/12 cases. In many pts HAT was diagnosed strikingly late. For early diagnosis careful monitonng of platelet counts are mandatory dunng hepadn therapy, Both Orgaran ~ and r-hirudin seem to be safe and effective in preventing further thromboembolism, P 179 To prospectively evaluate coagulation or fibrinolytic activation in children long-term anticoagulation with LMWH was administered a longitudinal study was perfomed. Within a 30 months period 48 children and adolescents (6 -19 years) suffering from malignant bone tumors (Ewing's sarcoma or osteosarcoma) received Enoxaparin (Clexane R/Rhone-Poulenc Rorer, France) for primary propylaMs. The majority of patients received 0.6 mg/kg bw Enoxaparin 12 hours before major surgery (limb salavage). In addition, ten neonates and children received Enoxaparin in the same dosage for secondary prophylaxis.

In both groups therapy was adjusted to 0.2 -0.4 IU/ml anti Xa activity. Median (range) duration of therapy was performed for l0 (6 -24) weeks. Before ('1"1), one (T2) and 3 weeks (T3) Within the context of the present study an investigation on the physiology of clotting was conducted in three groups each consisting of 12 patients, who had to undergo a total end-prosthetic hip implantation (TEPH) for the first time. Criterion for allocation to a particular group was type of intraoperative transfusion the patient received : infusion of whole blood, erythrocyte concentrate or fresh frozen plasma. At eight fixed times the activity or concentration of the components of the kallikrein-kinin and inhibitory systems was determined. The statistic evaluation ensued with the SPSS software package. For all three patient collectives an activation of both systems was to be observed with the TEPH-implantation whereby there were no statistically striking differences between the three groups. Thus in the case of the kallikrein-like activity an intraoperative rise of over 37% was registered, while at the same point in time there was only a 4% reduction in the prekallikrein activity. Postoperatively a 10% rise in the prekallikrein activity was to be observed. As a reserve inhibitor alpha 2-macroglobalin showed no significant change in activity during the period of observation whereas the acute phase proteins alpha 1-proteinase inhibitor and Cl-esterase-inhibitor yielded a postoperative rise of 135% and up to 100%. The beta-factor Xllainhibition showed a rise of 121% during the reconvalescent phase. The 65 year old female caucasian patient presented here, was admitted to a nearby district hospital in December '94 with the acute symptoms of pulmonary embolism. The source of the embolus could not be detected. High dose heparin was administered. PMI-I revealed a coronary artery disease and a peripheral artery occlusive disease of the left lower leg, yet blood pressure values of the lower ex~emities were only slightly below brachial pressures. The first episode of pulmonary embolism was followed by a second one two weeks later for which the patient received heparin again. Following this treatment the platelet count decreased significantly to 10/ni. With an acute thrombosis of the lower caval vein and with Leriche's Syndrome, i. e. occlusion of the abdominal antra from the bifurcation down to the ileofemoral arteries bilaterally, the patient was transferred to our hospital. On initial examination we saw a patient in no acute distress, no dyspnea, blood pressure 120/80 mmHg, pulse 94/rain, no peripheral edema or disturbances of sensitivity. Neither femoral, popliteal, nor pedic pulses were palpable on either side. A heparin associated thrombopenia was detected with the platelet aggregation assay and confirmed by the heparin induced platelet aggregation assay (HIPAA). The autoantibodies targeting the underlying PF4-heparin complex were determined later by an Elisa method in serum samples of the patient (Asserachrom HIA, Diagnostica St,ago). A temporary Anth&~r filter system was inserted transbmchially into the lower eaval vein and placed jnst above the thrombus. In the following days, 2 cycles of lysis therapy with 9 Mil U streptokinase were administered taking six hours for each cycle. Heparin was given in the mean time via the filter system in therapeutic dosages in order to prolong values 1.5 to 2 times the individual base line level. At the end of the second cycle, pulses were palpable inguinally, two hours later both popliteal and the right pedic pulse were present. The left leg showed better microperfusion than before. Computer tumoglaphy did not reveal an)-thrombotic material in the descending aorta and the iliac arteries. For continuous anticoagulation after lysis we used the heparinoid Orgaran® to overlap the period until full anticoagulation was achieved by coumarin. The heparinoid was tested before us non-reactlve in the HIPAA. With this prophylaxis the platelet count increased to normal values. HAT II related thrombosis is the most severe complication of heparin therapy, especially in patients with pre-existing thromoembolic disorders (DVT, DIC). We present to our knowledge the first case of HAT II during thrombolytic therapy (IT) with simultaneous hepatin administration. At admission a 16 year old girl showed complete occlusion of the pelvic veins of the right leg (R common and external iliac v., common and proximal superficial femoral v.). As in children no prospective studies on the thrornbolysis of DVT with rt-PA are available, we started TT according to the Frankfurt/M. regimen: rt-PA (Actilyse) bolus injection 0.4 nag/kg bw followed by continuous infusion of 1 mg/kg bw/d; simultaneous low dose heparin 100 -200 U/kg bw/d. Doppler ultrasonographie control on day nine after the beginning of Tr revealed a further distal growth of the thrombus into the popliteal vein. At the same time AT IR was decreased to 70% and there was a dramatic drop in platelet count to <50% of the baseline value (216 to 86 Gpt/1). The diagnosis HAT ]I was confirmed by positive HIPA-test. The only heparin preparation not cross-reacting with the antibody was the LMW heparinoid Orgaran. Heparin therefore was replaced by Orgaran: bolus injection 1,500 U/h followed by infusion of 200 U/h. rt-PA-therapy was not discontinued. AT RI substitution was performed at levels <70%.

Under Orgaran administration platelet count rose from 77 to 239 Gpt/1 within 5 days. On day 12 complete reperfusion was achieved and rt-PA was discontinued ,on day 14. The patient was discharged from the hospital on prophylaxis with phenprocomnon in good general condition At admission thrombophilia parameters (Protein C and S, APC) were within normal range. Heparin induced thrombocgopenia (HIT) is a serious but rare side effect of treatment with unfractionated heparin. Patients with HIT present with a rapid decrease in thrombocyte count and occurence of venous or arterial thrombosis under heparin treatment. Patients with arterial occlusive disease who need surgical intervention are of high risk to develop rethrombosis especially during the first few postoperative days while they are under i.v. heparin-treatment. In our study we investigated prospectively all patients who were admitted to a surgical unit because of arterial occlusive disease and received surgical intervention. 15 patients were screened on the 3rd and 6th postoperative day for heparin-antiplatelet 4-antibodies, decrease in platelet count and thromboembolic complications. 3 patients developed antibodies against heparin-platelet factor 4. One of the 3 patients developed a venous thrombosis in the operated leg in combination with a decrease in thrombocyte count. We conclude that heparin-platelet factor 4-antibodies are often produced in patients under i.v. heparin treatment. Only few of these patients actually suffer from clinically s3anptomatic HIT.

Medizinische Universit~itsklinik, Institut far Klinische Biochemie und Pathobiochemie, D-97080 Wtirzburg, Germany The important physiological and pathophysiological role of platetets in health and disease is well established. Platelets are also the primary target of many vasoactive hormones, factors and drugs. In addition, platelets provide a very important model system for studying agonist-and antagonist-mediated signal transduction events (1,2). Platelet activation by agonists is associated with shape change, extension of filopodia, generation of intracellular messengers, secondary agonists and activation of adhesion receptors and integrins including the fibrinogen receptor GP IIb/IIIa. Activation of protein tyrosine ldnases and calcium-dependent protein kinases appear to be of critical importance for the mechanism of platelet activation. In contrast, platelet inhibition by vasoactive factors and drugs is often mediated by the cAMP and/or cGMP signal transduction cascades. Recent data form our laboratory suggest that the focal adhesion protein VASP, a major target of the intracellular NO/cGMP and prostacyclin/cAMP effector systems, is an important link between signal transduction pathways and elements controlling cell motility (3-6). The implications of the recent advances in platelet signal transduction research for understanding the physiology, pathophysiology and pharmacology of human platelets will be discussed. domain. However, in vivo truncated "I'PO is much less potent than full length TPO due to its rapid clearance. This suggest that the C-terminal glycosylated domain acts to stabilize circulating TPO. TPO stimulates both proliferation of progenitor megakaryocytes and their maturation to platelet producing megakaryocytes in vitro. In vivo TPO induces dramatic increa~ses in megakaryocyte number and platelet production in animals, indicating that TPO regulates both thrombopoiesis and megakaryocytopoiesis and is the primary regulator of physiological platelet production. This later notion is supported by the phenotype of TI~ and cMpl knockout mice in which platelet levels are only 10-20% of normal while other blood lineages are unaffected. In animal models of myetosuppresion ,and marrow transplant, TPO reduces the platelet nadir seen in the~se models and significantly accelerates platelet recovery, TPO also has profound effects on red cell recovery in these models. These results suggest that TPO will be clinically u~ful for the treatment of thrombocytopenia in caucer patients who undergo chemotherapy or bone marrow transplant. The HAPARG-study ( .~a.emostatic Parameters as Risk factors in healthy volunteers) was sponsored by the German Ministry of Research and started in 1984. A previous study ( PARD, Intern. Angiology 5, 181-195,1986 ) had shown that spontaneously enhanced platelet aggregation as measured with the PAT-Ill-Test and a high fibrinogen level were significant risk factors for new vascular occlusions in diabetic patients. It was the aim of the HAPARG study to establish if spontaneous plateletsggregation and other hemostatic variables are independent risk factors for vascular occlusions also in healthy volunteers. Employees of HSchst AG, Frankfurt aged 40-65 years and personnel of the University of Mainz, aged 30-60 y. entered this prospective study. Besides anamnestic data on smoking, hypertension and diabetes blood pressure, the ankle/arm Doppler -index and an ECG were recorded and serum cholesterol, HDL, LDL, triglycerides, uric acid and glucose were measured. 1885 men and 988 women entered the study and were followed for 4-6 years. During this period 53 vascular occlusions ocoured ( 36 coronary and 9 cerebral events and 8 peripheral vascular occlusions). Only three of these events occured in women. Besides age and gender as expected risk factors the multivariate logistic stepwise regression analysis revealed as significant risk factors slightly elevated levels of blood glucose (p=0.001), smoking (p=0.0013), an elevated diastolic blood pressure (p=0,018),followed by spontaneous aggregation (p = 0.017) and higher HDL-levels (p=0.034) as significant risk factors for men. Higher fibrinogen levels just missed significance in this analysis (p=0.059). None of the other hemostatic parameters showed any significant correlation with the vascular events.To our knowledge this has been the first prospective trial in a large population of healthy individuals in which a platelet function parameter has been studied together with other possible risk factors. Spontaneously enhanced platelet aggregation seems to be an independent risk factor and may be an indicator of an ongoing active atherosclerotic process. Mild bleeding symptoms -petechia, prolonged bleeding after tooth extraction -were observed in a patient with normal coagulation tests but slightly disturbed phtelet function: decreased aggregation in response to ADP, PAl: and thrombin receptor-derived peptide (TRAP-6). Platelet count, agglutination with both ristocetin and botrocetin and thrombininduced aggregation were in the normal range. An abnormal membrane glycoprotein detected in electrophoretic studies of the patient's platelet proteins was shown to be related to GP llxx by immunoblotting. The variant GP differed even from the largest normal polymorphic GP Iba form A by an increase in molecular mass, Platelets of the patient contain the variant and normal C form in equal amounts. The quantity of GP Ib as detected by flow cytometry is normal. lmmunoblotting studies of proteolytic fragments of the abnormal GP indicated that the defect is located in the C-terminal part of the glycocalicin fragment. Measurement of the length of glycocalicin molecules after glycerol-spraying rotary shadowing electron microscopy demonstrated two populations of molecules in the patient sample: a normal on with an average size of 470 Angstr6m and a fraction with an average size 0f650 Angstr6n~ Preliminary results of molecular analysis of the GP Iba gene in the patient confirm the localization of the genetic defect by protein studies. In summary, the patient is heterozygous for a hitherto undescdbed molecular variant ofGP ~a. Barnes and co-workers have shown that simple triple-helical collagenlike peptides, comprising basically a repeat Gly-Pro-Hyp structure, are highly platelet reactive in polymeric form. They additionally have shown that the activity does not involve GPla/lla (integdn c~z~l). Platelets adhere under static conditions to these fibrillar peptides mainly in a bivalent-cation independent manner; the bivalent cation dependent adhesion can be inhibited by an antibody against GPflb/llla (integfin m~d33). However, platelets of a patient with type I Glanzmann's thrombasthenia secreted their ¢z-granule-and dense body content just like normal controls while activated with the triple-helical Gly-Pro-Hyp peptide.

The lack of an essential involvement of CD36 was shown with CD36deficient platelets, which respond to the peptides as readily as normal platelets when fibdnogen binding and CD62 and CD63 expression were measured. To study the involvement of the yon Willebrand factor (vWf) platetets from a patient with severe type 3 vWd (vWf not detectable in platelets and plasma) were investigated.

The vWf deficient ptatelets responded quite normally to the fibritlar collagen-like peptide. Addition of pudfied vWf to the vWf-deficient platelets before adding the paptide raised the platelet respond slightly

We conclude that neither ~3~ integrin, nor m~ integdn, nor CD36, nor vWf are the pdmary platelet receptor for the collagen-like peptide. There is evidence that native low density lipoprotein (LDL) can be oxidatively modified in vivo and the oxidized LDL may contribute to thrombogenesis and atherogenesis by damaging endothelial cells, eliciting vasocontractiola and stimulating blood platelets. By means of biochemical methods, electron microscopy, reflection contrast microscopy and computer image analysis, we find that oxidized LDL, compared to native LDL, affects platelets at least in the following five aspects. 1) Oxidized LDL alters platelet functional morphology in a time and dose dependent manner. 2) Oxidized LDL induces secretion of serotonin from both morphological resting platelets and shape changed platelets. 3) Oxidized LDL advances, accelerates and enlarges platelet adhesion. 4) Oxidized LDL causes cytodamage in platelets and cultured endothelial cells. 5) Oxidized LDL stimulates platelets and endothelium, which leads to a formation of microthrombi on the monolayer of human umbilical vein endothelial cells. Further experiments show that oxidized LDL inhibits the activity of Ca2+-ATPase in purified plasma membranes of platelets, and therefore increases the cytosolic calcium level in platelets. These results clearly indicate that calcium signaling pathway is intimately involved in the mechanism of platelet activation induced by oxidized LDL. Since oxidized LDL, a relevant platelet agonist, has been confirmed to be present in vivo, further studies on its role in platelet activation and plateletendothelium-interaction can help for better understanding thrombogenesis and atherogenesis. Recantly physical parameters such as sheer stress, hypo-or byperoxic conditions and hyperthermia have been shown to modulate endothelial cell dependent fibrinolysis. In this study we investigated whether yet another physie~ stimulus namely hypothermia might have an impact on the expression of components of the fibdnolytic system of human endothelial cells in culture. Confluent cultures of human umbilical vein endothelial cells (HLIVECs) were exposed to gC ° for 10, 20, 40, 80, or 160 rain, respectively. Control cultures were exposed to 37°C for the same time periods. Thereafter medium was removed, fresh medium was added and the cells were incubated for 24 hours at 37°C Conditioned media (CM) were harvested and plasminogen activator inhibitor-I (PAl-I) antigen was determined by a specific ELISA. Our data showed that HUVECs responded to exposure to 8°C with a time-dependent increase of PAI-i in CM. Maximum induction of PAl-! antigen was seen after 20 minutes exposure to 8°C (PAl-! antigen: 754_+.83 ng/105 cells for treated cells; 4202_17 ng/105 cells for control cells). The inereese in PAl-1 antigen was also reflected on the level of specific mRNA synthesis as evidenced by Northern blotting which showed a twofold increase in PAI-I specific mRNA in HUVECs exposed to 8°C as compared to control cells. In conclusion, our data gives evidence that hypothermic stress like several other forms of physiological or pathological stress, results in activation of endothelial ceils as evidenced by an increased expression of PAI-I. Our findings provide ~rther evidence that such activation of endothelial cells can be induced not only by biochemical mediators but also by physical stimuli. Nitric oxide (NO) is formed from intracellular L-arginine by two NO synthase (NOS) isoforms: a Ca+ +-dependent constitutive form (cNOS) and a Ca+÷-independent inducible form (iNOS) which is expressed after challenge of cells with immunologic or inflammatory stimuli. Endothelial cell derived NO is an important signaling molecule inducing smooth muscle cell relaxation and platelet inhibition, however its role in cell proliferation is poorly understood. The present study investigates the pattern of NOS isoforms and NO production in actively proliferating low density human umbilical vein endothelial cells (LD-HUVEC) and in high density monolayers of quiescent cells (HD-HUVEC). LD-HUVEC but not HD-HUVEC expressed iNOS (measured as Ca ++independent citrulline formation from L-arginine in cell lysates) with a maximal activity of 30-40 pmoles/min/mg protein, cNOS (Ca +÷dependent citrulline formation) was demonstrated in HD-HUVEC (0.02-0.2 pmoles/min/mg) but not measurable in LD-HUVEC lysates. lmmunoprecipitation of cell lysate with a mAb against murine macrophage iNOS decreased iNOS activity of supernatant by 70% in LD-HUVEC. Immunoprecipitation with an anti-ecNOS mAb did not modify iNOS activity in LD-HUVEC but abolished cNOS activity in HD-HUVEC. NO release (measured as nitrite + nitrate production in an 18 h incubation in the presence of 1 mM L-arginine) was 1.9 nmol/mg cell protein in the supernatant of HD-HUVEC whereas it was t0fold higher with LD-HUVEC. Measurement of intracellular cGMP after stimulation of cells with I-2 #M ionomycin (a sensitive index of eNOS activity) showed a 400-800% increase of basal levels in HD-HUVEC but only 50-150% increase in LD-HUVEC. On the contrary, sodium nitroprusside-induced cGMP increase was similar in non-confluent and confluent cells. The expression of iNOS and the high NO production in LD-HUVEC suggest a role of endogenous NO in mediating cell proliferation. Indeed, the NOS inhibitors L-NMMA and L-canavanine inhibited [~H]thymidine incorporation in LD-HUVEC by 60%. On the ++ nt NO r UCtlOn ar x r d m other hand eNOS and Ca -depende p od " e e p esse " differentiated HUVEC and correspond to the ability of the cells to regulate vascular tone and platelet activation. The processes of replication, maturation and motility of progenitor cells of different lineages in the bone marrow are tightly regulated, particulady by various cellular contacts and their modulation involving pericellular proteolysis. While mononuclear phagocytes and related cell lines are well characterized for surface localized plasminogen activation by receptor bound urokinase, megakaryocytes are poorly described in this respect. In the present study the expression of mRNA of urokinase receptor in megakaryoblastic cell lines CHRF-280, Dami and Meg01 was found at a low basal level and increased 5-10 fold after PMA-treatment. Urokinase receptor mRNA levels remained differentiation dependent elevated for up to 5 days. Functional analysis of uPAR expression on the cell surface was documented immunologically by FACS-anatysis and on the functional level by direct binding of radio-labeled amine-terminal fragment of urokJnase and mirrawed the increase in urakinase recptar mRNA level. As expected, antibody and ligand binding was sensitive towards treatment with phosphatidyl-inositol-specific phospholipase C, since the urokinase receptor belongs to the group of GPI-anchored receptors. In cultered megakaryocytes derived from bone marrow urokinase receptor mRNA was demonstrated by RT-PCR, as well. These results indicate a differentiation-dependent increase of urokinase receptor expression on cells of megakaryoblastic cell lineage suggesting the participation of urokinase dependent events during maturation process and intravasation of these platelet precurors. 

Regulation of AM gene expression may be coupled to oxidative stress through redox sensitive transcriptional regulatory factors. The aim of this study was to investigate the influence of selected antioxidants on TNFa (100 U/ml, 6 h)-induced surface expression of ELAM-I, ICAM-I and VCAM-I in human umbilical vein endothelial cells. AM expression was determined by quantitative flow cytometry and immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase method). The following antioxidants were tested: pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine (NAC), glutathionemonoethyl ester (GSHMEE), probucol and the estradiol-derived scavestrogens J824, J861, J901. Additionally, 17fi-estradiol was examined. The used concentrations were 1-5 mM for NAC and GSHMEE and 1-100 ~M for all other substances. According to their influence on TNF-induced AM expression, the tested antioxidants can be divided into three groups: 1) PDTC, NAC and GSHMEE inhibit the expression of all three AM, 2) J861 and J824 reduce VCAM-I and ICAM-I and increase ELAM-I, 3) J901 and probucol show no effect. 17fl-estradiol elicits a potentiation of TNFa-induced expression of all three AM. TNFa-induced VCAM-I expression was most effectively inhibited by PTDC and J861 (100 eM: 68% and 78% inhibition, respectively). The best inhibitors of ICAM-I induction were PDTC (100 ~M: 51% inhibition) and J824 (100 t~M: 47 % inhibition). J824 and J861 most effectively increased the ELAM-t induction (100/~M: 85 % and 62 %, respectively). The results confirm that antioxidant-sensitive mechanisms play a role in TNFct-induced AM upregulation. It is suggested that ICAM-1-and VCAM-l-expression is regulated differently from ELAM-I. The diversity of antioxidant effects might be due to distinct levels of redox control of transcription with an antioxidant-inhibited and an antioxidant-promoted step. Heamophiliacs who continue to produce tilers of F.V11I inhibitors no higher than 5 to 10 Bethesda Units (BU) when given continuous F,VIII replacement therapy are classified as low responders. Studies carried out on low responders to assess the effect of immune tole~lcc therapy fliT) in these patients. It was found that elimination ofiahibitors in low responders could be achieved using F.VLII at 20 to tOO U/kg given every 2 to 3 days over a period of 6 weeks. We consider that 1TT is justified in this group of patients. prevents rebooster effects and reduces elimimfion time of F.VIII inhibitors. It also reduces the enhanced mortality in patients with inhibitors and has been shown to be cost-effective. In view of the improved prognosis, we recontmend starting ITT in low respor, ders soon after the diagnosis has been made in order to minimize F.VIll exposure before isfi.tiating El'. ITr in low responders has a better success rate an.,d requires shorter U'eatment than ITT in high responders.

W.Schramm, Med.Klin~, Innenstadt, LMU, Mfinchen Following replacement therapie~ in hemphilm t_be developmem of inhibimrs to the p*ficat's deficient factor is the most serious sequelae. The incidence of inhibitors varies between 15 and 35 % of persons with hemophilia A. Inhibitor develop, mostly in paticm~ with sovere bemoph/lia A, early in Life and afmr few exposure days (9 -12 days). Using the Bethesdamethod the inh~itom can be derided in low (< 5 BU) and high msponder (>5 BU) Hemopb~iacs with high fitcrs have ~ually serious ~ problems. They are resistent to mg,,flary replacement therapy, The ~ goal in the treawnent is to control severn acum bleedin~ and to eradicate the inlu'bitor perrmnanfly and to induce tolea'ance. In the tream'tmt of acute blcedings in patients with hlhibitors factor VIII inhibitor bypassing ag~ts like activated prothro~ complex concenuxtes (FEIBA) or prothrombin complex concentrates (PCC) arc mostly used. The meehani~n of aefiou of theses concentrates is net fully investigated. Their effect is usually related to the high coment of activated clotting factors ~d phosphoUpids. Since some years acdwated recombinant Factor VII (F VII a) is used to treat patients with inl'dbitocs successfully in several clinical situations including surgery. In addition porcine factor VII1 is widely used in particular in the UK for the treatment of factor V].II inhibitor patients and could demonstrade good clir3cal results, In case of life threatening bleedings a temporary reducfic~ of inhihitors could be. ~hieved by using extem*,ivc plasma exchange (Protein A adsorption) and immune suppression with cyclophosphamid (~alm6 protocol). Follow~g the first description by H. Bmc~'~mn some modifications for tlm induction of irmnune tolerance in hemophilia A patients have ~en propet'ed. These schedule, can be derided into high, intemxxfime and low dosage roglrmms di:ffea'Jng in the dosage of factor VIII infused. Successful rates about 70 to gO % can be obtained with ~ and high dose regimens. But is has to be co~sidered that the~ expensive treA.t~nt regimens have a great physical and p .syc.hosocial impact to the benx~-li~s and thch" farm~e& The different immu~ mler-a.~ze mg~-'~ predominantly used in high rcsponder inhibitor. Most of the patients with low concentrations of inhibitors cm be managed with factor VIII in increased dosage. This is in agreement with the consensus recorrnr~rdadons for u'eatlncnt hemophiliacs in Germany fi'orn 1994. Before Vitamin K(VK) prophylaxis was generally accepted in Japan, the incidence of infantile VK deficiency was 1:4000 both idiopathic and secondary types. Since 1981, 4 nationwide surveys have been conducted. The current incidence rate is now about one-tenth that in early 1980. However, in a small number of eases, VK deficiency oceured despite prophylactic administration during the neonatal period.

In order to clarify the absorption,excretion and transplacental transport of VK in the perinatal period,following studies were carried out. t)Hepaplastintest(Normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)Correlations were made between mothers'and babies'Hepaplastin test values. 3)Transplacental transport of VK 2 was studied.

The general activity of VK dependent factors in pregnant women was much higher than in non pregnant women. As far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with Hepaplastin test value of less than 120% of the normal adult value, the value of the Hepaplastintest was less than 30 % of normal adult value in the cord venous blood°

We also demonstrated that VK passed through the placenta but only in small qualities. 61HIV-negative patients (median age 4]yrs, 13-70}, formerly treated with non-virusinactivated coagulation products, underwent hepatologic examination, including AFP screening and sonography. 31.suffer from severe, 20 from moderate or mild haemophilia A or B, 10 from other severe coagulation factor deficiencies. 52 had been treated with products of the Swiss Red Cross (SRC) only (28 with small pool cryoprecipitate}, 4 with foreign products only, 5 with both SRC and foreign products. Treatment intensity was variable with>20'000 IU/yr in 26,< 20'000 in ]6, < 1 treatment episode/yr in ]0, a total of only 1-3 treatment courses in 6 patients. 3 afibrinogenemic patients had prophylactic replacement therapy. HCV serology was positive in 56/6] patients (92%), in 47 with detectable HCV RNA (77%). The 5 persons who escaped HCV infection, with normal ALT-levels and without sonographic alterations, had low intensity treatment with small pool SRC preparations Only. ALT-levels were elevated in 33/56 Anti-HCV positive patients (59%). 26/56 had abnormal sonographic findings (46%). There was a clear correlation between elevated ALT-levels and abnormal sonographies: Of 33 patients with elevated ALT 23 had abnormal sonography, of 23 with normal ALT 3 had abnormal sonography. 8 patients had liver cirrhosis (6 with clinically overt hepatopathy), 4 (4/56 = 1%!) with hepatocellu]ar carcinoma (HCC) with elevated AFP-leveIs. Of these 4 patients 2 had intraarterial embolization with ]ipiodol-epirubicin; in 2 patients HCC diagnosis was made in a late stage. I patient with advanced liver cirrhosis underwent successful liver transplantation. 2 of the 6 patients with hepatopathy had severe haemophilia with temporary high alcohol intake, 4 had mild coagulatlon disorder with few treatment episodes. Possible precipitating factors were coinfection with HBV, high alcohol consumation and first exposure to HCV contaminated blood products in an advanced age, but not intensive replacement therapy. very similar results for F Vlll and vWF. Since the factor VIII level is kept steady above the level where there is an increased risk of haemorrhage, continuous infusion is haemostatically safer and more efficacious than bolus injections, Another advantage is a progressive decrease of clearence during the first days after surgery which leads to a substantial reduction of factor concentrate consumption by avoiding the innecessary peaks of bolus injections.

22 children with severe form of haemophilia A undergoing elective surgery received continuous infusions with different plasma-derlved and recombinant F VIII concentrates. Before surgery, patients got bolus injections to raise the factor VIII levels to more than 80 %. During continuous infusion factor VIII levels were measured two to three times a day and the infusion rate of 4 to 5 IU/kg/h could be reduced on the second or third day to 2-3 IU/kg/h. The clinical efficacy was excellent with no bleeding events. In 5 children with vWd also undergoing elective surgery continuous infusions with Humate pR were performed in the same way. No bleeding events were observed in these patients. None of the patients developed postoperative wound infections. The overall doses of F Vtll concentrate 'were about 20-30 % lower than those required during replacement therapy with bolus doses. 

lg8 FACTOR X FRANKFURT I : MOLECULAR AND FUNCTIONAL CHARACTERISATION OF A HEREDITARY FACTOR X DEFECT (GLA +25 LYS) Huhmann I., HOller B., Krinninger B., Turecek P.L, Richter G., Scharrer I., Forberg E., Watzke H. Univ. Klinik f0r Inhere Med.I, Abteilung for H~tmatologie und H~mostaseologie, W~en; Immuno-Ag, Wien ; Klinikum der J.W. Goethe-Univ. Frankfurt am Main, Abt. f. Angiologie. Factor X (FX) is a Vitamin K-dependent plasma protein which is activated either by FVIla/tissue factor or IXaNIIla. FXa is the main enzyme for conversion of prothrombin to thrombin. The congenital FX-deficiency (Stuart -Prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. Our propositus is a 24 year old patient presenting a mild bleeding tendency. His P'FI (36 sec) is within the normal range, the PT ( 73% of normal) is slightly reduced. The Factor X antigen level is reduced to 55% of normal. Molecular charactedsation of the genetic defect was performed by amplification of the eight exerts and exonintron junctions by PCR and subsequent direct sequencing of the products. In comparison to the normal sequence we could determine a single mismatch within exon II resulting in the substitution of +25 Gla (GAA) by Lys (AAA). The mutation abolishes a naturally occuring Mboll site in the DNA sequence of exon II. The status of the FX encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon II and restriction digest with Mboll. These family members were heterozygous with respect to the mutation in exert II. FX was isolated from plasma of the propositus by monoq ion exchange chromatography. Performing clotting assays with purified FX Frankfurt I we determined an activity of 89% of normal FX upon activation with RW, 77% upon intdnsic activation (aPTT) and 81% upon extrinsic activation (PT). This compares well with the results obtained from the patient plasma ( PT 56%, PTT 55% and RW 57% of normal) when the reduced FX-Ag-level of the plasma (55%) is taken into account. We therefore conclude that the substitution of Gla +25 to Lys results in a FX molecule which is severely defective in both the intrinsic and extrinsic pathway of blood coagulation. Bleeding after cardiothoracic surgery is still a frequent, important and sometimes life-threatening complication. Thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. This prospective study included patients undergoing cardiopulmonary bypass surgery. Blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. Blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, thrombin time, thromboplastin time, aPTT and levels of fibrinogen, ATIII and C-reactive protein; soluble fibrin (SF) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (FtDP) by an ELISA from Organon Teknika. N= 109 patients were examined (age: 64__+9 y). They lost 750+__460 ml blood (mean+SD) into the drains within the first 18 hours after end of surgery. A severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). Fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : FtDP levels exceeding 12 mg/I at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificity of 96% and a diagnostic efficacy of 91%. In contrast, soluble fibrin which correlated well with fibrinopeptideA (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n = 109). This observation does not match to the correspondence of SF with organ dysfunction during DIC: SF reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from BREDBACKA (1994). Other parameters were less predictive than FtDP and SF. Therefore, further examinations are necessary to determine the value of soluble fibrin for a risk prediction of bleeding complications or DIC. A differentiation of splits products deriving from either fibrinogen, fibrin or XL-fibrin will provide further insights into fibrin(ogen) metabolism. Heparin induced thrombocytopenia represents a multicomponent syndrome associated with the use of heparin and related drugs resulting in not only thrombocytopenia, but also arterial thrombosis of varying magnitude. The initial diagnosis ofthis syndrome is usually made by clinical observation and a drop in platelet count. Conventional diagnostic methods include platelet aggregation responses to patient's serum and ~4C serotonin release in response to patient's serum, aggregation/agglutination of patient's platdets in response to heparins and the detection of patients anti-heparin platelet factor 4 (HPF4-AB) ned-antibodies by using Elisa methodology. Several other individualized methods are also used to demonstrate platelet activation. To test the diagnostic validity of the platelet aggregation (PA) 14C serotonin release (SR) and the relevance of HPF~-AB 340 serum samples collected from patients with clinically eunfwmed eases of lilT syndrome were compared in parallel in various assay systems. The diagnostic efficacy of these tests varied from 60-74% with the PA test providing better results than others. When the PA test was compared with serotonin release, a poor correlation was noted (r=0.47). In contrast, the correlation between the PA and HP4-AB was somewhat better (r=0.66). In another study, blood samples collected from 50 patients treated with ahigh dose low molecular weight beparin for two weeks (80 mg O.D.) were tested. 20 of these patients showed a high titre of HPF4.AB without any decrease of platelet count. None of these patients were found to be positive in the 14C serotonin release assay. A third study included blood samples from DVT patients administered with IV heparin infusion, high dose SC LMW heparin (Certoparin) and IV LMW heparin for the management of DVT. None of these patient groups (20-34) exhibited any HIT responses, hmvever, the incidence of high HPF4-AB titre was found to be 53% in heparin, 36% in patients with LMW heparin IV and 26% in LMW heparin SC groups. PA and SR studies revealed 8% and 12% false positive ~ respeetively. These studies clearty suggest that the currently available ~ for laboratory diagnosis of HIT syndrome are of limited value, and caution should be exercised in the interpretation of the results obtained with these tests. Heparin-induced thrombocytopenia (HIT) is one of the major severe side effects during treatment with heparin. In postoperative medicine clinical studies demonstrated the prevalence of HIT with unfractionated over fractionated heparins. Few data are available from the non-ope1"ative medicine and from patients without thmmboembolism before heparinization. In a controlled prospective randomized study the safety and efficacy of low-dose heparin was compared with a lowmolecular-weight (LMW) heparin over 10 days in bedridden medical inpatients (Haemostasis, in press). 1968 patients were randomized and controlled for the development of thrombocytopenia. Thrombocytopenia was defined as a platelet count below 40.000lid at day 10. 4 patients developed thrombocytopenia in the heparin group and no patient in the LMWheparin group (p<0.05). None of the patients with thrombocytopenia developed a thromboembolic complication. In a second prospective case control study 90 patients with side effects on anticoagulants were treated with LMW-heparin once daily subcutaneously for a period of 1 month to 9 years. Platelet count was performed every 1 to 3 months. None of these patients developed thrombocytopenia during heparinization with LMW-heparin. It is concluded that HIT is a very rare complication in nonoperated bedridden medical patients. A decrease of platetet count may occur in about 0.5% of patients receiving low-dose heparin. The incidence of HIT with thrombosis during low-dose heparin and of HIT during LMW-heparin in non-operated patients is manyfold lower and remains to be determined.

Terminology: Instead of the term "hemorrhagic disease of the newborn (HDN)" the term VKDB should be used, since neonatal bleeding is often not due to VKdeficieacy and VKDB may occur after the neonatal period (i.e. after 4 weeks). Definition: VKDB is a bleeding disorder caused by reduced activity of VKdependent coagulation factors which responds to VK. Diagnnsis: in a bleeding infant a prolonged PT (INR > 3.5) together with normal fibrinogen and platelet count is almost diagnostic of VKDB. The diagnosis is proven, if VK shortens the PT (after only 30-60 minutes) and/or stops bleeding. Classification: Classification by age of onset into early (< 24 h~. classic fdav 2-7) and lale form (> I week <6 months), and by etiology into idionathic and ~ec0nd~'y. in secondary VKDB in addition to breast feeding other factors can be demonstrated, such as poor intake or absorption of VK and increased consumption of VK. VK-Prophylaxis: Benefits: Oral and intramuscular (i.m) VK (one dose of I nag) prevents equally well the classic form of VKDB. Lm. VK appears to be more effective in preventing the late form (times 15-> 50). The protection achieved by single oral prophylaxis (times 3-5) is improved by triple oral VK (times 15-30). Risks: Because of poten[ial ri~l~ associated with extremely high levels of VK and the possibility of injection injury, i.m. VK has been questioned as the prophylaxis of choice for normal neonates. Since VK is involved not only in coagulation but 'also in carboxviation with multiple effects, excessive deviations from the low physiologic concentrations, which prevail in the fully breast-fed healthy mature infant should be avoided. Proposal: Repeated (daily or weekly) small oral doses of VK are closer to physiologic conditions than single i.m. bolus doses, which expose neonates to excessively high VK levels. The incidence of intracranial VKDB can be reduced if the grave significance of warning signs is recognized (i.e, icterns, failure to thrive, feeding problems, minor bleeding, disease with cholostasis). Whether or not the more reliable absorption of the new mixed mieellar (MM~ nrenaral~i0n of VK can reduce the protective oral dose of VK-.prophylaxis has to be evaluated. Before Vitamin K(VK) prophylaxis was generally accepted in Japan, the incidence of infantile VK deficiency was 1:4000 both idiopathic and secondary types. Since 1981, 4 nationwide surveys have been conducted. The current incidence rate is now about one-tenth that in early 1980.

However, in a small number of cases, VK deficiency occured despite prophylactic administration during the neonatal period.

In order to clarify the absorption,excretion and transplacentel transport of VK in the perlnatal period,followlng studies were carried out. 1)Hepaplastlntest(Normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)Correlatlons were made between mothers'and babies'Hepaplastin test values. 3)Transplacental transport of VK 2 was studied.

The general activity of VK dependent factors in pregnant women was much higher than in non pregnant women. As far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with Hepaplastln test value of less than 120% of the normal adult value, the value of the Bepaplastlntest was less than 30 % of normal adult value in the cord venous blood.

We also demonstrated that VK passed through the placenta but only in small qualities. The point mutation G to A at nt 449 in exon V of the factor X gene (Gin 102 to Lys) has previously been found in two independent kindreds with FX deficiency. It occured in both families in an heterozygote state and was associated with two other genetic defect in the FX gene. We have identified another familiy in which this mutation occurs in a homozygote state. In this family the mutation is associated with the previously reported mutation Gla 14 to Lys which also occurs in a homozygote state. The PT and PTT of the proposita and her siter are markedly prolonged. The FX activity is reduced to <1% in the extrinsic system, to 30% in the intrinsic system and to 18 % after activation with RVV. The FX antigen is reduced to 20%. The coagulation profile of this family thus is identical with that of FX Vorarlberg despite the fact that the FX Vorarlberg kindred is only heterozygous for the mutation Glal02 to Lys.

Haplotype analysis could not rule out consanquinity with the FX Vorarlberg kindred. These data suggest that the mutation at nt 449 which leads to a fairly dramatic amino acid change from Glu to Lys would indeed represent a polymorphism. To further address this question we cloned the FX gene in an expression vector (pCEP 4) for transient expression in the human embryonic kidney cell line 293 and introduced the mutation at nt 449 by site directed mutagenesis. Hereditary deficiency of factor IXa, a key enzyme in blood coagulation, causes hemophilia B, a severe X-chromosomelinked bleeding disorder; clinical studies have identified nearly 500 deleterious variants. The x-ray structure of porcine factor IXa shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for fX activation by phospholipid-bound flXa and cofactor Villa. The 3.0 A resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (EGF)-like modules; the N-terminal Gla module is partially disordered. The catalytic module, with covalent inhibitor D-Phe-Pro-Arg chloromethyl ketone, most closely resembles fXa but differs significantly at several positions. Particularly noteworthy is the strained conformation of Glu-388, a residue strictly conserved in known fIXa sequences but conserved as Gly among other trypsin-like serine proteinase. Flexibility apparent in electron density together with modelling studies suggests that this may cause incomplete active site formation, even after zymogen activation, and hence the low catalytic activity of fixa. Most hemophilic mutation sites of surface fiX residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary flXa-fVIlla-fX complex structure: the stabilizing fVIlla interactions force the catalytic modules together, completing flXa active site formation and catalytic enhancement.

FACTOR X FRANKFURT I MOLECULAR AND FUNCTIONAL CHARACTERISATION OF A HEREDITARY FACTOR X DEFECT (GLA +25 ---, LYS) Huhmann I., HOller B., Krinninger B., Turecek Pi., Richter G., Scharrer I., Forberg E., Watzke H.. Univ. Klinik ftlr Innere Medi, Abteilung for H~matoiogie und Hamostaseologie, W~en; Immuno-Ag, Wien ; Klinikum der J.W. Goethe-Univ. Frankfurt am Main, Abt. f. Angiologie. Factor X (FX) is a Vitamin K-dependent plasma protein which is activated either by FVIla/tissue factor or IXaNIIla. FXa is the main enzyme for conversion of prothrembin to thrombin. The congenital F×-deficiency (Stuart -Prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. Our propositus is a 24 year old patient presenting a mild bleeding tendency. His PTT (36 sec) is within the normal range, the PT ( 73% of normal) is slightly reduced. The Factor X antigen level is reduced to 55% of normal. Molecular characterisaUon of the genetic defect was performed by amplification of the eight exons and exonintron junctions by PCR and subsequent direct sequencing of the products. In comparison to the normal sequence we could determine a single mismatch within exon II resulting in the substitution of +25 Gla (GAA) by Lys (AAA). The mutation abolishes a naturally occuring Mboti site in the DNA sequence of exon II. The status of the FX encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon II and restriction digest with Mboll. These family members were heterozygous with respect to the mutation in exon I1. FX was isolated from plasma of the propositus by monoq ion exchange chromatogrephy. Performing clotting assays with purified FX Frankfurt I we determined an activity of 89% of normal FX upon activation with RW, 77% upon intrinsic activation (aPTT) and 81% upon extrinsic activation (PT). This compares well with the results obtained from the patient plasma ( PT 56%, PTT 55% and RW 57% of normal) when the reduced FX-Ag-level of the plasma (55%) is taken into account_ We therefore conclude that the substitution of Gla +25 to Lys results in a FX molecule which is severely defective iP both the intrinsic and extrinsic pathway of blood coagulation. Bleeding after cardioth~)racic surgery is still a frequent, important and sometimes life-threatening complication. Thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. This prospective study included patients undergoing cardlopulmonary bypass surgery. Blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. Blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, throm. bin time, thromboplastin time, aPTT and levels of fibrinogen, ATIII and C-reactive protein; soluble fibrin (SF) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (FtDP) by an ELISA from Organon Teknika. N= 109 patients were examined (age: 64+9 y). They lost 750+__460 ml blood (mean_+SD) into the drains within the first 18 hours after end of sur. gory. A severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). Fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : FtDP levels exceeding 12 mg/I at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificily of 96% and a diagnostic efficacy of 91%. In contrast, soluble fibrin which correlated well with fibrinopeptide A (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n= 109). This observation does not match to the correspondence of SF with organ dysfunction during DIC: SF reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from BREDBACKA (1994). Other paramelers were less predictive than FtDP and SF. Therefore, further examinations are necessary to determine the value of soluble fibdn for a risk prediction of bleeding complications or DIC. A differentiation of splits products deriving from either fibrinogen, fibrin or XL-fibrln will provide further insighls into fibrin(ogen) metabolism.

This study was conducted as a randomized parallel -group clinical trial comparing the safety and efficacy of a low molecular weight heparin {LMWH} -Monoembolex SANDOZ and unfractionated standard heparin glFH) for the perioperative prevention of venous thromboembolie disease (DVT) following major surgms' in patients with gynecologic malignancy.. Three hundred and twenty 4 women (six drop outsl werr randomized and received either 3 times daily 5000 [l" s.c. UL.'i-i (SANDOZ Nuemberg Germany] (n = 164) or once a day t5000 I~'V'. Units s.c. Monoembolex (n = 160) plus two placebo injections. Heparin therapy was started the morning before opcrati(m and continued until the 7th postoperative day. Up to the 10th poatop, day the incidence of DVT was 6.25 % (n = 10; incl. 7 pulmona~ embolisms PE) in the LMWH group and 6.10 % (n = 10; incl. 4 PE} in the UFH group. The overall incidence of clinically hemorrhagic wound complications was significantly decreased in the LMWH group 16.3 % (n = 2hi compared to the UFH group 26.8 % {n = 44; p < 0.0051. The incidence of major hemorrhagic episodes was 9.4 % in = 151 in the LMWH group and 14.0 %/n = 23) in the UFH group. This difference was not statisticaUy significant. One case of fatal PE was observed in the LMWH -treated group. Five women deaths in the LMWH group were observed during the study and 3 in the UFH group. This study demonstrates that the perioperative treaunent of low molecular weight heparins is more safety than standard heparins in gynecologic -oncologic patients undergoing major surge .ry. However, the incidence of thromboembohc complications is simmilar in both treatment regimes. To explore the effect of targeting an antithrombin to the surface of a thrombus, recombinant hirudin (Hir) was covalently linked to the Fab' fragment of fibrin-specific monoclonal antibody 59D8 (Fab) resulting in a stable conjugate (Hir-Fab). In vitro, Hir-Fab was 9times more efficient than Hir alone in inhibiting fibrin deposition on experimental clot surfaces in human or baboon plasma (p<0.01). To validate these results in vivo, Hir-Fab was compared to Hir in a baboon model. The deposition of Ill-In-labeled platelets onto a segment of Dacron vascular graft present in an extracorporeal arteriovenous shunt was measured. Blood flow rate was 40 ml/min. One hour local infusions of 4500 ATU of either Hir-Fab or Hir resulted in deposition of 0.16 x 109 and 2.17 x 109 plate!ets, respectively. Equieffective dosages were 2000 ATU Hir-Fab and 9000 ATU Hir resulting in deposition of 1.06 x 109 and 0.93 x 109 platelets, respectively. Based on full dose response curves (n = 14), Hir-Fab was found to be > 4.5-fold more potent (based on activity) than Hir. Because of the small total amounts of antithrombins used and the short duration of these experiments, no significant systemic effects were observed. Thus, fibrin-targeted recombinant hirudin prevents platelet deposition and thrombus formation more effectively than uncoupled hirudin in vitro and in an in vivo primate model. Triabin, a 17 kDa protein from the saliva of the assassin bug Triatoma pallidipennis, is a new specific thrombin inhibitor (1). tt does not block the catalytic center but interferes with the anionbinding exosite of thrombin. The recombinant protein was produced with the baculovirus/insect cell system and used to study the inhibitory effect of triabin on thrombin-induced responses of human blood platelets and blood vessels. Aggregation of platelets in Tyrode's solution was measured turbidimetrically at 37°C. For the studies on blood vessels rings (2-3 mm) from small porcine pulmonary arteries were placed in organ baths for isometric tension recording. The integrity of the endothelium was assessed by the relaxant response to bradykinin. Like hirudin, triabin inhibited the thrombin (0.1 U/ml)-induced aggregation of washed human platelets at nanomolar concentrations (EC50 = 2.6 nmol/l); whereas the ADP-and collagen-induced aggregation were not suppressed. In PGF2c~-precontracted porcine pulmonary arteries, the thrombin (0.2 U/ml)-induced endothelium-dependent relaxation was inhibited by triabin in the same concentration range as found for inhibition of platelet aggregation. Higher concentrations of triabin were required fo affect the contractile response of endothelium-denuded porcine pulmonary arteries to thrombin (1 U/ml). In all these assays, the inhibitory potency of triabin was dependent on the thrombin concentration used. These studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of the thrombin-mediated cellular effects. Dept. of Medicine, University Hospital Benjamin Franklin, Free University of Berlin, Dept. of Medicine and Dept. of Surgery, Heinrich-Heine:University Dusseldod After standardized training in home prothrombine estimation using the CoaguChek system, 150 consecutive patients (p) who had St. Jude Medical aortic or mitral valve implantation were allocated to two random arms; 75 p were asked to control the INR themselves every third day. In the remaining 75 p anticoagulation was managed by the home physician without recommending an interval for these controls. All 150 p were monitored during the education period to a target therapeutic range of INR 3.5-4.0. P were asked to contact their home physician immediately if the INR was measured 0.5 below or above the target range (INR-corrider 3.0-4.5). All p had out-patient re-examinations every three months. Thrombotic, thromboembelic and hemorrhagic complications were documented by the p using special documentation cards. The following findings were documented during the follow-up period: 4.49 0.9 0 The results of this randomized study demonstrate a significant improvement in the management of oral anticeagulation by home prothrombine estimation. Significant (p<0.001) more INR measurements were found inside the target therapeutic range. Moreover. bleeding and thromboembolic complications could be reduced (p = 0.038) in the study group with home prothrombine estimation. Life-threatening thromboembolic and hemorrhagic complications were not observed in p who were on home prothrembine estimation, while three such events (2.72 %/year) were documented in group A. Local vascular injury following PTCA exposes circulating platelets to prodmmbogenic stimuli. By binding to platelet GP IIbllIIa fibrinogen crosslinks platelets, which represents the final common pathway of platelet aggregation. Fradafiban (BIBU52ZW) is a non-peptide compound with effective, reversible inhibitory effects on fibrinogen binding to GP IIb/II/a on human platelets. In the first double-blinded, prospective phase II study three escalating doses of BIBU52ZW as a continuous 24 h-i.v, infusion were tested in comparison to placebo in 65 patients with stable angina pectoris undergoing elective PTCA. The mean receptor occupancy with 20rag, 40ms and 60ms per hour were 71.974, 84.5 % and 87.9% at 24 hours, respectively. As compared to placebo breeding time was significantly prolonged (7 vs 20rain) during fi-adaIiban infusion with a weak dose-dependency. Platelet aggregation in platetet rich plasma ex vivo with collagen (2.0 and 4.0 gg/ml), ADP (2.5 and 5.0 gmol/ml) or Ca-ionophor A 23187 (2.5 and 5.0gg/ml) was significantly and dose-dependently inhibited as compared to placebo. Using the two upper doses of fradafiban, we observed major bleeding complications in 8 patients requiring blood transfusions or vascular surreal repair. In these patients, too, maximal antiplatelet effects could be documented. These data sugest that BIBU52ZW is an effective fibrinogen receptor antagonist in patients. The requirement of ad hoc receptor occupancy determination or platelet function monitoring for safe and effective clinical use should be evaluated. In a placebo controlled interaction study 9 healthy volunteers were randomized to receive either a 24 hour infusion of PEG-Hirudin ( 0.02 mg/kg/h) after an i.v, bolus of 0.2 mg/kg + placebo, or 325 mg/day acetylsalicylic acid (ASA) for three days followed by a placebo infusion or the PEG-Hirudin infusion + ASA. Each volunteer received all three treaments. There was a washout period of at least 14 days between the infusions. At short intervals aPTT, activated clotting time (ACT), ecadntime (ECT), alia-activity using the chromogenic substrate 2238, Collagen-induced aggregation, Platelet adhesion and platelet induced thrombin gene,ration time (PITT) were measured, Bleeding time (Simplate) was studied before drug administration, on day three before the infusion and 6 hours after start of the Infusion.The Infusion of PEG-Hirudin after 4 and 8 hours led to a mean hirudin plasma level of 1.8 pg/ml. ASA markedly inhibited Collagen induced aggregation as expected. The mean bleeding time was prolonged under the influence of PEG-Hirudin from 5.2 to 6.22 min, after ASA from 5.8 -18.2 min and after the combination of PEG-Hirudin + ASA from 5.4 -33.7 min. In each volunteer the bleeding time was longer under the combination than after ASA alone. In two volunteers receiving PEG-Hirudin + ASA the bleeding time measurement was stopped after 60 rain. None of the coagulation parameters or platelet function tests correlated with the prolongation of the bleeding time. However the bleeding time was excessively prolonged in those volunteers who had a marked prolongation under ASA alone.The combination of hirudin at a higher dosage with ASA probably is associated with a relative high risk of bleeding. Either the hirudin dosage should be reduced if the combination seems feasabie or ASA should be given after the end of hirudin treatment. Fibrinogen with the STA/Stago and the MLA/Dade systems correlated well, but neither system correlated well with the ACL/IL system. AT III, protein C, protein S, and anti-Xa heparin assays using Stago reagents performed as expected for normals and low abnormals on the STA. Factor levels on the STA/Stago system were less sensitive than factor levels obtained with the Dade reagents on the MLA or Fibrometer. Using the STA/Stago system, thrombin time results correlated well with the APTT and heparin levels. The thrombin time was not associated with additional manipulation for assay preparation, nor any cross-contamination of reagent or sample, since on the STA reagents do not come into contact with tubing. The STA was not sensitive to hemolytic, icteric or lipernic samples for clotting assays artd showed the same sensitivity as the MLA for chromogenic assays. The overall data comparisons, high throughput, minimal operator intervention for reagent/assay change and ease of operation warrant further evaluation of the STA hemostasis analyzer.

A. Wehmeier, D. S6hngen, C. Rieth Klinik for H#,matologie, Onkologie und klinische Immunologie der Heinrich-Heine-Universit~it D0sseldorf

Hirudin selectively inhibits thrombin by direct interaction. Because the effect of hirudin is independent of antithrombin III and other factors, it seems an attractive alternative to current anticoagulants. However, it is uncertain whether hirudin influences plateletassociated thrombotic disorders and how it compares with conventional and LMW heparin. We investigated the effect of 2 recombinant hirudin preparations (Rhein Biotech, Dt3sseldorf) on platelet function tests: in vitro bleeding time, adhesion to glass beads, aggregation in platelet-rich plasma and whole blood. Hirudin was used in concentrations of 0.1-100 I.tg/mi, and was compared to trisodium citrate (0.38%), conventional heparin (50 IU/ml) or LMW heparin (Fraxiparin, 500 IU/ml). Both recombinant hirudins showed normal activity in thrombin neutralization tests, and prolongation of thrombin time and aPTT. However, in vitro bleeding time was not prolonged by hirudin, but was more than doubled by addition of conventional and LMW heparins. Platelet retention to glass bead columns was reduced by hirudin in a dose-dependent manner to about 40% but was more effectively reduced by both heparin preparations and citrate. Hirudin had an inhibitory effect on p!atelet aggregation in PRP induced by thrombin, collagen, and predominantly epinephrine but not ADP and ristocetin. In whole blood, a small effect could only be observed with hirudin concentrations of >25 ~g/ml as compared to citrateanticoagulated blood.

In summary, thrombin inhibition by recombinant hirudin has little effect on in vitro platelet function tests in comparison to heparins and calcium depletion. The role of endothelin (ET), prostaglandins and the coagulation system in the pathogenesis of acute renal failure is still to be defined. In anaesthesized pigs the effects of i.v. infusion of ET (3 /~g/kg) alone (group 1, n=6) and after pretreatment with the potent thrombin-inhibitor hirudin (0,5 mg/kg)(group 2, n=6) on haemodynamics, coagulation parameters (factor VIII, antithrombin III, precallicrein, fibrin monomers, aPTT) and prostaglandins were investigated. Plasma renin activity (PRA)-, creatinine clearance-, urine volume-measurement and blood gas analysis were performed hourly. ET-infusion caused an initial BP-reduction and marked HR-reduction followed by a transient BP-elevation and HR-reduction. Activation of platelets can be directly measured by flow cytometry using monoclunal antibodies. In an in vitro study the effect of the thrombin inkibitors argatroban, efegatran, DuP 714, recombinant hirudin and PEGhirudin on platelet activation induced by various agonists was studied in whole blood. Blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregatiun of platelets. For anticoagulation of blood the thrombin inhibitors mentioned above were used at a final concentration of 10 ~tg/ml each. Blood samples were then incubated at 37°C either with saline, r-tissue factor (rTF), arachidonic acid (AA), adenosine diphosphate (ADP) or collagen. At definite times (1, 2.5, 5, 10 rain) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured by means of fluorescent monoclonal antibodies to platelet surface receptors GPIIIa (CD-61) and P-selectin (CD-62). The agunists used induced a platelet activation of 37.4 + 15.2 % (rTF), 65.1 + 12.1% (AA), 19.3 + 7.4 % (ADP) and 27.1 + 12.8 % (collagen). Flow cytometric analysis showed that all thrombin inlaibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. In contrast, after induction of platelet activation with the other agonists an increased percent CD-62 expression was found showing a strong platelet activation with a maximum at the same times as in non-anticoagulated blood.

In conclusion, the results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. The formation of active serine proteases including thrombin may be effectively inldbked by these agents. The observations further suggest that, while thrombin inkibitors may control serine proteases, these agents do not inhibit the activation ofplatelets mediated by other agonists. This work was supported by the grant BMFT 07NBL01.

ANIMAL EXPERIMENTAL STUDIES ON THE PHARMACOKINETICS OF PEG-HIRUDIN E. Bucha, A. Kossmehl, G. Nowak Max-PIanck-Gesellschaft e. V., Arbeitsgruppe "Pharmakologische H~imostaseologie", Jena

Hirudin, when complexed with polyethylene glycol (PEG), increases its molecular weight from 7 to 17 kDa, thereby preventing extravasation of this drug. PEG-hirudin is distributed almost only in the intravascular blood space. In addition, its increased molecular weight retards the renal elimination. The elimination half-life of hirudin in rats (58 + 12 min, as determined) is increased five-fold (244 ± 18 min). With the same hirudin dose applied, the blood level of hirudin is increased 19-fold, measured in the 13-elimination phase. In the urine of rats, 2 -3% of the hirudin activity were recovered following hirudin administration, but 48% could be detected after PEG-hirudin had been applied. After subcutaneous administration of PEG-hirudin, the trnaxwalue is reached at 380 rain (r-hirudin: 65 min); the Cmax-value is increased 3-fold, compared to that of r-hirudin (2.5 pg/ml). 24 hours later, still one fifth of the maximum concentration (Cma,) is present in the blood, and the renal elimination is still retarded. In the urine of rats, 12% of the hirudin activity applied were recovered in the 24-h urine sample. With intact renal function, following subcutaneous administration, PEGhirudin is abte to produce a constant blood level of hirudin over a long pedod. Thrombin inkibitors such as r-hirudin (RH), argatroban (A), efegatran (E), and PEGhiradin (PH) are currently undergoing extensive clinical trials in such cardiovascular indications as PTCA, AMI, and treatment of unstable angina. A rapid assessment of the anticoagulant actions of these agents is, therefore, crucial to assure their efficacy and safety. Currently, ACT and APTT are used to measure the anticoagulant effect of these agents. We have utilized a dry reagent technology based on the motion of paramagnetic iron oxide particles (PLOP) to measure the antithrombin effects of various thrombin inhibitors (CV Diagnostics, Raleight, NC). The heparin monitoring card has been modified to measure antithrombin agents in various anticoagulant ranges for (A)0 (E), (RH), and (PH). Blood samples drawn from patients treated with (A) and (RH) have been evaluated and concentrations of these agents have been calculated using an external calibration curve. In the in vitro setting, citrated whole blood or citrated frozen plasma can be used to evaluate the anticoagulant effects of these agents. The results obtained are comparable to the ACT which is conventionally used for the monitoring of these agents. Both (RH) and ( 

period. We would like to present a case of heparin-induced-thrombocytopenia (HIT) in a 47 years old woman who underwend open heart surgery. She suffered from a combined aortic valve disease and leading stenosis. Laboratory analysis showed constant low platelet counts (50/nl) without heparin application, so that an idiopathic thrombocytopenlc purpura was suspected. But platelets also decreased after heparin application. Heparin-antibodies were found in the heparin induced platelet activation assay (HIPAA). Treatment with corticosteroids and immunoglobulines, respectively, showed no improvement but the patient unfortunately developed a pneumonia with legionetla pneumophila. Therefore, the only suitable anticoagulant for the necessary aortic valve replacement was Hirudin: A bolus injection of r-Hirudin of 0,75 mg/kg b.w. was administered 10 min. bevore start of the extracorporal circulation (ECC), the heart-lung machine (HLM) was primed with 6 mg r-Hirudin and another bolus of 5 mg of r-Hirudin was administered. Additionally 10 mg of r-Hirudin was applicated to the cell-saver-reservoir. During the period of ECC Ecarin clotting time and aPTT values were taken every ten minutes for monitoring r-Hirudin concentration. The postoperative anticoagulation was performed with a constant infusion of r-Hirudin starting eight hours after the end of ECC and monitored by aPTT. Due to mechanical aortic valve the further anticoagulation was performed with phenprocoumon, starting 3 days postop. The therapy with Hirudin showed no side-effects. Hirudin, threrefore seems to be a suitable anticoagulant in patients with high risk for bleeding complications like this. Doses fi:om 2-30 mg/kg gave similar post-op blood loss measurements without s dnseresponse (4-15 oc/kg) (less blood oozing than a historical heparin control but equivalent post-op blood loss; 10 q-3 ec/kg). Doses >30 mg/kg showed more intra-op blood loss than the lowe~ doses, but equal post-.op blood loss. The bleeding time test was less elevated than for heparin. Platelet counts and hematoerit did not vary except for hemodihition on pump. Liver enzymes did not vary significantly pre-op to post. ACT values showed ARG was eliminated (dose-dependently) by 1 hour post-op. Dogs were hemodymamieally stable during the peri-operative period, and overall gave predictable responses to ARG (as opposed to variable responses to heparin). In a substudy it was demonstrated that hypothermia did not affect the activity of ARG, nor did varioos formnlations. This dose finding study strongly suggests that ARG may be a safe and effective alternative to heparin for patients undergoing CPB. This is particularly important for the growing population of patients with HIT who require cardiac surgery, for which no anticoagulant alternative is presently available. Three recent clinical tdals with r-hirudin (TIMI 9, GUSTO 2 and HIT) have shown that the risk of severe haemorrhagic side effects was strongly associated with high aPTT-levels. The large intedndividual variability of the aPTT and the lack of a linear dose-effect ratio, however, limits its value for reliable monitoring of the anticoagulant effect of hirudin since even severe overdosage due to impaired renal elimination may not be detected with this assay. We have therefore evaluated the ecadn clotting time (ECT) as descdbed by Nowak and Bucha (Thromb. Haemost. 1993; 69: 1306) under conditions which allow conclusions on its reliability in the clinical situation.For this, citrated venous blood obtained from healthy volunteers, patients with unstable angina pectoris, and patients treated with marcumar was supplemented with different concentrations of PEGhirudin. Measurements of aPTT and ECT were made in duplicate.

In contrast to the aPTT, the ECT showed a close, linear relationship with PEG-hirudin plasma concentrations in the range of 100 and 10 000 ng/ml. The lineadty of this relationship was not affected by the presence of unfractionated or low molecular weight hepadns in concentrations of up to 10 pg/ml. The ECT was not affected by fibdnogen concentrations 60% below normal. A somewhat higher slope but no change in linearity was found in plasma from marcumar-patients with quick-values between 20 and 32%. No significant differences were found between values measured in citrated blood or plasma or using different coagulation timers. The most potent thrombin inhibitor containing a benzamidine moiety is NAPAP (K i = 6 nmol/I). Unfortunately, the pharmacokinetic properties (fast elimination by hepatic uptake and biliary excretion, poor enteral absorption) are unsuitable for the use of NAPAP as an oral anticoagulant. The application of choice of a synthetic thrombin inhibitor would be the oral one, Therefore, we looked for other lead structures. With the Nc~-arylsulfonylated piperazides of 3-amidinophenylalanine we found a new group of derivatives which inhibit thrombin with Ki-values in the nanomolar range. The piperazides exert anticoagulant activities with high selectivity, leaving activated protein C and components of the fibdnolytic system unaffected.

In rats, the piperazides are rapidly eliminated from the circulation (tl/2 ~ 10 min) upon i.v. administration, too. After oral administration, the systemic bioavailability is low. Upon intraduodenal administration of high doses widely varying blood levels were seen, depending on the mode of administration. To cladfy the importance of a possible hepatic first pass effect we studied in more detail the pharmacokinetics of the N~-(2naphthylsulfonyl)-3-amidinophenylalanine N'-acetylpiperazide in rats using HPLC-analysis. Like other benzamidines the piperazide is excreted via the bile to a high extent. Enteral absorption rates of about 20 % are found after blocking the hepatic uptake and biliary excretion. Hence, a hepatic first pass effect appears to be the main reason for low systemic bioavailability after orallenteral administration. At the same time, fast elimination from the circulation by hepatic uptake is the main problem for maintaining effective blood levels with benzamidines. Therefore, the elucidation of the structural elements influencing the absorption and elimination processes of these types of inhibitors is necessary. The piperazides of 3-amidinophenylalanine bear the possibility to easily introduce a wide variety of substituents on the second nitrogen of the piperazine moiety. A 69-year-old female patient with diabetic nephropathy increasingly developed signs of allergisation combined with dyspnea, erythema, pruritus, and circulatory insufficiency two months after start of heparin-anticoagulated haemodialysis und initial surgical application of a double lumen venous catheter. In addition, growing thrombocytopenia was observed involving a drop in platelets by 50%, compared to the initial values. The haemodialytic efficiency was reduced by massive thrombosis of the dialyzer and subsequent repeated interruption of treatment. At the end of May 1995 heparin antibodies were detected and the HAT diagnosis was confirmed. Immediately afterwards, haemodialysis treatment was continued, applying hirudin as anticoagulant. Using steam-stedlised Haemophan dialyzers and 0.14 mg/kg r-hirudin (Iketon, Italy), the minimum therapeutic blood level of hirudin (0.4 pg/ml whole blood) was reached. This provided therapeutically relevant blood level conditions during a 4.5h haemodialysis. More than 80 regular haemodialyses were run without problems. In all hirudin-anticoagulated haemodialysis treatments the ecarin clotting time was used as the method of choice for bedside blood level and dosage control. After the 34th haemodialysis, the frequency was reduced from 3(4) to 2 haemodialyses a week. Accordingly, the hirudin dose was increased to 0.2 mg/kg. The creatinine clearance increased continuously from initially 2.6 to 10.4 ml/min after the 13th week of hirudin-anticoagulated haemodialysis. Platelet count and haemodialytic efficiency normalized. We could demonstrate that the regular use of hirudin as anticoagulant along with dialyzers impermeable to hirudin enables very good results in haemodialysis treatment in heparin-associated thrombocytopenia, Hirudin is suited for use as anticoagulant in problem patients with hepadn-induced allergy when combined with a drug monitoring method fit for bedside use. Capillary electrophoresis methods provide a fast measurement of proteins. Thus we developed for pharmacokinetic measurements of r-hirudin and peg-hirudin capillary electrophoresis methods. For the measurement of r-hirudin we used fused silica capillary and a borate buffer. This buffer was used to detect r-hirudin, but could not be used to measure PEG-hirudin. For simultaneous measurement we used a neutral capillary to prevent protein absorption to the capillary wall. The buffer was a 20 mM tricine buffer (pH = 8.0 field strength 500 V/cm). It resolved r-hirudin from peg-hirudin at 214 nm using reverse polarity.

A linear correlation between the peak area and the concentration was found between 80 pgtml and 10 mg/ml for hirudin (r 2 = 0.99) and between 2,5 and 10 mg/ml for peg-hirudin (r2= 0.99) was found By coinspiking of human plasma and urine with r-hirudin and peghirudin the two proteins were completely resolved. A linear correlation between the peak area and the concentration was found. The method separates r-hirudin from peg-hirudin and may be applied to biological systems to measure the concentration of r-hirudin. Triabin is a thrombin inhibitor from the saliva ofT. pallidipennis structurally unrelated to any protease inhibitor known and which probably functions by an interaction with the anionbinding exosite of thrombin. We used Sf9 insect cells infected with recombinant Baculovirus to produce sufficient triabin for a detailed biochemical characterization. The activity of the protein purified from cell lysates was assessed in a fibrinogen clotting assay and was found to be similar to that of the natural protein. A 4-fold prolongation of thrombin-clotting time and APTT was achieved with 22 nM and 600 nM triabin, respectively. A kinetic analysis of the thrombin-catalyzed fibrinopeptide A release from fibrinogen showed that triabin is a tight-binding inhibitor. Using the graphical method of Dixon, the Ki was determined to be 3 pM. Introduction: Thrombocytopenia is a common adverse effect of heparin therapy, in type II HIT platelet decrease induces severe complications. We here present two special cases of type II HIT. Case report I: A 66 year old male patient with DVT of the left leg was treated with therapeutic doses of heparin. From the first to the 12th day of therapy, platelet count decreased from 122000 to 54000/ui. HIT was confirmed by HIPA-test, heparin therapy was s~opped and treatment with the heparinoid Orgaran n was started. During the following days, arterial thromboses in the right A. femoralis occurred. Several thrombe~tomies were not successful and although Orgaran ~" was stopped because of suspected crossreactivity, amputation of the right leg could not be avoided. During the following days under Hirudin-treatment platelet count normalized and no further complications occurred. Case report 2: A 74 year old female patients suffering from hip fracture was treated by surgery with TEP-operation and received prophylactic heparin treatment. After 6 days, platelet count decreased from initially 170000 to 8000/ul and DVT of the right leg was diagnosed. On the same day, severe bleeding into the left leg was observed and hemoglobin concentration was diminished to 7.8 g% (before surgery 16.0 g%). HIT was confirmed by HIPA te~t, heparin was stopped and treatment with Orgaran started. Thrombocyte count normalized and no further complications occured. Conclusion: HIT type II can cause severe bleeding as well as thromboembolic complications. Because of possible cross-reactivity between heparin and Orgaran~,, Hirudin should be given in HIT patients. Currently thrombin time (TI), aPTT, activated clotting time (ACT) or anti Ila -activity (alia), measured by a chromogenic substrate test are used to monitor hirudin treatment or prophylaxis. The "13" responds very sensitive to hirudin plasma levels end thus requires variable thrombin concentrations. APTT appears to be more adequate, however, it shows large interindividual variations and does not respond sensitive enough to higher hirudin concentrations. ACT is a simple whole blood clotting assay, but it is strongly influenced by the blood collection technique. The ecadn clotting time (ECT) is a new clotting assay, recently described by Nowak and Bucha (Thromb.Haemost 1993, 69,1306) . It measures the clotting time of citrated blood or plasma after prothrombin activation by ecarin, a snake venom of echis carinatus. EC.T shows a linear dependence on different hirudin concentrations over a wide concentration range ( e.g. 0.1-5 pg/ml). In a clinical interaction study healthy volunteers were administered Hirudin, ASA or both. 15 male volunteers received an i.v. infusion of PEG-Hirudin (0.02 mg/kg/h) for 24 hours after an initial i.v. bolus of 0.2 mg/kg to compare the sensitivity and reliability of ECT with aPTT, l-r end ACT. The ACT was measured on the Hemochron 801, USA, ECT on a fibrin timer, aPTF using the APTT lyophylized Silica reagent by IL and alia on an ACL (IL-Milan) with the chromogenic substrate 2238. All tests were performed in duplicate. ECT was more sensitive to different hirudin concentrations than aPTT, or ACT. The ECT results were better correlated with the alia-activity than aP'l"r and ACT. The lower detection range for ECT is 0.05 pg/ml Hirudin. ECT is a very sensitive, simple and reliable test for the monitoring of hirudin treatment and prophylaxis. Recombinant and synthetic inhibitors of thrombin such as hirudin, efegatran and argatroban are currently in various phases of clinical trials in several surgical and medical indications. The therapeutic effects of these agents are usually monitored by APTT whereas in cardiovascular indications, cefite ACT and Hemotech® ACT are used. The reliability of both APTT and the ACT tests in predieting the safety of various thrombin inhibitors has been heavily debated. Furthermore, some of these inkibiturs are administered simultaneously to heperinized or coumadinized patients and the obtained APTT and ACT results do not lady refleot the effects of these agents. Fcafin is a snake venom derived fi'om Echis carinatus which converts prothrombin into mesothrombin, targeting the Arg~3-Ile TM bond between the A and B chains of prothrombm. While thrombin inhibitors are capable of inhibiting mesothrombin, ATIII/beparin complex does not have any effect. Using purified ecarin, Nowak and Bucha (1993 Thromb Haemost 69:1306) proposed to assay hirudin. Since thrombin inhibitors exhibit similar mechanisms of thrombin inhibition, ecarin clotting time (ECT) was evaluated to test its diagnostic efficacy in various experimental and clinical settings. Lyphilized eoarin was obtained from Knoll AG, Ludwigshafen, Germany). Concentration dependent clotting times for himdin, efegatran and argatroban were obtained in a range of 0-15 p.g/ml. All of the antithrombin agents produced a concentration dependent prolongation of ECT and showed va~Angpotendies inthe order ofefegatran> argatreban> hirudin, on a gravimetriebasis. On a molar basis, the anticoagulant order of potency was found to be hirudin> afegatran> argatroban. Utilizing the ECT, the effect of these inhibitors on patients undergoing bolus or infusion therapy, resulting in a concentration level of ~ 10 gt g/rnt, have been measured. Unlike such global tests as PT and APTT, patients receiving simultaneous heparin or oral anticeagulants can be monitored for antithrombin specific prolongation ofthe ECT. Plasma samples from heparinized (APTT 45-90 sec) or coumadinized ('PT 15-25 see) patients, supplemented with argatroban or hiredin did not show any differences m the ECT. A medified ecarm ACT comparable to the celite ACT has also been developed. Initial results demonstrate that this test is not affected by aprotinin, heparin and reduction of the prothrorabin complexes in the INR range of 1.5 -3.0. These results indicate that ecarin based clotting times provide SlX~etlie ~lts of circulating levels of thrombin inhibitors, which can provide reliable information to optimize their safe(y and efficacy. R-hirudin is a highly potent and selective inhibitor of the serine proteinase thrombin. After intravenous administration, r-hirudin is eliminated exclusively with the urine. Its plasma half-life is very short, 1-2h. PEG-hirudin is a derivative produced by coupling polyethylene glycol (PEG) to a specially designed recombinant hirudin mutein. PEG-coupling results in a considerable prolongation of the plasma half-life of PEG-hirudin, compared to r-hirudin. After intravenous administration of r-hirudin into rats, a very small amount of ,,hirudin-like" activity (2 -4% of applied activity) was recovered in the urine. In contrast, after PEG-hirudin had been administered, more than 30% of the applied activity could be recovered in rat urine. These results suggest differences in the renal metabolism of PEG-hirudin and r-hirudin. Within the scope of pharmacokinetie studies in rats we investigated the appearance of biologically active metabolites of PEG-hirudin after kidney passage in urine. Affinity chromatography on immobilised thrombin was used as a quick and gentle method in searching for biologically active hirudin metabolites in rat urine. But it had to be completed by anion-exchange and/or reversed-phase chromatography to ensure that all active metabolites were detected. The isolated biologically active metabolites were purified by reversed-phase HPLC and were biochemieally characterized. In previously reported studies we found a hlrud n derivative consisting of the amino acids 1-50 as the main metabolite in rat urine following intravenous administration of r-hirudin. This metabolite was not detected in the urine after administration of PEG-hirudin, confirming the suggestion of a different renal metabolism. Carrageenans are high molecular weight sulfated polygalactans of plant origin (derived from red algae) with anticoagulant properties. In previous studies we investigated the anticoagulant activity of lambda-carrageenan, a highly sulfated type of carrageonans. Unlike heparin, lambda-earrageenan exerts its anticoagulant activity primarily through direct inhibition of the serine proteinase thrombin. Only a part of its antithrombin activity is indirectly mediated through antithrombin III. To investigate relations between molecular weight and biological activities, tambda-carrageenan has been hydrolysed and fractionated. The molecular weight has been determined with the aid of size exclusion HPLC using dextrans as molecular weight standards. The degree of sulfation has been determined by anion-exchange HPLC. We have obtained low molecular weight lambdaearrageenans ranging from 10,000 dalton to 100,000 dalton with degrees of sulfation of 13 -17% and 33 -38%. The anticoagulant and antithrombin activity of low molecular weight carrageenans have been determined using coagulation assays and purified systems, and we have compared their activities with those of heparin and other sulfated polysaecharides. Further, we have investigated the ability of lambda-carrageenan and its low molecular weight derivatives to inhibit the activity of human blood phagocytes. The activity has been determined by measuring the cellular chemiluminescence in a mieroplate himinometer using a himinol-dependent assay and zymosan as phagocytosis activating agent. We have used an assay in human whole blood and assays with isolated human mononuclear and polymorphnuclear cells. The anticoagulant activity and also the ability of carrageenans to inhibit the activity of human macrophages decrease with decreasing molecular weight and decreasing degree of sulfation. The natural ocouring, yellow pigment curcumin is the major component of tumeric and is commonly used as a spice and food-coloring agent. Since curcumin has been reported to have anti-tumorpromoting, antithrombotic and anti-inflammatory properties, we studied, whether curcumin acts on the transcription factors AP-l(Jun/Fos) and NF-~:B in cultured endothelial cells (EC). When EC were cultured in the presence of curcumin, Electrophoretic Mobility Shift Assays (EMSA) demonstrated, that binding of endogenous AP-1 to its DNA recognition motif was suppressed. Inhibition was due to direct interactions of curcumin with the DNA-binding motif for AP-I. Enhanced AP-1 binding, induced after TNFa stimulation of EC, was decreased in cells pretreated with curcumin. This resulted in reduced transcription and expression of Tissue Factor, known to be controlled by AP-f and NF-~B. Nuclear Run on assays proofed, that curcumin directly reduced the TNFa mediated transcription of genes, regulated by AP-1, as TF, endothelin-1 and c-Jun. Thus, curcumin did not only suppress APl(Jun/Fos)-binding, but also inhibited TNFa induced Jun transcription, Transient transfections with Tissue Factor promotor plasmids confirmed, that inhibition by curcumin was dependent on intact AP-I sites. Beside its effect on AP-l-binding, curcumin reduced the radical dependent activation of NF-KB due to its antioxidant properties, however, this inhibition was indirect and less prominent. The relevance of the in vitro data was confirmed in vivo in mice bearing Meth-A-sarcoma. When mice received curcumin before TNFa was injected, tumors showed reduced AP-1 activation. Simultanously fibrin/fibrinogen deposition decreased, most probably due to reduced Tissue Factor expression. Thus, curcumin inhibits AP-t activation and expression of endothelial genes controlled by AP-t in vitro and in vivo. (Jung, 1991) . Additionally, haemorhenlogical parameters (plasma viscosity, erythrocyte aggregation) were measured. In all patients aPTT, bleeding time, platelet adhesiveness, von WiUebrand f~ctor and factor VIII concentration and activity were determined. The patients with von Willebrand disease showed characteristic morphological changes of capillary geometry. Tortuosity of nailfold capillaries was markedly increased as well as the diameter of capillariez on the arterial and venous side. Plasma viscosity was significantly low. Multiple parameter analysis concerning to Galen and Gambino (1983) and using the parameters ,,plasma viscosity below 1.25 mPas", ,,torquation index higher than 5", ,,erythrocyte column diameter bigger than 16,9 gin" showed a positive predictive value of 100 %. Capillary diameter and capillary tortuosity have a positive predictive value of 91,2 %. Additionally, a reduction of the vasomotorie reserve and/or a decreased erythrocyte velocity in the capillaries below the reference range was found in most of the yon Willebrand patients. It was quite remarkable, that 16 of 40 of the yon Willebrand patients showed significant capillary bleedings. These findings confirm some former observations (e.g. O'Brian 1950) and preliminary reports of our group (Koscielny 1994). Polymerase chain reaction (PCR)-based quantitation of mRNA transcripts is an important tool in the investigation of the underlying molecular defects in inherited platelet disorders, such as the Bernard-Soulier syndrome. However, for the exact quantitation of mRNA a number of methological requirements has to be met. First, a standard (s) mRNA must be synthesized which is able to undergo the same processing as the target wild type (wt) mRNA. Secondly, the quantitation step following the PCR must differentially recognize standard and target DNA, and thirdly, the assay must be precise with respect to both inter-and intraassay variability. In order to satisfy these requirements we constructed a s-GPIb mRNA which is identical to the wt-GPIb mRNA except a 13 bp long primer recognition site at its 5" end allowing differentiation between the PCR amplified wt-or s-GPIb cDNA through incorporation of a fluorescein or biotin labelled 5" primer. Both standard and w[ GPIb mRNA showed identical amplification kinetics in the PCR reaction. The amplified DNA was quantified using an DNA binding assay. In this assay binding of amplified DNA to GCN4 fusionprotein-coated microtiterplates is measured. Since the GCN4 binding motif is incorporated into the wt-and s-GPIb cDNA through an identical 3" primer, competition between s-and wt-cDNA during amplification has been analyzed. At a given concentration of 250 nM of GCN4. primer no competition between the sDNA and wt-DNA for the primer was observed during 25 PCR cycles. The sensitivity limit of the assay performed in this way was 250 amol wt-GPIb~, DNA, and intraassay variability reached from 1.66% to 6.72% calculated for 100 fmol and 5 fmol DNA, respectively. To sum up, combination of RT-PCR with the amplified DNA binding assay and usage of an internal standard mRNA allows sensitive and accurate quantitation of GPIba mRNA in human platelets. Since uPA and thrombin are main conrtibutors to the process of proliferation and migration of vascular smooth muscle cells (VSMC), which is part of the pathogenesis of atherosclerosis. We are currently assessing the role of spatial expression of uPA and thrombin receptor (TR) on cells with human carotid artery plaques (n=10). We have used a double immunolabeling approach, combining anti-uPA and anfi-TR antibodies. To identify the different cell types, we used the following antibodies: anti a-smooth muscle actin (a-SMA) for smooth muscle cells, Ulex europaeus agglutinin I (UEA I) for endothelial cells, inflammation cell cocktail (CD68+CD45) for monocyte/macrophage and lymphocytes and an anti-proliferation cell nuclear antigen antibody (PCNA) to stain proliferating cells. in the carotid atherosclerotic plaques, uPA immunostaining was distributed focally, preferentially in the fibrous cap and some cells of the foam cell rich region (FCRR). It was present in distinct patterns: cytoplasmic staining. TR staining was distributed similar to uPA staining. With double staining combining anti uPA antibodies with anti-TR antibodies, cellular co-localisation of both uPA and TR was demonstrated. These cells were identified as smooth muscle cells by -SMA. Inflammatory cells were mainly localized within the FCRR, they only stained for uPA. In conclusion: Our data demonstrates that uPA and TR are coexpressed in VSMCs in human carotid artery atherosclerotic plaque tissue. We therefore conclude, that the mitogenic activity of uPA is associated with the thrombin signalling pathway. In the Proficiency Test of the ,,Deutsche Gesellschaft flir Klinische Chemie" (DGKC) 1/95, 5 lyophilised plasma samples (Immuno AG) were sent to participants: a normal plasma and 4 plasmas from persons under oral anticoagulation (OAC-plasmas. INR 1.9 to 3.7). The participants (N=552) returned the PT times obtained and in most cases (N=355) also the ISI value for the thromboplastin used (ISI of pack insert). The INR was calculated using the PT of normal plasma and the ISI of pack insert (method I). Two additional methods for INR calculation were compared with method I. According to the concept of calibrated plasmas (Houbouyan et al., t993), a calibration curve was constructed using the normal plasma and the 30AC-plasmas. The INR calculated using the PT •fn•rma¿ plasma and the laboratory-specific ISI value given as 1/slope of the calibration curve (method II) or was read off directly (method HI). For INR values, calculated by the 3 methods from the participants data (N=355), outlier elimination (2SD, iterative) was performed. The INR mean values for all 3 calculation models remain in a narrow range. Using calibrated plasmas (method 1I and m), less outlier were eliminated and CV's obtained were smaller than using the conventional procedure ( I ).

Obviously, the INR inherited problems, such as accurate ISI value, PT value of normal plasma and instrument/laboratory influences on ISI, can be reduced using calibrated OAC-plasmas.

PRACTICAL APPROACH AND EDUCATIONAL CONSIDERA-TIONS OF HOME PROTHROMBIN TIME ESTIMATION A. Bernardo, A. Bernardo, C. Halhuber Herz-Kreislauf-Klinik, Bad Berleburg, Germany

Specific training is necessary for the patient to achieve reliable and reproducible results in prolhrombin time measurement. The training scheme is based in many respects on experience with similar training courses for home control and management of diabetes and asthma. The education program is divided into a theoretical and a practical part. The theory part has group sessions of twenty patients of a time. The practical course is reduced to a maximum of five patients. The sessions are conducted by a medical doctor and by specialized medicaf/technical assistants. On average eight hours of theoretical education and two hours of practical training are sufficient. The contents of the theoretical lessons are:

• need for anticoagulation after heart valve replacement, • potential interaction between anticoagulants and other medication, • accurate recording of the measured prothrombin time results, • techniques of prospective determination of the necessary amount of anticoagulant, • calculation of the individual doses, • potential pitfalls and mistakes, • corrections in case of over-and under-dosage, • early recognition of thromboembelic and/or bleeding complications. An alternative is a full-day intensive course which can be held during the weekend. Our recently reported (1) observation that oral anticoagulant treatment causes an increase of heparin cofactor II (HC II) activity in plasma is now confirmed by a more extensive study. In 43 thrombophilic patients who were on vitamin K antagonist therapy (Marcumar R) we found a median HC II level of 142 % as compared to 119 % for 72 thrombophilic patients without any therapy (p < 0.002" ) and 104 % for 59 healthy controls (p < 0.001" ). Moreover we observed that the increase of HC II level was significantly correlated with increasing INR-values (r = 0.63, p < 0.001). Follow-up observations on some patients showed, however, clear differences in the levels of HC II activity after onset of vitamin K antagonist therapy. Thus, some patients responded rapidly with a significant increase in activity ("strong responders") while others showed only slight changes ("weak responders").

In conclusion, the determination of HC II activity may result in an improved estimation of the risk of bleeding, especially in high intensity treated patients (INR > 3.5). After intracoronary Stent implantation an aggressive oral anticoagulation (OAC) therapy is mandatory. To find out whether coagulation activation occurs after coronary stent implantation during high dose OAC therapy markers of plasmatic coagulation and D-Dimer were measured. Patients 5 male patients (average age 57 years) were examined. Blood samples were taken before and right after stent implantation and during the following week. Patients got 30 mg phenprocoumon during the first three days and additionally heparin and acetylsalicylic acid (ASA) were given. Methods PTZ, aPTT, TZ, protein C, TAT-complexes, FI+2 and D-Dimer were measured. Results D-Dimer levels increased steadily between day 0 and day 7. TATcomplexes showed a slight increase from day 0 (2.4 Bg/I) to day 3 (15.3 ~tg/I). On day 7 TAT levels were down again (2.0 p,g/l). Fl+2 (day 0:1.0 ng/ml) also showed a slight increase on day 3 (1.3 ng/ml). Protein C decreased steadily from day 0 (108%) to day 7 (15%). Conclusion During the initial phase of OAC therapy a coagulation activation is reported but no significant elevation of TAT or Fl+2 was found. This result shows that additional heparin and ASA therapy was sufficient to avoid systemic coagulation activation. The increase of D-Direct should be interpreted as a si~=m of local fibrinolytic reaction due to stent implantation. Three methods for the determination of Prothrombin Time from capillary blood in patients under oral anticoagulation have been investigated. Two methods were run on CoaguChek® monitors (Boehringer Mannheim) from capillary whole blood. After fingerpuncture the first drop of blood was applied to the well of a CoaguChek® test strip directly from the finger-tip, whereas the second drop was sucked into a non-anticoagulated plastic capillary (Hirschmann) and immediately applied to the test strip -and vice versa to eliminate any influence of first and second drop of blood. The third method was Hepato Quick (Boehringer Mannheim) which was determined out of citrated capillary blood from an earlappuncture. 66 specimen of patients under oral anticoagulation were investigated. The method comparisons between each of the CoaguChek® methods and the laboratory method show good results and the correlation between the CoaguChek® methods is excellent. Mean differences to the lab methods are -0.1 INR in both cases. No mean deviation was detectable between the CoaguChek® methods. Scattering of CoaguChek® versus Hepato Quick was +/-0.6 INR in the range 1 to 4 INR except for three outliers and one patient with fluctuating results in the lab method which could not be resolved. Introduction: Haemorrhagic coumarin skin necrosis is a severe complication during initial phase of oral anticoagulant therapy. Histological examination shows thrombotic occlusion of small vessels, but little is known concerning the pathophysiologic background of the bleeding component. Recently, we described protein Z deficiency in patients with bleeding complications of otherwise unknown origin. Thus, we were prompted to measure protein Z in patients with coumarin skin necrosis. Patients: 4 patients (I man, 4 women; age: 35±10 years) suffering from haemorrhagic coumarin skin necrosis were examined. All patients had normal liver protein synthesis function, none was under oral anticoagulant treatment during this study. Method: Protein Z antigen test, Diagnostika Stago, France. Results: 4 out of the 5 patients examined had diminished protein Z levels ( 700, 820, 1080, 1700 ug/l) in comparison to normals (2900 ug/l). In one of our patients, protein Z was normal (3020 ug/l). Conclusion: Low protein Z levels are additional risk factors for haemorrhagic coumarin skin necrosis. Oral anticoagulant therapy is the treatment of choice in patients with need for long-term anticoagulation. Since oral anticoagulants interfere with the function of vitamin K, it is not clear whether stable oral anticoagulation can be achieved in patients with need for continous substitution of fat-soluble vitamins including vitamin K. We report about a 59-year-old man who had experienced progressive hypertrophic obstructive cardiomyopathy over the preceeding 21 years. Atrial fibrillation has been first diagnosed 18 years ago. Latter on, recurrent ischemic attacks and embolism of the right arteria iliaca occurred. In 1993 the patient received extirpation of the ileum and subtotal amputation of the jejunum because of mesenteric infarction. The resulting short bowel syndrome requires continous substitution of fat-soluble vitamins. Since vitamin K free preparations of fat-soluble vitamins for parenteral use are not available, prophylaxis of thrombosis has been performed with unfractionated hepadn. As a consequence of the longterm treatment with hepadn the patient developed severe osteoporosis. Therefore, the decission 1:o discontinuate heparin therapy and initiate oral anticoagulation has been made. Because of its shorter halflife warfarin (coumadin) was used instead of dicoumarol. Over a 4 weeks lasting induction phase INR values were controlled daily. A dosage regime starting with '10 mg warfarin at the day of vitamin application (day 1) followed by 3.75 mg on day 2 and 1.25 mg on days 3, 5, and 6, respectively, was found to be optimal to maintain INR values within the target range (INR: 2.0-3.0). In order to minimize the risk of hemorrhage the vitamin administration was changed to the subcutaneous route. During an observation period of 6 months neither any bleeding or thrombotic complications nor a vitamin deficiency occurred. These data indicate that stable oral anticoagulation can be achieved despite extreme variation of vitamin K plasma levels. Portable monitors for home monitoring of INR are well established for adults on oral anticoagulants. Patient's compliance is improved as well as long term outcome. Experience concerning accuracy of the procedure in children is limited. 32 INR determinations were performed in parallel from venous and capillaryblood samples of an infant on phenprocoumon, starting at the age of 4 months. The COAGUCHEK® monitor from Boehringer Mannheim was used. Choosing an arbitrary range of agreement of ,qNR 0.5 for both determinations, 81% of the measurements were within the defined range. 5/6 outliers were due to low INR resulting from difficulties in capillary blood sampling. The degree of agreement increased when the procedure was performed at least once a week. In conclusion: INR determination with a portable monitor may be helpful in home monitoring oral an.ticoagulant therapy in young children. A dose adjustment should be done only on the base of INR determination of venous blood -if it is considered the gold standard -to avoid over-anticoagulation. A stable anticoagulation is one of the most difficult tasks in attending patients with heart-valve-prosthesis. If prothrombin times are out of the therapeutic range, the risk of bleeding or thromboembolism increases disproportionately. For this reason any improvement in anticoagulant control and/or management can have far reaching consequences in decreasing complications, in extending longevity and in improving quality of life. For the first time a clinical trial was started in 1986 and continues until today at the Cardiac Rehabilitation Center Bad Berleburg, Germany with patients mainly after heart valve replacement. The patients were trained to measure their own prothrombin time and to adjust their own dosage of the oral anticoagulant. Within six years 600 patients were trained: 216 patients could be followed up with regard to their selfdetermined prothrombin times. The results were within the therapeutic range in 83.1% of the measurements (n=14.812) taken by the patients themselves. On average, the patients who determine their prothrombin time themselves did so at a weekly interval. Neither major bleeding nor thromboembolic complications could be observed in the 205 patient-years of home prothrombin estimation. It is to be hoped that the usual rate of complications can be reduced when patients determine their prothrombin time themselves at a close interval, resulting in more constant values in the therapeutic range and slight corrections of the anticoagulant dose. Home prothrombin estimation promises better quality of life and has a considerable potential to achieve this goal. Circulating plasma thrombomodulin (TM) is a novel endothelial cell marker, which may reflect endothelial injury. TM acts as thrombin receptor which neutralises the fibrin-forming effect of thrombin, and also accelerates the formation of the anticoagulant protein C/S pathway. TM therefore belongs to the anticoagulant defence system against thrombosis. Increased TM levels have been described in various diseases such as ARDS, thromboemboembolic diseases, TTP, diabetes, LE and CML reflecting alterations of the vascular system at the endothelial level. To find out to what extent cardiac catheterisation imtates vascular endothelium, TM concentrations (Stago, Asnieres, France: x 10 3 IU/ml) were investigated prospectively in 58 infants and children (three days -16 years). Blood samples were drawn before the intervention, immediately at the end and 24 h later, snap frozen (-70 °C) and investigated serially in dublicate six weeks -3 months later. The results (median and range values) are shown in the Enhanced TM concentrations immedately after the operative intervention, followed by normalisation within 24 h, indicates that cardiac catheterisation in pediatric patients rather leads to a short lasting irritation of the vascalar endothelium than to severe irreversible endothelial damage. Recently in an al=Wl" based method Dahlb~ick et al described in vitro resistance to the anticoagulant effect of activated protein C (APC) in thrombophilic adult patients. APCR is in the majority of cases associated with the Arg 506 Gin point mutation in the factor V gene. Concerning the special properties of the neonatal hemostatic system (low vitamin K dependent coagulation factors, physiological prolongation of the PT and aPTF) we adjusted this aP'IT based method (Chromogenix, M~,lndal, Sweden) to neonatal requirements: APCR was measured in 120 healthy infants according to Dahlb~ck. The results were expressed as APC-ratios: Clotting time obtained in a 1:1, 1:5 and 1:11 dilution with factor V deficient plasma (Instrumentation Laboratory Munich. Germany) using the APC/CaCI2solution divided by clotting time obtained with CaC12 in the same I:1, 1:5 and 1:11 dilution. In addition, plasma of 24 neonates with septicaemia were investigated and data of 18 infants aged birth -three months with Arg 506 Gin +/-were shown. The Arg 506 GIn mutation of the factor V gene was assayed by amplification of the DNA samples by PCR followed by digestion of the amplified products with the restriction enzyme Mnl I. Results were confirmed by SSCP -analysis or by direct sequencing of DNA from patients with APCR. Results are shown in the 1.6(1.4-1.95) Neonates and infants were considered to be APCR when the aPTT ratio was < or = 2. Concerning the special properties of the neonatal hemostatic system, our data show concordance with the PCR method in neonates and infants only, when the aPTT based method was performed in the I: 11 plasma dilution. Case report: We report on an 8-year old boy with severe hemophilia B and frequent screaming at night. EEG showed spike wave activity, starting from the temporal lobe, but generalizing within seconds. Complex partial seizures were diagnosed and therapy with carbamazepine was initiated. As no improvement was seen NMR was performed. This revealed lesions within the right frontal cortex. Higher doses of carbamazepine were not succcssfull as was therapy with phenytoin and pfimidone respectevely. The patient is now treated with carbamazepine and valproate. He still suffers from one short seizure per day. Because of his seizures we started prophylactic replacement therapy with 600 I.E. factor IX twice per week. Discussion: In 1992 Wilson et al. first detected brain abnormalities in 25 of 124 children and adolescents with hemophilia A or B who were negative for immunodeficieney virus (1). The most common findings (14/25 patients) were small, focal, nonhemorrhagic white matter lesions of high signal intensity on T2weighed images. Similar lesions have been reported in children with sickle cell cerebral infarction (2) . Only three of these 14 patients had seizures, all of those having a documented history of intracranial hemorrhage. Our patient has similar lesions as those described by Wilson et al. But no history of intracranial hemorrhage is documented. Even if tuberous sclerosis might be a differential diagnosis, we think that the abnormalities are related to hemophili a or its treatment, because the patient has no further signs of this disorder. Conclusions: 1. In patients with hemophilia and seizures NMR might be useful as a high sensitive method for the detection of gray and white matter changes. 2. Further studies should be initiated to determine the prevalence of pathological conditions in the brain of hemophiliac patients. Disseminated intravascular coagulation (DIC) is a rare, but foudroyant disease occuring in GRAM-negative sepsis like meningococcal septicemia. Despite the avallibility of potent antibiotics, mortality in mertingococcal disease remains high ( about 10 % ), rising to 40 % in patients presenting with severe shock and consecutive DIC. As the clinical course and the severity of manifestations of systemic meningococcal infections varies there is a need for early diagnosis of the infection and stage of coagulopathy in order to reduce the high mortality rate. Few and rapidly available parameters are needed to classify the wide spectrum of clinical and laboratory findings in patients with DIC. The parameters include partial thmmboplastin time, pmthmmbin time, plasma levels of fibrinogen, fibrin monomers and dimers, fibrin degradation products and the thrombocyte count. Monitoring the course of hemostaseologicai findings in 26 pediatric patients with systemic meningococeal infections we observed a change of coagulation parameters as early as in the first stages of the infection: A prolongation of partial thromboplastin time to an average of 69. 1 sec (range 22 -150 sec, norreal 30 -45 sec), a decrease of prothrombin time to 45.7 % (range 13 -71 %, normal 70 -100 %) and of antithrombin III to an average level of 16. 8 U/ml (normal 20 -29 U/ml ) was found 1 to 4 (-6) hours after admission.

The consecutive development of hemostaseological parameters mentioned above permitted to define the stage of coagulopathy and thus to induce a stage related therapy. Primary treatment consisted in control of shock by liquid substitution, compensation of metabolic acidosis, correction of clotting disorders ( AT III and heparin in stage of pre-DIC ; AT III and fresh frozen plasma in case of advanced DIC ) and treatment with g-lactam antibiotics ( e. g. cefotaxime or ceftriaxone ). An early assessment of the coagulation disorders in meningococcal disease can be based on few coagulation parameters, thus an appropriate treatment may be arranged to prevenl the patient from a fatal outcome of meningococcai septicemia and protect him from the development of a WATERHOUSE-FRIDERICHSEN-syndrome. This study was designed to prospectivdy evaluate coagulation and flbrinolyfie activation in 60 children (neonate -16 years) during cardiac catheterisation with low dose flush heparin (10 IU/ml saline). APTT (Instrumentation Laboratory: see), anti Xa activity (Xa; Chromogenix: IU/ml), prothrombin fragment Ft.2 (F1.2; Behring Werkc Marburg: nmol/l) and D -Dimer formation (D-D; Bnhring Werke/vhrburg: ug/l) were investigated before (T1), at the end (T2) and 24 h after cardiac catheterisation (T3). In addition, to evaluate the influence of inherited thrombophilia in all patients resistance to activated protein C (APCR), protein C, protein S and antithrombin were investigated. During catheterisation median (range) hepadn was administered in a total dose of 60 (17-206) IU/kg bw. In addition infants < 6 months of age (arterial catheterisatiun only) or patients with known thrombophilia received 300 -400 IU/kg hepafin for fmther 24 hours. The results (median and range) are shown in the Ft.2 was sigificanfly elevated above the pediatric boundary immediately after the intervcation and nearly reached baseline values 24 h later. In contrast no cfinically relevant fibrinolytic activation was seen: D -Dimer formation increased within the pediatric boundary immediately after the catheter and returned to basdine levels 24 h later. Three children showed resitance to APC. tn one child stroke occurred before. Not knowing the result of APCR in the remaining two patients only one neonate received further prophylactic heparin. The third neonate without heparin prophylaxis suffered from venous occlusion within two days after the intervenfon~ In addition, no protein C, protein S or antithrombin deficiencies were found. Although administration of low dose flush heparinisation during cardiac cathetefisation could not prevent short -term coagulation activation, no thrombotic events occurred in children without inherited thrombophilia. If fnrther prophylactic hepariuisation in children with A~R, protein C, protein S or antithrombin deficiencies may prevent vascular occlusion requires a more intensive study.

A.Sandvoss, W.Eberl, M.B0rchert

Introduction: Capillary leakage, edema and hypovolemia are common complications in preterm infants especially if birth weigth is below 1.500 g. Septicemia, asphyxia and immaturity seem to be most important risk factors. To determine the influence of C 1-Esterase Inhibitor (CIlNA) in preventing contact phase and complement activation we investigated C11NA concentrations in normal and symptomatic preterm infants. Methods: Activity of CIlNA were measured by chromogenic substrate method (Behringwerke), CIlNA concentration with radial immunodiffusion (Behringwerke,Germany). Results: CllNA-activity in asymptomatic preterm infants (n= 14) was 65+/-15% of normal at birth. Healthy newborns showed activities of 80+/-20%. CIlNA reached normal adult values 2-4 days after birth. Preterm infants with respiratory distress syndrome(n = 14) showed lower activity on day 2-5, patients with additional septicemia (n=15) had decreasing C1 INA-activities in the first three days of life. Individual course of CllNA-activity and thrombocyte count correlated in the group with IRDS with and without septicemia. In children with capillary leakage onset of diuresis went parallel with raising CllNA-activity. Markers of contact phase (F Xlla) and complement activation (C 5al were investigated in single cases and evidence for involvement of both systems was found. Conclusion: Contact activation and complement system play an important role in capillary leakage in preterm infants. CIlNA regulates both systems. Activity of CIlNA correlates with clinical course, substitution therapy is possible and may improve outcome of these critical ill patients. Antiphospholipid antibodies (APA) interfere with hemostasis probably by inhibition of protein C or prothrombinase complex. Thereby, APA might lead to thrombosis or increased bleeding. However, incidence and clinical importance of APA has not yet been investigated in children. Therefore, we assayed plasma samples of 220 children, aged 0,1 to 19 years (mean 7 years) by ELISA detecting IgG-and lgM-antibodies directed against eardiolipin, phosphatidyl serine and phosphatidic acid. In patients with increased bleeding, thrombophilia or prolonged clotting tests a detailed coagulation analysis was performed. According to their diagnosis children were devided into 5 groups: I. autoimmune diseases, II. infections, III. metabolic diseases, IV. other diseases, V. healthy children. Results: APA were found in 69/220 patients. In the respective groups we demonstrated APA in the following proportions: 1. lgG-isotype: Activitiy of C1 Esterase Inhibitor (C11NA) is reduced in preterm infants especially if birth weigth is below 1.500 g and respiratory distress syndrome and/or septicemia is present. Capillary leakage with generalized edema, hypovolemia and hypotension is resulting in imbalance between inhibition and activation of contact phase and complement system. iln four patients we investigated seven courses of substitution ;with commercial C1 Esterase Inhibitor preparation (Berinertr,Behringwerke), case reports are given. All patients had clinical symptoms of capillary leakage, all had septicemia accompanied by either respiratory distress, disiseminated intravascular coagulation or mutiple organ failure. jEfficiacy of substitution therapy is dose related, supranormal iactivities of CIlNA are necessary, reflecting raised consumption of inhibitor in ongoing disease. Clinical effects on diuresis, catecholamine need and especially on thrombocyte counts are demonstrated.

OR ARTERIAL THROMBOEMBOLIC EVENT IN CHILDREN E. Lenz, C. Heller, W. Schr6ter*, W. Kreuz Johann W. Goethe-Universit~itskinderklinlk, Frankfurt a. Main, Germany * Georg-Augast-Universit/itskinderidinik, G/3ttingen, Germany

Venous thrombosis as well as arterial thrombo-occlusive events are rarely observed in childhood, but can lead to life-threatening situations and longterm sequelae in these Patients. After the initial stage of treatment (thrembolysis or thrombectomy) the pediatrician has to decide how to efficiently prevent re-thrombosis in the individual patient. Anticoagulation after venous thrombosis is generaUy recommended for 6 months after the event; if an underlying thrombophilic condition has been detected in the patient anticoagulation has to be considered lifelong. When evaluating antithrombotic therapies for children it is of importance to consider whether the anticoagulatory effect is mainly necessary in the venous or arterial vessel system. The hemorrhagic risk and side effects of the different anticoagulatory preparations have to be taken into account, especially when treating small children.

Only limited experiences exist concerning the suitability of the preparations for long-term anticoagulation in children and general recommendations on the ideal dosage in pediatric patients are still missing. We want to disscuss different types of anticoagulants (such as coumarins, unfractionated heparin, low molecular weight heparin (LMWH) and inhibitors of platelet aggregation) their mode of action, their suitability for pediatric patients and their side effects and relevance of these side effects especially in children. From the experience in our own pediatric patients, we would like to report on the indications, which can be given to administer these different preparations, the dosage regimen we recommend and the Laboratory tests to monitor save and efficient re-occlusion prophylaxis in our patients.

In this context we would like to present our data on 8 patients with either thrombosis or arterial infarction due to a thrombophilic condition, who had all contraindicatioas to oral anticoagulation by coumarins. Because prophylaxis for re-thrombosis was mandatory in these patients, LMWH was given for long-term anticoagulation in a dally subcutaneous dosage of 100-150 anti-Xa U/kgbw. Monitoring was done by Anti-Xa-test (0,4-0,8 anti-Xa U/ml). Under this regimen none of the patients developed re-thrombosis or bleeding complications. Alopecia was seen as a side-effect. This study was designed to prospectively evaluate coagulation and fihrinolytic activation after cardiopulmonary bypass with aprotinin (2x17000 U/kg bw) in 42 infants and children aged 0.1 -15 years, and to correlate these findings to the clinical outcome. Prothrombin fragment F 1.2 (F1.2; Behring Werke Marburg: nmol/l), antithrombin-serinesterase -complex (ATM; Stago: ng/ml), D -Dimer formation (D-D; Behring Werke Marburg: ug/l), tissue-type-plasminogen activator ag (t-PA; Chromogenix: ng/ml), plasminogen activator inhibitor 1 antigen (PAI; Chromogenix: ng/ml) and Cl-inhibitor (C1; Behring Werke Marburg: x 10-3 g/l) were investigated before the operation (T1), at the end of the operation (T2), and on postoperative days 1 (T3), 4-6 (T4) and 7-9 (T5), respectively. The results are shown in the table (median and median absolut deviation): T1 T2 T3 T4

"1"5 NV FI.2 0.9 +/-0.5 1.7 +/-0.9 1.4+/-1 1.8+/-0.8 1.6+/-0. The platelet (PL) function defect induced by thrombolytic agents has been attributed either to the degradation of PL surface receptors or to the anti-aggregatory effect of FgDPs. In contrast to other plasminogen activators scu-PA is intimately Inked with PL: they can rapidly incorporate exogenous seu-PA, release it upon stimulation and bind the proenzyme. Recently we have reported that exposure of PRP to recombinant scu-PA (2.5-t00 uM) in timed interval 1-30 min resulted in dose-dependent inhibition of PL aggregation. Timecourse changes of the process were followed by the biexpotential kinetics: a rapid initial inhibition during the first 3-5 rain with the moderate suppression of PL aggregation in the 30 min period. When tcu-PA (25-100 nM) was exposed to PRP in the same conditions dose-and time-dependent inhibition of PL aggregation was also observed. Since the effect was obtained no earlier than t0 min after exposure of tcu-PA to PRP, and the threshold dose was higher. Comparable inhibition of PL aggregation was obtained with 25nM of scu-PA versus 100nM of tcu-PA and the llbrinogen depletion by the end of the 30 min period was 2% and 30% respectively. It's likely that tcu-PA and its precursor have different mechanisms of action on the PL aggregatory function. In a recent study we have shown that recombinant rscu-PA inhibits platelet (PL) aggregation in PRP. To exclude the possible influence of rscu-PA/plasma interfere on this process the aggregation of washed PLs was under the investigation. PLs were washed according to modified Mustard's method, suspended in buffer and adjusted to 250,109/1. The resuspended PLs were exposed to 5-100 nM of rscu-PA for 30 min at 370(;. At time points 3, 5, 15 and 30 min the aggregation with 0.6 IU/ml of thrombin was measured. It was found that the exposure of PLs to rscu-PA (20-100 nM) for 3 mAn resulted in marked inhibition of their aggregation. Since after 15-30 mAn of incubation with 20-50 nM of rscu-PA the inhibitory effect on PL aggregation became less pronounce or even disappeared. When 5 nM of rseu-PA was used the inhibition of PL aggregation became significant only by 15 rain of exposure period and didn't change for 30 man of investigation. The observed results may be cormeeted with uptake of rscu-PA by PLs from surrounding buffer as well as with individual variations of PL response to the same concentration of rscu-PA. Loss of glycosylation may result in a reduced platelet (P) survival and perhaps altered function. We analyzed the structural and functional effect of specific deglycosylation (combinations of N/O-glycosidase and neuraminidase treatment) of P and isolated P GPIb. Washed and formaldehyde-fixed P were digested as follows: 1) With neuraminidase (0.125U/ml) + O-glycosidase (3.1mU/ml) + N-glycosidase (1.25U/ml), 2) with neuraminidase alone (0.2U/ml), 3) with N-glycosidase (2U/rnl) and 4) with neuraminldase (0.2U/ml) + O-glycosidase (5mU/ml). All reactions were performed in the presence of protease inhibitors (PMSF, Leupeptin, SBTI), After washing x2 the P and identically treated controls were analyzed by flowcytometry with the antibodies 6DI (Mab: a-GPIb), 7I-L2 0Vlab: a-GPIIIa), and the lectins wheat germ agglutinatinln (WGA, for NeuNac) and peanut agglutinin (PNA, for [3DGal(1-3)-GalNac) which confirmed effective and specific deglycosylation by the respective enzymes (but gave only minor differences with 6DI and 7H2). The botrocetin (13) and ristocetin (R)induced agglutinations showed aRer treatment 1) (all enzymes) a full inhibition of R-induced agglutination but only a mildly reduced B-induced agglutination (70% of normal). Treatment 2 and 3 (neuraminidase alone, and N-glycosidase alone) affected both agglutinations only mildly (70-80% of normal).Treatrnent 4) (O-deglycosylation) however showed a major inhibition of R-agglutination down to 30%, while B-agglutination interestingly was almost fully retained. The results of the rotary shadowing electron microscopy of purified GPIb suggested a collapse of the normally stretched, glycosylated, GPlb, not only after the treatment with all three glycosidases, but also .after O-deglycosylation alone. We conclude that Oglycosylation is most important for ristocetin-induced platelet-von Willebrand factor-interaction and responsible for the typical stretched shape. The phenomenon of in vitro platelet aggregation and consequent pseudothrombocytopenia (PTCP) in the presence of calciumchelatization by Na-EDTA and sodium-citrate was studied in blood samples of a patient. Initial platelet counts electronically measured were 20000/ul blood anticoagulated with Na-EDTA and sodium-citrate. Normal platelet counts were found in heparin-anticoagulated blood and in capillary blood. Immunoglobulines of the IgG and IgM subclass were identified in the patients plasma. By incubation of the patient's serum with platelets of healthy individuals, platelet-clumping occurred in the presence of Na-EDTA and sodium-citrate but not in the presence of heparin. The platelet membrane glycoproteins (gp) Hb/llIa, IX and IIIa/VNR g-chain were involved in the antigen antibody reaction as demonstrated by specific antibodies and flow-cytometry. On platelet surface permanent calcium-exchange and -replacement is dependent on external calcium concentration. Calcium depletion induced by calcium chelators as Na-EDTA and sodium-citrate might conformationally change platelet surfaces and induce formation of neoantigens. The decrease of gp llb/Illa platelet surface antigen to 10% (normal >75%) indicated the important role of the gp IIb/IIIa receptor at PTCP. The saliva of Tdatoma pallidipennis, a triatomine bug, was found to contain a protein called "pallidipin", that specifically inhibits collageninduced platelet aggregation but not adhesion or shape change. To investigate the mechanism of action of recombinant pallidipin the influence on platelet fibdnogen binding after activation by collagen type I in different concentrations was measured by flow cytometry. The same concentrations of pallidipin that inhibited the coUagen-induced platelet aggregation completely did not cause any inhibitory effect on fibdnogen-binding in the PRP from the same donor measured contemporaryly.

Collagen type I-induced platelet aggregation of CD36-deficient platelets from two different unrelated blood donors was inhibited by the same concentration of pallidipin that inhibited aggregation of control platelets. There was no inhibition of collagen-induced fibdnogen-binding in the CD36-deficient platelets as well.

Pallidipin did not cause inhibition of collagen-induced membrane expression of CD62 and CD63 of control and CD36-deflcient platetets as measured by flow cytometry. However eadier studies had shown an inhibition of collagen-induced ATP and {3Tg secretion by pallidipin. Therefore we compared the effect of pallidipin in unstirred and stirred PRP samples. While pallidipin had no effect in unstirred samples it showed strong inhibition of pTg secretion in stirred samples.

We therefore conclude that pallidipin does not act on collagen-induced aggregation through CD36 and that the inhibition is a post fibdnogenbinding event. Pallidipin does not influence the first steps in secretion, which are independent from cytoskeleton and platelet-platelet contact, but inhibits the following steps.

17-HYDROXY-WORTMANNIN DOES NOT iNHiBiT THE TRANSPORT OF 1NM-GOLD LABELLED FIBRINOGEN IN RESTING PLATELETS. E. Morgenstem, B. Kehrel and K.J. Clemetson Medical Biology, Saarland Univ., Homburg, Germany, Haemostasis Research, Univ. Muenster, Germany and Theedor-Kocher-lnstitut, Univ. Bern, Switzerland. Wortmannin, an inhibitor of phosphoinositide 3-kinase and of myosin light chain kinase blocks reactions of the activated platelet. To obtain informations about the role of the contractile cytoskeleton in receptor-mediated transport of resting platelets, the effect of 17-hydroxy-Wodmannin (HW) on the endocytosis of fibrinogen from the surface of resting platelets was studied. Gel filtered platelets (GFP) were incubated for 10 min at 37°C with HW (3x10-6M) or with Iloprost. Controls and GFP preincubated with HW or Ilopmst were incubated with 1.4nm-gold labelled fibrinogen molecules (Fg-Au; final concentration 40p.g/ml) at 37°C. The experiments were stopped after 5 or 30 min by rapid freezing. After freeze substitution in acetone with 4% osmiumtetroxide, sedal sections were prepared. The sections were examined after incubation with ascorbic acid (5% in H20) for 30 rain at 20°C (to reduce metallic osmium) and silver-enhancement using Danscher's (1981) method (to visualize the Fg-Au). Examination of ADP stimulated platelets in the presence of 40Fg/ml Fg-Au shows that the ligand is able to mediate aggregation. The examination reveals, that Fg-Au was present in a low density on the platelet surface, in higher density in the surface connected system (SCS), in coated pits and vesicles and separated smooth vesicles (representing endosomes?) as well as in the matrix of alpha-granules. After 30rain, the number of labeled granules was increasing. Labels on the surface and on the mentioned cytoplasmic membranes were observed during the whole period of incubation. HW or Iloprost did not alter the resting GFP and the mentioned qualitative ultrastructural findings in both preparations did not show differences to the controls. We conclude from the results with HWthat the regular contractile function of the cytoskeleton is not necessary to transport the Fg-Au in resting platelets. METHODS: EDTA anticoagulated whole blood was incubated with thiazole orange and analyzed with a flow cytometer. Young platelets were defined by having a high fluorescence from thiazole orange (normalized to platelet size). Platelets were also incubated with fluorescent antibodies to GPIb, GP lib/Ilia and GMP-140 (two colour method). RESULTS: Surface expression of GPIb was the same in young and older platelets. Results for GP lib/Ilia and GMP-140 (in resting and activated platelets) will be presented. CONCLUSION: Young platelets can easily be detected using thiazole orange and flow cytometry. There is no differential expression for GPIb. Further results will be presented. The influence of erythrocyte and thrombocyte content on the release of ATP by different agents in whole blood specimens was tested. The measurement had been performed in the Lumi-Aggregometer using the principle of the luciferin-luciferase reaction. Altogether 39 blood samples were diluted gradually before induction of the release reaction by arachidonic acid (1,25 mmol/I final concentration), ADP (30 IJmol/I) and collagen (1,0 and 5,0 tJg/ml). The peak of the obtained curves was transformed into percent values of the maximal deflection by the undiluted sample (= peak in relation) and into ATP concentrations (= absolute peak) after testing the ATP standard in parallel for each dilution step separately. The peak in relation increases by increasing dilution with all inducers. It was identic with the ATP standard and with collagen, somewhat lower with arachidonic acid and much higher by ADP. A luminescence-optical effect may influence all these results. The absolute peak decreases by dilution under arachidonic acid and collagen as it was expected by the decreasing thrombocyte content of the samples. Under induction by ADP no decrease of the absolute peaks by increasing dilution of the samples was abserved. This can be explained only by liberation of ATP from the erythrocytes. The ATP standard is essential for the quantification of the release reaction. ADP doesn't suit for it. Collagen with a final concentration of 1 pg/ml was proven as the best inducer. Platelet aggregation induced by several agents has been photometrically investigated in disc shaped rotating cuvettes coated with vessel wall tissues obtained from human umbilical cord, either endothelium or smooth muscle cells or extracellular matrix or combinations of them. In addition, effects of endothelium incubated with several cytokines on platelet aggregation have been studied. Endothelial cells strongly inhibited aggregation depending on their cell count and the concentration of the inducer. Smooth muscle cells showed the same effect but very less marked. In presence of extracellnlar matrix spontaneous aggregation occured. Endothelium could inhibit this spontaneous aggregation when present in the same cuvette, smooth muscle cell could not. Incubation of endothelium with several cytokines increased its anti-thombotic properties. For example, at a platelet count of 3x105/id in the PRP, 10 -6 M ADP led to maximal aggregation in uncoated cuvettes, in presence of 5,5x106 endothelial cells aggregation was completely abolished, in presence of 2,75x10 "6 cells aggregation was decreased to 40%. Smooth muscle cells diminished the aggregation effect of 0,1 NIH Thrombin to 67% when only one side of the cuvette was coated and to 63% when both sides were coated. Endothelium could not inhibit aggregation induced by 2,5 x 10 -6 M ADP but endothelium incubated with 500 U/ml TNF-a or 30 U/ml Intedeukin-lfl or lmM L-Nitro-Arginin for 24 h did completely inhibit aggregation. Platelets become sticky and adhere to surfaces or to another without contracting and secreting. During maturation of megakaryocytes finally platelets lost their genomic nuclear message. Only mitochondrial DNA of platelets can be identified. We focused our attention on the impact of mitochondrial DNA and the mitochondrial transscriptive mechausisms during platelet activation in normals. Materials and methods: Leucocyte free (Nagentte chamber, flow cytometric analysis) platelet rich plasma or platelet concentrates a_~er hemapheresis were filtered by PALL 100 leucocyte filters. The influence of different anticoagulants (commercially available Sarstedt tubes containing citrate, heparim EDTA and 500 ATU/ml hirudin Wacker) was examined. Activation was due to a 60 nun. hemapheresis procedure ( 3-5fold increase of CD 62, CD 63) and ex rive stmaulation due to 4 NIY U/ml thrombin, 0.025 m CaC12 or combmatious. The guanidiurn method for total RNA preparation were used according to T. Brown: Current protocols in molecular biology 4.21-4.9.14,1991. Different primers of mitochondrial genome (e.g. cytochrome b and ATPase) were prepared using PCR and mitochondrial transscription was examined using Northern-Blot-technique. Results: 1., There is less activation of mitochondrias using hirudin anticoagnlation, but a 2fold increase of mitochoindrial RNA content in heparinized samples. 2., Stimulation with thrombin leas to an increase to 5.5 E-l0 RNA btg/platelet, compared to 4.7 -4.8 E-10 RNA ~tg/platelet under unstimulated conditions.. Conclusion: There is evidence for the importance of platelets mitochondrial DNA and mitochondrinl transsefiption in regulation of cytosceleton and platelet activation. Thrombospondin-1 (TSP-1) is a large homotrimeric glycoprotein originally identified as a platelet alpha-granule component. The investigation of its putative role in a variety of pathophysiologies like haemostatic disturbance, malignancy and wound healing requires specific laboratory reagents. Monoclonal antibodies are one of the most powerful of these reagents. Therefore, we purified human TSP-1 from thrombin-stimulated platelets using affinity chromatography to generate monoclonal antibodies in mice. A subclass IgG 1 monoclonal antibody designated 48.42 was purified from ascitic fluid and further characterised. Western blot experiments demonstrated that this antibody reacted only with the unreduced molecule whereas the TSP-1 subunit chain was not recognised. No cross-reactivities with human fibrinogen, fibronectin, vitronectin and von Willebrand factor were found. Preliminary results indicate that the monoclonal antibody 48.42 can be used to investigate TSP-1 function in several assays including immunocytochemistry and cell adhesion as has been demonstrated for HL-60 cells. In addition, a sandwich enzyme immunoassay was developed using goat-antihuman TSP-1 IgG and derivatised monoclonal antibody 48.42 (peroxidase, biotin) as a sensitive method for detection of TSP-1 in human body fluids. In the following study the expression of the platelet antigen (CD62P) and the leukocyte antigen (CDllb) were measured in whole blood, in addition to platelet-leukoeyte adhesion (rosette formation) by means of multicolour fluorescent labelling (CD45, CD14, CD42a). The measurements were carded out both in freshly drawn whole blood which had been antieoagulated with different agents, and in stirred samples of whole blood under controlled conditions (37°C, 1000 rpm, different stirring times). The results are presented as the percent positive events in each gate (platelets, leukocytes -PMNL, monocytes, lymphocytes and rosettes -plateletpositive events in the PMNL, monocyte and lymphocyte gates), whose mean fluorescence is given in addition to an index comprising the product of the percent positive events and their mean fluorescence. Stirring (max 15 rain) induced an increase of CD62P on the platelet surface of ca. 10%, without any change in the mean fluorescence. Under these conditions increased CDllb on PMNL and monoeytes could be detected. An increase in the rosette formation could also be measured (greater index), in that the percent of monocytes which were platelet-positive increased with no change in the mean fluorescence of the positive events, whereas PMNL showed an increased mean fluorescence, but not an increased number, of platelet-positive events. The time-dependent changes in rosette formation on stirring could be further increased by addition of ADP. These results show that it is possible to measure rosette formation, and also the influence of effector agents (inhibitors or activators of platelets or leukocytes) on rosette formation, in whole blood using flow eytometry. 17 ITP patients undergoing splenectomy were observed after 1-30 years following operation and divided into 2 groups. First group consisted of 8 patients with normal platelets count and absence of haemorrhagic syndrome. Second group was formed of 9 ITP-patienfs with episodes of thrombocytopenia recovery following certain time period after splenectomy. In the aim to study the cellular immunity there were carried out immunophenotypical investigations of blood samples using immunofluorescence method with monoclonal antibodies application. The increase of B-cells, expressing CD22, CD37, HLA-Dr-antigen has been revealed in the 2nd group. Quantity of SRFC, CD3 +, CD5 + cells in the blood of recovered patients was lower than in patients of the first group. This group was also characterized by statistically significantly increased level of CD4 + cells while the CD4/CD8 ratio was equal to 1.0 :i: 0.3 % (0,5 + 0,1% in patients of the second group, respectively, p>O,05}. Also the relatively high expression of activating antigens in patients with thrombocytopenia recovery after splenectomy was stated. Among infectious complications in all patients observed were predominantly found various types of throat infection, mainly with unsatisfactory treatment possibilities. We have observed the OPSI-syndrome in 2 patients, being featured with marked tiredness, breath loss, intolerance of hard physical working, diminished ability to maintain physical activity. Extracellular matrix (ECM) produced by human endothelial cells closely resembles the vascular subendothellal basal lamina in its organization and chemical composition. Thus it contains collagens, fibroneetin, von Witlebrand factor, thrombospondin, fibrinogen, vitronectin, laminin and heparin-sulphate. Platelets carry different receptors on their membrane surface with specific binding capacities for one or more of these extracellular matrix proteins, such as glycoprotein (GP) IIbIIIa, GP Ib/IX and GPIIIb. Incubation of platelets with ECM results in platelet adhesion, degranulation, prostaglandin synthesis and aggregation. We studied patients whose platelets showed either a receptor defect in GPIIbIIIa or GPIIIb or a storage pool disease. Adhesion experiments were performed using siliconised glass, collagen coated surfaces, immobilized fibrinogen as well as human subendothelial matrix. Platelet adhesion of patients with Thrombasthenia Glanzmann (receptor defect of GPIIbIIIa) resulted in a total lack of binding to silieonised glass and immobilized fibfinogen. Adhesion to collagen was almost normal in spite of the fact that only single platelets sticked to the surface and no microaggregates were observed. The adhesion to ECM was diminished and also no aggregates were detected. Patients with a receptor defect in GPIIIb showed normal platelet adhesion to siliconised glass and immobilized fibrinogen but binding to collagen and ECM was markedly reduced, while platelets with a storage pool defect sticked to siliconised glass but failed to adhere to ECM. By centrifugation of citrate blood (250 x g, 10 min) erythrocytes and leucocytes go to the bottom, whereas plasma and thrombocytes stream in the upper part of the probe. So the thrombocyte count doubbles in the platelet rich plasma in contrast to the platelet count in the whole blood volume. If the thrombocytes are more or less activated, they adhaere on erythrocytes, leucocytes or aggregate end are not able to stream upwards. The quotient between thrombocyte counts in PRP and whole blood is a measure for thrombocyte activation. We chequed the value of this screening in different groups of patients with arterial occlusions disease (AOD), chronical venous disease (CVD), diabetes mellitus (Dm] and in healthy control persons (Control). Variation coefficient of the method is 3.7 (PRP) and 4.4 (TC) respectively (Coulter counter). Differences to the control group are significant. Changes in the patient groups in dispensaires follow up 5 years are also significant.

NICARDIPIN -INDUCED IMMUNTHROMBOCYTOPENIA P. Eichler 1, C. Hinrichs 2 , g Greinacher l i.Institut fur immunologic und Transfusionsmedizin, Ernst-Moritz-Arndt-Universitat Greifswald, 2. Deister-S0ntel-Klinik, Bad M0nder

Drug-dependent immune-thrombocytopenias are a rare but clinically important variant of immune-thrombocytopenias. Patients are at risk to suffer from severe bleeding complications. Especially in patients receiving multiple drugs, diagnosis of drug-dependent immune-thromboeytopenia is often difficult. We report the case of a 71 year old male patient who received Allopurinol, Captopril, Digitoxin, Furosemid, and Nieardipin. The patient presented with hematomas (pit. count < 10 G/l) and later developed bone marrow dysplasia.

In an ELISA using whole platelets and patient serum, a weak reactivity in the presence of furosemid, but a stronger reactivity in the presence of nicardipin (Antagonil, CIBA-Geigy) could be demonstrated. The reaction pattern is given in the The enzyme-immunological determination of soluble fibrin (SF) proved to be highly sensitive and specific. This SF-ELISA detected fibrin hacking fibrinopeptide A (FPA) via the monoclonal antibody 2t35 specific for the neoepitope generated on the Aa-chain after the split of FPA. Lill et al. recently introduced a new assay modification which utilizes the same antibody as the old one but takes advantage of a pretreatment of plasma specimens with KSCN. This strong chaotropic ion is used to dissociate the various fibrin complexes possibly hiding fibrin epitopes. It was the aim of this study, therefore, to compare the two SF-ELISA modifications (with and without KSCN-pretreatment of specimens) . In order to examine the dynamics of thrombin-induced fibrin(ogen) metabolism we made course observations in patients with a certain form of septicemia. Both assay modifications detected fibrin(ogen) derivatives which differed considerably in kinetics (n= 160 samples from 10 courses). The former SF-ELISA (no KSCN) correlated well with prothrombin fragments, thrombin-antithrombin !11 -complexes and with the release of fibrinopeptide A ( r > 0.96, n= 151). Results of the new SF-ELISA with KSCN pretreatment of patients' plasma, however, correlated conspiciously well with D-dimer levels (r > 0.94) but distinctly less with the markers of thrombin generation (-0.12 < r < 0.29). This good correlation with D-dimer levels was unaccountable since the D-dimer maximum occured significantly later than the peak of markers of thrombin generation (p < 0.05). Therefore, KSCNpretreatment of fibrin specimens seems to lead to a change in the specificity of the fibrin assay despite usage of the same catching antibody. Different half-iifes of differently composed fibrin complexes should be considered in trying to explain the findings. Nevertheless, the results of the former assay without KSCN-treatment correlated much better with the well-known dynamics of thrombin-induced fibrin generation during hemostasis activation than the data from the new assay modification. Consequently, further examinations are necessary to specify the effect of KSCN on soluble fibrin complexes and the resulting assay specificity. A rapid assay for the determination of the Primary Hemostasis Potential (PHP) of whole blood has been developed (Kundu et al, 1995) from the original method of Kratzer and Born. The new system employs a disposable test cartridge which holds the sample (citrated whole blood) and all components for the tests at the same time. The test procedure is very simple. The cartridge is loaded with -500 p.l citrated whole blood and is inserted into the Platelet Function Analyzer (PFA 100aaw). The test is started automatically after a preincubation phase of 2.5 rain. The reaction starts with the contact of the whole blood and the capillary which is connected with a collagerdephinephrin coated membrane with a small aperture inside the test cartridge. Under constant negative pressure the sample is aspirated and through the contact ofplatelets and vWF with collagen adherence and aggregation begins. The adhesion and aggregation process leads to the formation of a platelet plug which obstructs the flow through the aperture. The result of the PHP is reported as closure time (CT). Additional parameters such as bleeding volumes are possible as well. First results show good reproducibility, normal values in the range of up to 150 sec. and a good discrimination of healthy donors from patients with congenital or acquired platelet dysfunctions. The system detects aspirin induced thrombocyte function defects and von Willebrand disease. In ease of an abnormal result in the collagerdepinephrin system a second type of cartridge with a collagerdADP coating can be employed. In the majority of cases aspirin induced dysfunctions are normalized and could thus detect aspirin use. The proposed system may be a valuable tool for routine assessment of the primary hemostasis potential in a routine citrate blood sample laboratory. inducing mental stress in 20 young healthy male volunteers aged 20 to 40 ),ears with no previous history of thmmbophilia or a hemorrhagic diathesis was performed by a first time parachute descent from an altitude of 1000 meters. The purpose of this investigation was to find out whether there are any changes in the corpuscular and plasmatic fractions of peripheral blood. We were especially interested in elucidating changes in the procoagulatory and/or fibrinolysis systems. Venous blood samples were obtained directly before and directly after the jump. Flight time from the departure of the airplane to the landing of the parachutists was approximately 20 minutes. The maximum time that elapsed between the two blood withdrawals were 45 minutes. In a preliminary study with different voinnteem, certain fluid imbalances had been observed. Absolute numbers of leukoeytes (6.9 vs. 9. l/n0, erythrocytes (4.6 vs. 5.1/pl), and platelets (246 vs.276/nl) significantly increased (p < .001), as well as the hemoglobin concentration from 145 to 156 g/L (p < .018). Even though fluid imbalances before and after the jump had practically been excluded by measuring nearly identical hematoerit values (.41 vs..42), we noticed a marked drop in aPTr (27 vs. 23 sec) and a significant increase in factor VIII ~tivity. As a direct stress response, we found a rise in fibrinogen concentration (2.4 vs. 2.8 g/l) which is one of the shortest acting acute phase proteins. Concerning reactive fibrinolysis, D-dimers showed an increase in concentration from 115 lag/L to still normal values of 192 lag/L, which was not significant due to low numbers of values (p = .086). We observed similar changes in fibrin monomers and prothrombin fragments Fl+2. From other investigations on the kinetics of the activation of the procoagulatory system we know that maximum activil7 is not reached until 24 hours after initiation of activation.These investigations studied perioperative changes in different kind of operations which served as a control group concerning the degrees of tissue damage and resulting coagulation disturbances. To better understand these phenomena we plan to induce mental stress in a laboratoq' environment to further exclude unknow~a influences on the mechanisms which can activate the procoagulatory and fibrinolytic systems. TRIODENA (T) 30/40/30 ug EE, 50/70/100 ug gestodene) were tested for their effect on hemostatic parameters. Three groups (n=20) of healthy female volunteers were treated for 6 months with one of these OC. Blood was taken before treatment (day 24-28 of pretreatment cycle, 0) and on days 18-22 of the 3 ~ (I) and 62 (II) treatment cycle. Indications of an activation of blood coagulation and fibrinolysis were detected as the plasma levels of prothrombin fragment F I+2 and of fibrin split product D-Dimer and plasmin antiplasmin complexes were found elevated during treatment. The following main regulatory components of blood coagulation, activators and inhibitors, were investigated: factor VII antigen FVIIAg, FVII clotting activity FVIIe, circulating activated factor VII cFVIIa and antithrombin 3 AT3 activity, total protein S antigen tPS-Ag, free protein S antigen fPS-Ag, protein S activity PSact, circulating thrombomodulin eTM FVIIAg, FVIIe and cFVIIa significantly increased during treatment; cFVIIa: 0: C 32.4 mU/ml A prethrombotic condition characterized by elevated levels of circulating soluble fibrin has been claimed to be a predisposing factor for accumulation of coronary thrombotic material in acute myocardial infarction. The present study includes 161 patients with clinical suspicion of myocardial infarction. Blood samples were drawn by the primary care physician, upon arrival in the hospital, and after 2, 6, 12, and 24 hours of hospital stay. Patients with myocardial infarction were identified by typical course in 12 lead ECG, and upon sequential determination of troponine T, myoglobin, CK, and CK-MB. Patients with primary CPR were excluded from evaluation. Soluble fibrin was measured by Enzymun®-Test FM (Boehringer Mannheim). Patients with acute myocardial infarction display soluble fibrin levels within the normal range (< 5 ~tg/ml) during the initial two hours after onset of symptoms. There was no significant difference between patients with myocardial infarction and patients with coronary heart disease without myocardial infarction. Slightly elevated levels were found in patients with atrial fibrillation, reflecting intracardiac fibrin formation. In patients without fibrinolytie treatment, a slight increase of soluble fibrin levels with a maximum after approximately 8 hours is observed. Most patients with fibrinolytic treatment display a considerable increase in soluble fibrin, with maximum levels immediately after infusion of the fibrinolytic agent. Four patients with pulmonary embolism showed soluble fibrin levels in the range of 40-300 [.tg/ml, which remained in the same range during the entire observation period. In conclusion, circulating soluble fibrin is not increased in patients with acute myocardial infarction and does not appear to be a predictor of acute coronary events. High levels of soluble fibrin in patients with fibrinolytic therapy may reflect release of fibrin from thrombotic material, but also de novo generation of fibrin due to release of active thrombin from thrombi not necessarily located in the coronary vessels. Detection of elevated levels of soluble fibrin in patients with acute chest pain should result in careful examination for signs of pulmonary embolism or aortic aneurysm. The possibility to determine activated coagulation factors opens the question if data provide evidence of an activated coagulation or fibrinolysis and if this has a prospective value. We investigated patients with confirmed thrombosis, postsurgical septieaemia and also after liver transplantation. In all patients factor VIIa, XII, XIIa and also the fibrinolytic parameters t-PA, PAI-1, PAP, Plasminogen and a2-AP were determined. In addition, F1+2 and APC-resistance with heterocygote factor V-Leiden-mutation and confirmed thrombosis. We found increased factor VIIa which showed partly also an increased Fl+2. Patients with other pathological results such as a reduced t-PA and/or increased PAI-1 showed a low incidence of elevations in factor VII or F1+2. The activation of factor XII seems to be of minor importance in patients with thrombosis. A different picture is found in septic and transplanted patients. Obviously factor XII-activation is of major importance in this group. A deterioration of the clinical symptoms is correlated with an increased factor XIIa which is paralleled by a decrease of factor XIIactivity. The investigation of fibrinolysis parameters such as PAI-1 and PAP demonstrate a fibrinolytic disturbance of the balance. Statistically significant are differences in septicaemic patients both in the surgical and in the internistical group in contrast to polytrauma patients. In patients with liver transplantations significant changes are apparently related to rejection of the transplanted organ together with a deterioration of the clinical picture. The possibility to detect activated coagulation factors may be a tool to detect changes in the hemostasis system at an early stage and to use this for an improved therapy. Control of long-term oral anticoagulation is usually performed by serial determinations of the prothrombin time. However, the assessment of effective anticoagulation versus the potential risk of bleeding complication is difficult to achieve. Molecular markers of blood coagulation activation might add valuable information in individual cases. We investigated 48 patients with thromboembolic manifestations (deep vein thrombosis n=22, pulmonary embolism n= 13, myocardial infarction n= 13) for one year beginning with admission to the hospital. TAT, prothrombin fragments F 1 +2, D-dirner and fibrin monomer concentrations were analysed. All markers were significantly increased at the time of initiation of anticoagulant therapy thus reflecting a prethrombotic situation. Patients suffering from venous thromboembolism demonstrated higher concentrations of TAT and F 1 +2 in comparison to myocardial infarction (34.6 vs 12.3 pg/1, p=O.009; 2.8 vs 1.3 nmol/I, p=0.0025). F 1 +2, TAT and D-dimer concentrations decreased gradually over the first 14 days of anticoagulant therapy reaching values within the established normal ranges in all cases. F 1 +2 and TAT concentrations reflect the activity of the coagulation system during long-term anticoagulation whereas analysis of fibrin monomer yielded partly controversial results. We conclude that F 1 + 2 and TAT appear to be superior to fibrin monomer for the individual control of oral anticoagulant therapy. The influence of thyroid failure on haemostasis is controversial. Mainly hypoceagulable states have been described in clinically overt hypothyroidism. Since hypothyroidism has been associated with an increased risk of atherosclerosis, we studied a wide range of haemostatic factors in untreated female patients with subclinical (B, n=42, age 59+13) or overt (C, n=8, age 55-zcJ) hypothyroidism, as well as in hypothyroid women under 1"4 treatment (D, n=8, age 57+9) and euthyroid controls (A, n=80, age 50+14). Simple screening tests (prothrombin time, activated partial thromboplastin time, fibdnogen), procoagulant factors (FVII, FVIII, von Willebrand factor), coagulation inhibitors (antithrombin Ill, hepadn cofactor II, protein C, protein S) and fibdnolytic factors (plasminogen, antiplasmin, plasminogen activator inhibitor, tissue plasminogen activator) were measured. Results Factor VII activity (VII:C), factor VII antigen (VII:Ag) and their ratio were found increased in hypothyroid patients. Factor VIII activity showed the same tendency, whereas von Willebrand factor ramained unchanged, as did all other parameters with exception of free protein S, which declined in overt hypothyroidism and in T4 treated subjects. These differences tended to diminish after exclusion of 26 women with estrogen replacement therapy for menopause, but the ratio VII:CNII:Ag, as well as FVII:C still remained significantly higher in hypothyroid patients.

Conclusions: Subclinical and overt hypothyroidism are associated with significantly higher levels of factor VII:C and VII:Ag. The disproportionate increase in VII:C compared to Vll:Ag, as shown by their ratio, might reflect the presence of activated factor VII (VIla), which in turn indicates a hypercoagulable state. This pattern becomes more pronounced with the concomitant estrogen replacement after menopause. Exocytosis following platelet activation leads to translocation of CD62P (P-selectin), CD63, and thrombospondin, from cytoplasmic granules to the cell surface membrane, where these molecules, serving as activation markers, can be detected by flow cytometry. We here report detectability of these molecules preformedprior to platelet activation -inside the cytoplasm of resting platelets. Two different methods are compared, i. e. using either methanol or the Fix&Perm kit (An der Grub) for cell membrane permeabilization. In addition, interleukin(IL)-Ice is shown to be present in platelet cytoplasm after methanol treatment, but not after permeabilization using Fix&Perm. Whenever cell surface positivity for a specific marker coincides with intracellular presence, blocking of the surface membrane sites prior to membrane permeabilization is required in order to obtain fluorescence intensity attributable to cytoplasmic staining. Our data demonstrate the feasibility of the methods presented for the detection of intracellular platelet molecules. This technique should also provide a means for estimating the relative quantity of intracellular platelet antigens, provided the permeabilization procedure does not lead to antigen leakage or destruction. Physical exercise activates the clotting as well as the fibrinolytic system as indicated in numerous investigations of exercise by running and by bicycle ergometer but not by swimming. The positive effect of an endurance training in coronary sport groups is induced also by influences on the hemostatic system. The influences are suppression of the clotting activation by the acute exercise and by an increased fibrinolysis response. Different hemostatic parameters, therefore, were analyzed before and after swimming of male coronary patients (n=33; median ag~ 61 years, achieved heart rate: 68/min). Indicating plasmatic clotting activation there was a significant increase in molecular markers TAT and F1+2 among the coronary patients (TAT from 2,1 to 3,4 pg/1; FI+2 from 0,92 to 1,1 nmol/1). The degree of clotting activation among the coronary patients was less than that observed in a group of young volunteers in a former investigation. This must be explained by existence of the coronary heart disease or by the higher age in the patient group. Indicating an activation of fibrinolysis t-PA activity increased significantly in coronary patients (from 0,14 to 0,5 IU/ml) resulting in an unchanged balance between coagulation and fibrinolysis. From this findings of the hemostatic systems no increased risk of the coronary patients by swimming can be derived. A prerequisite, however, are precautions l±ke to devoid exercise in the anaerobic range, exclusion of major heart failure and of cardiac arrhythmias before begirming of the swim training. The principle of the Fontan operation consists in anastomosing the right atrium to the pulmonary arteria, thus bypassing the right ventricle and using the only functional single ventricle as a pump for the systemic circulation. There are only few data about the influence of the changes in hemodynamics on coagulation and fibrinolysis. We investigated the coagulation system in 20 children and young adults aged 4 to 21 years in a general examination 4 to 61 months after Fontan procedure. Besides other abnormalities of the coagulation system, there were significantly increased values for the thrombin-antithrombin-III-complex (TAT) in 12 Patients (60%). As a marker for an activation of the fibdnolytic system we found elevated plasmin-alpha2-antiplasmin-(PAP-) levels in 14 patients (70%). Less frequently, the concentrations for the prothrombin-fragments 1 and 2 (F1 and 2) (7 patients, 35%) or the d-dimer (2 patients, 10%) were increased. We didn't find significant differences in a clot-lysis-assay between Fontanoperated patients and an age-matched control group. There was no significant correlation between activation of coagulation and clinical situation or diameter of the pulmonary arteria. Whether the present data can help to estimate the risk for a thrombo-embolic complication following Fontan procedure, still has to be investigated. The results of the clot-lysis-assay suggest, that for lysis of thrombi the same dose of rt-PA should be used as for other patients.

A 2nd GENERATION FUNCTIONAL PROTEIN S ASSAY P. van Dreden* and E. Adema** * Serbio, Gennevilliers France, ** Boehringer Mannheim, Tutzing Germany A second generation protein S test was developed with improved sensitivity to protein S and better reagent stability. The test result was found to be unaffected by APC-Resistence (10 patients, heterozygote for the mutation with a aPTI' + aPC ratio between 1.4 and 1.9), Heparin up to 2 IU/ml and F VIII activity between 1 and 250%.

In the test, diluted sample is mixed with protein S deficient plasma, activated factor V, activated protein C, phospholipids and an intrinsic pathway activator. This mixture is incubated for 3 minutes. During this time, the activated protein C inactivates part of the F Va. The extend of F Va inactivation depends on the protein S concentration. After 3 minutes CaCI2 is added and the time untill clot formation is measured.

The clotting time is a linear function of the protein S concentration between 10 and 140% protein S. For the three preproduction lots the difference in dotting time between 10 and 100% protein S was 43-54 seconds. This compares to 30-40 seconds typically obtained with the old test. Within run precision (n= I0 on STA) is cv= 2 -7% on the basis of Protein S. Day to day precision (n=10 on STA) was found to be cv= 4 -11%, again calculated on the basis of protein S concentration. The cv of 11% was obtained for an AVK plasma with 13% protein S; it corresponds to a standard deviation of only 1.5% in protein S.

The insensitivity to interferences, in particular APC-Resistence and better precision and stability are expected to improve the quality/reliability of a protein S determination. In this study we evaluated the use of hormonal contraception on the parameters protein C, protein S and PAl. Samples from 71 women with, without hormonal contraception and in menopause were assayed by coagulometric (protein S clotting test (Behdngwerke, Marburg, FRG) or chromogenic methods (protein C activity test and PAl reagent from Behringwerke, Marburg, FRG) in double determination and were compared with the reference ranges. In addition thromboplastin time (Thromborel S reagent) and fibrinogen (Multifibrin) from Behringwerke, Marburg, FRG, and aPTT (Actin FS reagent from Dade corp., Unterschlei6heim, FRG) were determined.

In women using hormonal contraceptives (p<0,01) and in menopause (p<0,05) protein S activity was significantly reduced compared to other women (<45 years) while protein C acitivity did not change. In menopausal women a higher susceptibility to thrombosis was supported by an increase of aPTT (p<0,05) and fibronogen (p<0,01). While there was no change for PAl, plasminogen was significantly lower in women using hormonal contraceptives and in menopause (p<0,05). We could not observe a higher turnover of coagulation and fibdnolytJc system with hormonal contraception. Noteworthy was the occurence of low (<200 mg/dl) and borderline fibrinogen (max. 220 mg/dl) in 40,9% of women res. in 22,8% of women (together with borderline aPTT) who had an individuell risk for arterial disease.

Protein S Protein C Fibdno~en aPTT Plasminog~ without HCC 109,1-+13,6 78,3-+14,1 255,0-+14,0 36, [2] [3] [4] [5] 4 24, [0] [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] 2 with HCC 85, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] 2 78, 5±12, 0 253, 8.+24, 1 35, [2] [3] [4] 8 14, 9 menopause 90, 3+97, 6 87, 4±41, 4 307, 05:57, 6 39, [8] [9] [10] 0 12, 6 HCC= hormonal contraception

Hemostatic parameters in a patient undergoing bone marrow and subsequent liver transplantation due to veno-occlusive disease C. Salat 1, , E. Holler t,3, HI. Kolbl, 3, B. Reinhardt l, R. Pihusch 1, P. G0hring 2, S. Poley 2, E. Hiller 1 l=Med. Klinik IIi, 2 = Institut flit Klin. Chemie, Klinikum Grosshadern der Ludwig-Maximilians-Universit~tt Mfinchen, 3=H~tmatologikum der GSF A 40 year old patient suffering from ALL received allogeneic bone marrow transplantation (BMT). After an uncomplicated early posttransplant period the patient was dismissed after 4 weeks. A bilirubin rise with subsequent liver failure was observed during the following weeks. According to biopsy proven hepatic veno-occlusive disease (VOD) liver transplantation was performed on day 79. Unfortunately the patient died on day 140 due to aspergillosis. We monitored levels of protein C (PC) and S (PS) as well as PALl during the pre-and posttranspiant period. PAL1 level was normal (<43 ng/ml) during the first 4 weeks after BMT but increased with the manifestation of VOD (317.5 ng/ml on day 47). It reached its peak immediately before liver transplantation (547.6 ng/ml) and returned to normal levels within the next few days. PC levels which were normal before BMT decreased prior to clinical diagnosis of VOD and were normal after liver transplantation. PS levels lay within the normal range at all timepoints. VWF was elevated before BMT (240%) and remained relatively stable during the whole investigatonal period ranging from 170 to 260%. It is assumed that VOD is initiated by an endothelial cell injury -possibly due to radiochemotherapy -and subsequent hypercoagulability. Our results indicate that the "endothelial cell marker" vWF is not helpful in predicting VOD. The kinetics of the investigated parameters underline the significance of PC and PAI-1 as described by others and our group earlier, whereas PS does not seem to play a role in the pathogenesis of VOD. The Budd-Chiari syndrome (BCS) is characterized by hepatic venous outflow obstruction that may be caused by the precipitation of a thrombus. It frequently coseggregates with other major diseases like myoloproliferative diseases or defects in the haemostatic system (antiprotein C and protein S deficiencies e.g.). Only recently, the factor V Leiden mutation (FVLM) has also been associated with BCS. We hypothesized that defects in the thrombo-modelling associated anticoagulant pathways (TMAAP) are a major risk factor for the precipitation of BCS. We screened our cohort of 27 patients (pts) with BCS for the presence of defects in the TMAAP and identified 3 pts with protein S deficiency (PSD). These pts were screened for the three point mutations in Exon 1 (Codon-25; ins T), Exon 15 (Codon 636; A-->T) and in Intron 10 (G-->A + 5) of the PS alpha-gene that have been demonstrated by Bertina et al to coseggregate with PSD. Restriction enzyme analysis and confirmation-sensitive gel electrophoresis for the detection of single-base differences in doublestranded PCR-products were employed. All living family members of the indicator pts were also screened for heterogeneties in the three point mutation as described. No single abnormality in these genes despite presence of PBD in those family members was found. In addition, pts and family members were also screened for FVLM. One pt and two of his family members, in addition to PSD, were subject to FVLM. The other two lots and their family members were not subject to FVLM. In contrast to the first family, despite PSD, those two pts suffered from Morbus Crohn and acute myeloid leukaemia as risk factors for BCS. We conclude: PSD is one major risk factor for the precipitation of BCS. To precipitate this disease, one additional risk factor is required. PSD may be caused by genomic defects in the protein S gene other than those described by Bertina. Only a few publications describe a thromboembolic disease due to dramatically reduced protein S levels being associated with viral or bacterial infections, Autoimmune mechanisms are suspected but the aetiopathogenesis is still under discussion.

We report on a 5 year old boy who developed purpura fulminans of the left leg during varicella infection. On the fourth day of infection the disease started with pain and haemorrhagic efflorescence localized at the left taft. On admission the boy suffered from a purpura fulminans with central necrosis measuring 15x8 era. Suspecting a hereditary thrombophilic disease we started therapy with protein C concentrate and recombinant tissue type plasminogen activator. The fellowing coagulation investigation showed a severe deficiency of protein S (total protein S-antigen < 5 U/ml, free antigen not measurable) in combination with Factor V Leiden mutation. Other thrombophilie and coagulation parameters did not show deviation from normal range. After 4 weeks we saw a slight improvement of the total protein S antigen up to 50 U/ml. The free protein S antigen was still undetectable. During the following weeks the patient recovered slowly and the protein S activity and antigen normalized. Because of skin necrosis thromboembolie prophylaxis was initiated with low molecular weight heparin (Fragmin®, 100 IE/kgbw/die) and continued for 6 months. Under this therapy there were no further thromboembolic events.

These results suggested an autoimmune protein S deficiency in a patient suffering from chickenpo×. An analyses of autoantibodies at the time of diagnosis showed a slight increase of the antieardiolipin antibodies (IgG 16,1 IU/ml, IgM 15,1 IU/ml) which normalized during hospitalisation. We suspect an antibody to protein S probably caused by similar presented viral antigens. We suppose that autoimmune mechanism during different infections in combination with a heterzygous APC-resistance may be a potential risk factor for developing thrombotic disease. In the central nervous system mRNA encoding for prothrombin and thrombin receptor is present and astroglial cells in culture process and secrete thrombin. Moreover, effects of thrombin on brain cells including change of neudte outgrowth and astrocyte shape are described, but the molecular mechanisms are unclear. We investigated the effects of human <z-thrombin and a six amino acid thrombin receptor activating peptide (TRAP-6, SFLLRN) on [Ca2+]i, phosphoinositide hydrolysis and protein kinase C activation in the rat glioma C 6 cell line which is a widely used in vitro model for studying glial functions. Stimulation of C 6 cells with both c{-thrombin and TRAP-6 resulted in transient [Ca2+]i mobilization, [3H]inositol phosphate response and activation of the PKC isoenzymes c{,I],7,5, and s. These results suggest that ~-thrombin and TRAP-6 activate at least partially the same intraceltular signaling pathways in rat glioma C 6 cells which is an evidence for involvement of "tethered ligand" receptor in thrombin induced signaling in glioma C 6 cells. Further investigations in this field are in progress including both receptor binding studies and more detailed analysis of thrombin receptor mediated signaling network and the cellular response of thrombin induced transmembranal signal transduction pathways in C 6 glioma. Thrombin is a potent mitogen in vascular smooth muscle cells (SMC). The mitogenic effect of bovine thrombin and human thrombin receptor-activating peptide (huTRAP-14) was investigated in bovine coronary artery smooth muscle cells. Confluent cells (passage 4-8) were incubated for24 h in serum-free medium with thrombin (10 nM) or TRAP (10-100 pM). The mitogenic response was assessed using [SH]thymidine incorporation. Results demonstrated that thrombin induced a three-to fourfold increase in [3H]thymidine incorporation compared to the control, thus being as active as the potent mitogen PDGF-BB (20 ng/ml). The mitogenic effect of thrombin was dependent on its proteolytic activity. When the active site of thrombin was irreversibly inhibited by D-Phe-Pro-Arg-chloromethylketone (PPACK) no increase in [°H]thymidine incorporation was observed even at the high concentration of 250 nM PPACKthrombin. Thrombin-induced [3H]thymidine incorporation was significantly reduced by the reversible direct thrombin inhibitor Nc£-(2naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide (NAPAP, lpM) . In contrast to thrombin TRAP-14 at a concentration up to 100 pM did not increase [3H]thymidine incorporation in the absence or presence of the aminopeptidase inhibitor amastatin (10 IJM). In order to demonstrate whether huTRAP caused a stimulation of the receptor and signal transduction in bovine SMC the activation of protein kinase C (PKC) was measured as translocation of the cytosolic to the membrane bound fraction. It was found that both thrombin (10 nM) and TRAP (10 pM) induced a translocation of PKC after 20 rain. In conclusion, thrembin and TRAP activated PKC in coronary smooth muscle cells but only thrombin acted as a mitogen. Endothelial cells once stimulated with thrombin are resistant to subsequent stimulation directly after the first. But after a recovery period of about 90 min the cells are sensitised again for activation by thrombin. In our experiments this resensitisation couldn't be inhibited by actinomycin D or cycloheximide nor by the phosphatase inhibitor ocadaic acid. Therefore an intracellularly pool of thrombin receptors has been proposed possibly co-localised with Golgi vesicles. Brefeldin A (BFA) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organisation of the Golgi apparatus. Because of this we examined the effects of BFA on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. The vWf release from WeibeI-Patade vesicles was used as physiological parameter of activation of thrombin receptors by thrombin. The addition of BFA (20 pg/ml -20 ng/ml) to endothelial cells blocked the receptor response regeneration, whereas the first stimulation was not influenced by BFA at the concentrations used. These results give further evidence that the proposed storage pool of thrombin receptors is Iocalised in vesicles of the Golgi apparatus.

Randomized Comparison of Double Bolus Reteplase (r-PA) and Front-Loaded Alteplase (rt-PA) in Patients with Acute Myocardial Infarction (RAPID II) C. Bode, RW. Smalling, J. Kalbfleisch, G. Berg, DJ. Odenheimer, E. BShm, WD Weaver and the RAPID II Investigators. Med. Klinik III, University of Heidelberg, Germany

The study was undertaken to assess the efficacy of a double bolus regimen of 10 + 10 megaunits of reteplase, a recently developed deletion mutant of tissue-type plasminogen activator, relative to front-loaded (GUSTO-regimen) alteplase. There was no age limit and patients were recruited up to 12 hrs after onset of symptoms. The primary endpoint "patency at 90 minutes" was centrally assessed in a blinded fashion.

Infarct related coronary artery patency (TIMI grade 2 or 3) and complete patency (TIMI grade 3) at 90 min for reteplase vs. alteplase were 83.4% vs. 73.3% (p=0.03) and 59.9% vs. 45.2% (p=0.01), respectively. At 60 minutes, the incidence of both, patency and complete patency was also significantly higher in retep ase vs. aiteplase treated patients: 81.8% vs. 66.1% (p=0.03). and 51.2% vs. 37.4% (p=0.01). Reteplase treated patients required fewer additional coronary interventions within the first 6 hrs of treatment (13.3% vs. 26.5%, p=0.01). There were no significant differences between reteplase vs. alteplase regimens in 35 day mortality (4.1% vs. 8.4%), severe bleedings (12.4% vs 9.7%) • or hemorrhagic stroke (1.2% vs. 1.8%).

Reteplase, when given as a double bolus of 10+10 MU to patients with acute myocardial infarction, achieves significantly higher rates of early patency and requires fewer acute coronary interventions than front-loaded alteplase without an apparent increased risk of complications. Lysis therapy with UHSK of deep vein thrombosis (DVT) of the lower extremity commoniy results in a relatively high patency rate. In our patients, patency was achieved in approx. 70-80%. These results were supported by several studies. In DVT of the pelvic veins, however, this kind of aggressive therapy is excluded due to the risk of iatrogenically induced pulmonary embolism. On the other hand, the need for an effective therapy is evident because of the usually occtnring complete occlusion of the pelvic veins and the following severe clinical symptoms 0Phlegnmsia cerulca dolens etc.).

Until September "95, 44 consecutive patients (16 males, mean age 41 ranging from 18 to 63 years) with DVT of pelvic veins were Included in this study. After clinical and phiebographic/computer tomographic (CT) diagnosis, a temporary filter system was placed intravenously either by a wambmchial (34 pat.) access route, lransfemorally (6 pat_) or Wansjngularly (4 pat.) under mdiological control. The open filter was positioned above the thrembus in the lower caval vein: 26 times an Antbeor system, 11 times an Anglocor and 10 times a Cook filter were used. In the following three days up to 3 cycles of lysis therapy with 9 Mli U of streptokinase were administered, each cycle lasted six hours. Heparin was given simultanously via the filter system in therapeutic dosages in order to prolong alrIT values 1.5 to 2 times the individual base line level. Effective lysis was controlled by a second phlebography/CT, and the filter system removed through the introducer sheath valve. After lysis therapy all patients were put on coumarin with overlapping heparinization. Successful lysis (complete recaanlisation without remaining thrombi and lysis of more than 50 per cent of the original thrombus) could be achieved in 62% (29 patients). In two cases a fatal pulmonary embolism occurred. 5 times great parts of a thrombus Were captured by the filter and could be removed with the system. Due to an insufficient boparin therapy we detected a newly developed thrombosis of the lower cavul vein twice after lysis. This complication could be resolved by additional administration of streptokinase. In nearly all cases we found extensive hematomas on the catheter insertion sites. 4 patients with transfemoral applications had to be transfitsed with red packed cells due to retroperitoneal hematoma. One filter system was broken after use. Lysis of DVT of the pelvic veins with UHSK and with the protection of a removable temporary filter system situated in the lower caval vein enables us to carry out an effective therapy. The filter systems, however, have to be ameliorated in order to better prevent fatal pulmonary embolism. Heparin should be administered at least in the remainder of the day of lysis. The insertion site should be the enbital vein because compression for bleeding complication is easily feasible at this location. We report about seven of our patients (pts.) with BCS treated by fibrinolytic therapy (5 female, 2 male, age 27, 6 years + 8). Only one case was of unknown origin while in four pts. myeloproliferative syndromes, in one protein C deficiency and in another one Morbus Behcet was detected In five pts. chronical and in two acute BCS was diagnosed. As complications were found: In three pts• thrombosis of vena iv.) cava, in one ofv. porta and v. lienalis, in one more of v• porta and v• cava. We treated just one patient with urokinase (3 x 800.000 IU/d for 18 days). Four pts. were treated with rt-PA in concentrations of 25 to 50 mg/d (for a period of up to 14 days) and continuous heparin infusion. High doses of rt-PA (80 to 120 mg/d for one or four days) were given in one case of acute and one of chronic BCS. Partial (after high-dose rt-PA nearly complete) recanalisation of thrombotic hepaticel veins was only achieved in the two acute cases of BCS. No significant recanalisation was proven in hepatical veins of chronic BCS. Three times complete fibrinolysis of v. cava thrombosis (two after highdose therapy) and one partial lysis could be attained. In one patient partial lysis of v. porta and in another one of v. lienalis was found. Furthermore complete recanatisation of v. porta followed local rt-PA lysis during surgical intervention. In our two pts. with acute BCS significant improvement of hepatical venous flow could be measured after fibrinolytic therapy• In chronic BCS hepatical venous flow did not improve while the complication of v. cava thrombosis could eventually be treated successfully. Probably due to fibrinolytic therapy one case of esophageal varix bleeding occurred. The benefit of flbrinolytic therapy of BCS seems to be influenced by the dur~ion of the disease• According to our results fibrinolytic therapy should be considered in cases of acute BCS as well as in the above mentioned thrombotic complications• Recent studies suggest that transport of thrombolytic agents through the clot can play a major role in determining the reperfusion time needed for thrombolysis. As the mechanisms of this phenomena are rather unclear we studied fibrin (0.4-1.5 ~tM) clot lysis by plasmin (30 nM) presented outside or inside the clot in purified system (Tris-HC1 buffer, 37°C). The clots was formed by addition of CaC12 (30 mM), thromboplastin (1 U/ml) and human thrombin (6 NIH/ml)t The lysis degree was determined either by counting of radiolabelled fibrin degradation products or by time of total lysis. Obtained kinetic parameters of this reaction: kest=99.4 rain -1, KM=I.9 ~tM (plasmin inside clot); kcat=l.36 rain -1, KM=I.3 lzM (plasmin outside clot). This data indicate that eatalytie efficiency of plasmin from inside the clot is 50-fold higher than from outside as measured from kcat/KM. Furthermore we studied plasma clot (0.25 ml) lysis in original plasma (3 ml) containing SK (5-100 nM) and seu-PA (5-100 riM) in the final concentrations from inside or outside the clot. The results are presented in the The similar results have been got when the fibrin clots were formed with batroxobin instead of thrombin. It may be concluded that fibfinolysis is a surface-dependent process. It's likely that the higher level of plasminogen activators in blood would prevent thrombus formation, while physiological clots would stay resistant. For the improvement of the thrombolytic therapy in children more clinical data are needed. Therefore we report on our experiences with rt-PA presenting the clinical course in 11 patients (age range: 7 days to 16 years) with venous thrombosis (n=9) and purpura fulminans by meningococcosis (n=2). The patients were treated with rt-PA (Actilyse R) for 10,5 hours to 13 days. The venous thromboses were localized in lilac/femoral veins (n=4), brachiocephalic/jugular/subclavian veins (n=3) and the cava superior vein (n=l). Central venous catheters were causative in 2 cases. The dosage range was between 0.2 to 0.5 mg/kg for the initial dose and 0.8 to 2.0 mg/kg/d for the subsequent continous infusion. Ten patients received simultaneously heparin. Complete clot dissolution occured in 6 cases, partial clot dissolution in 1 patient. In 4 patients with a history of the first clinical signs of thrombosis of 3 weeks or more the lysis was not successful. Concerning the side effects only small bleeding episodes on former venipuncture sites were observed in 5 patients. Systemic effects such as the decrease of the plasma flbrinogen concentration were rare too. In conclusion, the thrombelysis with rt-PA presents an effective and safe therapy in children at: the rt-PA dosage used. In case of a history of the thrombotic event of more than 3 weeks a thrombotytic therapy should not be carried out. D.Dimer is widely used in clinical situations associated with thrombotic diseases. The most popular application is the exclusion diagnosis of deep-veinthrombosis and pulmonary-embolism and the assay is performed in emergency. D.Dimer is also frequently measured for evaluating the thrombotic risk in the post-operative period. Quantitative, flexible and sensitive assays are required. We have developed a new automatic method which works on the coagulation walk-away instrument, STA. This assay was developed with two monocloual antibodies specific for D.Dimer coated on microlatex particles and it uses an immuno-turbidimetric technology. Absorbance is measured at 540 nm and is a direct relationship of D.Dimer concentrations. The assay works with 50 ~tl undiluted plasma and is performed in less than 10 minutes. The dynamic range is from 0.2 to 16 [~g/ml and no prozone effect is observed up to concentrations higher than 100 ~tg/ml. The detection threshold is of 0.2 [lg/ml. The intra-assay reproducibility is below 5% and the inter-assay reproducibility is below 6%. Recovery studies of purified D.Dimer added to normal plasma were between 94 and 105%. No interference of heparin, bflirubine, lipids or of rheumatoid factor up to concentrations of 100 U/ml is observed. There is no effect of high fibrinogen concentrations (> 10 g/l). When compared with conventional ELISA techniques (Asserachrom DDi), the assay demonstrated a correlation coefficient of 0.97 on 131 samples from normal individuals and hospitalised patients with elevated D.Dimer concentrations. Slope was of 0.97 and intercept was of -0.07. This new assay offers a full flexibility for individual testing as the calibration curve is stable for at least one week on the instrument. It is then well adapted for all the applications of D.Dimer measurements in coagulation laboratories. 16 children between an age of 3 days and 11 months ( median 6 weeks ) with thrombotic or embolic occlusion of major vessels were treated with rt-PA for thrombolysis. The affected vessels were both sided renal veins or one sided renal vein and V. cava inf. in 8 cases, the V. cava superior in 3, the V. cava inf. plus renal veins plus aorta in 1, the left ventdcle in 1, the aorta in 1, the A. femoralis in 1 and the V. portae in 1 case. 10 out of 16 occlusions were associated with an indwelling catheter. Underlying dieseases were sepsis (4), prematudty (3), vitiurn (2), asphyxia (1), short bowel syndrome (1), HUS (1), diabetes (1), CMV (1), exsiccosis (1) and M. Hirschsprung (1). Thrombolysis was performed with an bolus of rt-PA (0.1-0.2 mg/kg) followed by continuous infusion (0.8-2.4 (-9) mg/kg/24h, median 1.8 mg/kg/24h). Low dose hepadn (100 IE/kg/24h) was given dudng full dose hepadn (aPTT 1,5-2 times normal) after the thrombolysis. In 5 pts. rt-PA was administered locally through the catheter and in 11 cases systemically. In 13 patients the vessels could be recanalised completely, in 2 partially, in 1 patient the therapy had to be discontinued. In 2 vessels a reocclusion occurred. Bleedings were noted in three patients, all from recent venous puncture sites. The results encouraged us to start a multi-canter trial which has been approved by the ethical committee and is open for recrural. The aim is to compare efficacy and safety of rt-PA with urokinase, the only recommended standard in the management of critical major vessel obstruction in newborns and infants. The design is a randomised, notblinded trial with a cross-over option after three days in cases without success. Study end points are recanalisations, major bleedings and number of cross-overs. Inclusion criteria are age under 1 year, lifethreatening vessel obstruction, age of thrombus up to 10 days, no precaeding fibdnolytic therapy. Exclusion cdteda are cerebral hemorrage, pedventricular leukomalacia, surgery dudng the last 7 days and CNS injuries during the last 2 months. Although our knowledge on inherited thrombotic coagulation disorders has greatly expanded within the last years, there are still man}, patients with recurrent venous thrombosis in whom no obvious predasposition can be identified.Thus we decided to include also so-called rare defects associated with thrombosis in our routine thrombophilia screening programme, such as FXII deficiency. FXII is an important element m the intrinsic pathway of fibrinolysis and there is evidence for an insufficient fibrinolytic activity in FXII deficient pts..Up to date only few and controversial data exist about the frequency of FXII deficiency in pts. with thrombophilia. Cons~uently the aim of our study was to evaluate the association between FXII deficiency and juvenile venous thrombosis in a great population. Patients and methods: 1554 pts. (851 female, 703 male, aged I to 61 ys, median age 38.2 ys) with venous thromboembolism before the age of 45 ys were studied. One-stage clotting activity assay of FXII (FXII:C) was performed on ACL using FXII deficient plasma from Instrumentation Laborato~. FXII antigen concentration (FXII:Ag) was measured by electroimmundiffusion using reagents from Behfingwerke, Enzym research respectively. The normal ranges are tl~. routine reference values obtained m our labratory from 80 healthy subjects (40 males, 40 females, median age 26.2 ys); 95% range: FXII:C 53-135%, FXII:Ag 57-132%). Results: 122/1554 pts.were classified as FXI1 deficient (F 60, M 62), giving a prevalence of 7.8%. Severe FXII deficiencies with FXII:C below 1% were observed in 7 pts..ll5 pts= proved to have moderate FXII deficiency with FXIhC ranging lrom 2 to 51% and FXII:Ag ranging from 1 to 53%. In none of them inherited deficiencies of other well established thrombophila risk factors could be detected. None of the FXII deficient pts. had positive lupus anticoagulant tests. Familial FXII deficiency was found m 9 cases. Discussion and conclusion: The precedences of FXII deficiency amongpts, with venous thromboembolism was previously described to be 7.5-10%. Supporting these data, we have shown a praevalence of FXII deficiency of 7.8 %. In comparison to the frequency of other well established thrombophila risk factors we consequently have observed a relatively high prevalence of FXII deficiency m our study group.These data, from the largest such study reported, strongly indicate that FXII deficiency may not be a rare deficiency and may be more frequently associated with thrombosis than currently suspected. We describe a family with an exceptionally rare, i.e. plasminogen, deficiency, combined with subnormal activities of coagulation factor XII (Hageman factor). The first thromboembolic event, pulmonary embolism in the proposita was diagnosed at age 35. Since that time, 'spontaneous' venous thromboembolic events verified by phlebography and perfusion/ventilation lung scan recurred once every year despite oral coumarin therapy, whose intensity varied over an exceptionally wide range despite tight control The patient was repeatedly given succesful thrombolytic therapy with streptokinase or recombinant tissue plasminogen activator. Her plasma plasminogen chromogenic activity was 51-59 % compared to a normal plasma pool (reference range 70-130 %), plasminogen antigen was diminished to the same extent. The patient's factor XII exhibited only 28-55 % activity in a factor-deficient plasma assay as compared to a normal plasma pool. Other known risk factors for recurrent venous thromboembolism were not present : no evidence of malignancy, no obvious precipitating events, normal values of antithrombin III, protein C, protein S, fihrinogen, thrombin time, platelets, lupus-like anticoagulant, aPTT prolongation after addition of activated protein C. The proposita's mother had died at age 60 from pulmonary embolisnt No coagulation studies are available. The proposita's sister was first diagnosed deep leg vein thrombosis at age 17, since that time recurrent episodes of venous thromboembolism have been diagnosed also in an other hospital. This sister's plasminogen activity was 120%, but factor XII activity was reduced to 55 %. Three brothers of the proposita were examined, too, all in their 3rd decade of life. None of them recalled symptoms of or treatment for thromboembolic disease. In one brother, factor XII activity was normal (100-105 %), but plasminogen only about 50 %. In the 2nd brother, factor XII was very variable (64, 42 and 94 %), plasminogen was in the lower normal range, In the 3rd brother, factor XII was about 50 % (repeatedly), plasminogen was normal. Current knowledge about the risk of thromboembolism with both enzymes is limited, the optimal management remains controversial.

Msrgit Serbsn,Maria Cucuruz,Dan Madras,Carmen Petrescu, Natalie Rosiu,Rodica Costa III rd Psediatric Clintc,Universtt V of Medicine,

The unsatisfactory efficiency of entihepetitis B vaccination in our haemsphiliscs suggested the control of the immune status in 52 HIV negative patients,by establishing through flowcitomstrie with monoclonsl antibodies the lymphocyte subsets (CD3,CD4,CDS,CD&/CD8 ratio and CD19) and by seric tmmunoglobulins levels; the immunological parameters have been correlated with the serological markers of hepatitis infections (HAY, HBV,HCV ebd HDV) as well as on dependence with the treatment (blood,plasma,crysprecipitate,fector VIII/IX concentrate) and the quantity of their consumption (UI/k9 weight/yesr).The interpretation of the results pointed out • significant lower level of CD3,CD& (p<O,O01) st the group of hsemophiliscs treated with blood,plasma and cryopraclpitate; sdditionsly,in this group of patients it was found s significant drop of the CD19 counts (p< 0,001) without an immunoglobulin-deficiency. The same behaviour was found at the group of haemophiliacs carriers of HB~ and HCV markers.The superpositisn of hepatitis infections on the treatment with blood and native blood products in most of the cases has let unanswered the question related to the mechanism of the immunodeficiency: the hepatitis B or C infections or the negative impact of the itarative important alloantigenlc load ? Illumination with l~tM methylenblue (MB) is used for inactivation of enveloped viruses in human plasma. Via a photooxidative mechanism viral structures as well as plasmaproteins especially fibrinogen can be damaged. Fibrinogen is an important mediator in the cell-cell interaction of platelets. The four RGD sequences (Arg-Gly-Asp) of fibrinogen can be recognized by the activated fibrinogenreceptor GPHb/IIIa . After binding to GPIIb/IIIa one molecule fibdnogen can link two platelets. The aim of this study was to investigate the influence of MB treatment on the fibrinogen activity in human plasma. Furthermore we were interested to demonstrate if altered fibrinogen inhibit platelet aggregation by blocking the GPIIb/]IIa receptor. Human plasma was treated with 1-50~M MB. After illumination fibdnogen was isolated by fl-alanin precipitation. Purified fibdnogen was analysed by one-and twodimensional gelelectrophoresis. Influence of modified fibrinogen on platetet aggregation was measured with gelfiltrated platelets. Possible receptorblockade were examined in an 125j-fibrinogen competition assay. Densitometric scanning of reduced SDS-pages showed a decrease of the a-subunit as well as an increase of high molecular aggregates with increased MB concentrations. This effect was not detectable for fibrinogen isolated from plasma with 1BM MB. This results were confirmed by two-dimensional gelelectrophoresis. IBM MB treatment showed no receptor blockade in aggrement with normal platelet aggregation after stimulation with ADP and fibrinogen.

PROPOSED VALIDATION OF A QUANTITIVE QUALITY-ASSURED PCR METHOD F. Dorner, J. Eibl, G. Zerlauth Immuno AG

We have developed a highly sensitive and reliable quantitative method, based on the polymerase chain reaction (PCR) and on the reverse transcriptase polymerase chain reaction (RT-PCR), to screen for the nucleic acids of Human Immunodeficiency Virus type 1 (HIV-I), Hepatitis B virus (HBV) and Hepatitis C virus (HCV) in plasma and plasma products. The detection of PCR products is carried out by laser induced fluorescence, the procedure is therefore termed "laser induced fluorescence PCR" (LIF-PCR). The procedure allows the precise quantitation of genome equivalents due to the use of two calibrators that are co-amplified by the same set of primers as the target template in one and the same test tube. A high degree of reliability is achieved by co-precipitation of the extract, co-amplification and co-determination of the calibrators together with the genome equivalents to be determined. In this report we present validation data for the LIF-PCR and discuss them in the light of the recently published ICH-Guidelines on the validation of analytical procedures. 

Taking into account the pecularitles of the replacement therapy of haemcphiliacs in Romania the study aimed at evaluating the dimension and profile of blood-born infections in these patients; 142 hsemophlllacs,elasslfled in 3 groups: A-ulth no exposure to blood or bloodproducts, B-treated ~ith factor-concentrates and C-ulth plasma,cryoprecipItate + factor-concentrates in their therapy have been investigated (Elisa method) for serological markers of Hepatitis A,B,C end D, Cvtomegalovirus (CMV) and HIV I and HIU 9 infection. The infections profile of our haemophiliacs~hes some peculsrities. The hepatitis infection is the most extended,lnvolving more than 75% of the patients. The frequency is for Hepatitis -bZ,Z~ , U -"/~,~ , G -bb,7% end O -24% : the multiple infection was diagnosed in more than 50% of cases. The prevalence uas significantly hlgher in the group C. We studied blood count and relevant coagulation system parameters of autologous blood donors. Indications for autologous blood donation were divided in 4 categories: a group of diverse (A, n=13), reduction mammoplasty (B, n:5), prostatectomy (C, n=IO) and total hip replacement (D, n=74). We performed bleod count (BC), thromboplastin time (TT), partial thromboplastin time (aPTT), fibrinogen (Fib), F VHI Act, vWF RCo, vWF Ag, F XIII, AT III, D-dimer-fragments (DD) and fibrinmonomers (FM). All parameters were analyzed as initial value, BC, TT, aPTT, Fib, F XIII and AT III were measured additionally at each donation. The percentage of initial pathologic data was (1) were included in the study. The inclusion criterittm was: an endegen(y.~ red cell reserve (ERCR) of less than 400 ml in men and less than 540 ml in women. ERCR is the volume of blood that can be withdrawn up to tim hematocrit of 0.34. The selected dosages of rhEPO (200, 400 and 800 IU/kg body weight biweekly over 4 weeks) were based on the calculated blood volume and the ERCR. A unit of whole blood, separated into an autologens red cell concentrate and one unit of fresh frozen plasma, was collected when ]mnoglobin concentration was at least 11.5g/100 ml (or hematocrit was at least 0.34) up to an autologons predeposit of 800 ml red cell volume (RCV). Additionaly, platelet concentrations and serum ferritin levels were measured~ Results: At the initial clinical examination the ERCR was 333 ml in women and 307 in men. This resulted in a weekly dosage of 2x32.000 IU rlaEPO in women and 2x25.000 IU rhEPO in men. 7 patients donated 5 red cell each (858 ml RCV on an average) within 19.1 days. The targeted RCV could not be reached in 3 patients because the operation was reschednled. The collection from one patient had to be aborted because of angina pcetoris complains. Tic following side-cffczts occurred: episodic hypcrteJlsion (2 tim~), chest pain (5), fafigne (8), inflnenza-lihe syndrome (3). Platelet count increased from initially 203 4-68 x 10'//d (I00 %) during therapy to a maximum level of 260 + 93 10V~ (128 %) without any clinical signs of thrombeembolism. In the control group (autologous blood donation weekly 3 or 4 times without erythrupoiedn stimulation, n=12) platelets increased from initially 230 __+ 73 10'/FI (100%) to 255 + 71 10V#I (110%) I1]aximlzm Colldttsions: The adlninist/'ation of lhEPO in the above described manner tO autologous donors undergoing heart surgery is well tolerated. It allows to collect a sufficient number of autologous blood units and the preparation of a predeposit at short notice. Essential thrombocythemia is a chronic myeloproliferative disease with a benign clinical course that is mainly complicated by microcirculatory or thrombotic vascular disorders. It is at present uncertain which patients need (lifelong) therapy and whether cytoreduction is necessary. We retrospectively evaluated vascular complications in 128 patients (33 men, 95 women) with a median age of 61 years in relation to therapy. Three major treatment groups were analysed: 17 patients treated with acetylsalicylic acid (ASS) alone, 29 patients given alkylating agents, and 62 patients treated with interferon alpha (IFN). There was no significant number of patients treated with hydroxyurea. IFN-treated patients were younger (median age 54 years) than patients in the other groups, but platelet counts before therapy were similar (median 0.84-1.13 x 1012/I). Before therapy, 47% of patients treated with ASS, 45% treated with alkylating drugs, and 32% treated with IFN had bleeding events, microcirculatory or thrombotic complications. Dudng therapy, median platelet count was 0.9 x 1012/I in patients on ASS, 0.68 x 1012/I in patients treated with alkylants, and 0.48 x 1012/I in IFN-teated patients. The rate of patients with complications during therapy was 18% (9.5%/patient year) for ASS, 14% (6.3%/patient year) for patients on alkylating drugs, and 15% (6.2°/dpatient year) for IFN-treated patients. We conclude that all three treatment regimes seemed to have a beneficial effect on the incidence of vascular complications in essential thrombocythemia. The results give a rough estimate of complication rates to be expected and may form the basis for randomized clinical trials. Adhesion of activated platelets to leukocyte* has been shown to be mediated by at least two molecules on the platelet surface: P-selectin (CD62) and fibrinogen (Fg) bound to the GP IIb/IIIa complex. Interaction of platelets with teukocytes via CD62 is believed to stimulate the production of reactive oxygen species (ROS) in leukocyte*. The role of platetet-bound Fg as a signalling molecule in platelet-leukocyte interaction is controversially discussed: stimulatory as well as inhibitory effects on leukocyte ROS production has been described. In the present study we measured ROS production in undiluted samples of citrated whole blood (WB) as well as in suspensions of erythrocytes and leukocytes in platelet-rich plasma (platelet-rich WB) or platelet-poor plasma (ptatelet-poor WB). ROS production was determined by measuring luminol-enhanced chemiluminescence (CL). Stirring WB samples at 1,000 rpm for 10 min resulted in an activation of leukocyte* (increase of CL, upregulation of CD1 lb) and platelets (platelet aggregation, upregulation of CD62). Stirring-induced CL was significantly higher in platelet-poor WB when compared to platelet-rich WB. Higher CL in plateletpoor WB than in platelet-rich WB was also found when ADP (moderate stimulation of platelets and leukocyte*) or FMLP (strong stimulation of leukocyte*) but not when PMA (strong platelet and leukocyte stimulation) was added. Dextran sulphate that is known to block CD62-mediated interaction ofplatelets with leukocyte* caused a dose-dependent inhibition of stirring-induced CL in WB samples. In contrast, when binding of Fg to the platelet surface was blocked by RGDS or the Fg receptor antagonist GR144053, stirring-induced CL, but not CL in unstirred samples, was significantly enhanced. GR144053 also reduced adhesion of platelets to neutrophils, but did not affect CD62 exposure. The results indicate that interaction of platelets with leukocyte* in whole blood via CD62 and Fg may have opposite effects on leukocyte ROS production: stimulation by CD62 and inhibition by platelet-bound fibrinogen. Late reocclusive processes represent one of the most commonly seen serious adverse events after coronary angioplasty. The pathophysiology of both of these processes is multifunctional and represens complex time dependent pathophysiologic events involving both the humeral and cellular processes. To test the effects of low molecular weight heparin (Certoparin, Sandoz AG, Numberg Germany) against late occlusive processes, four groups (Groups A-D) of rats (7-10) were anesthetized and the external common carotid arteries were exposed. All groups received a 200 u/kg of unfraetionated heparin through the femoral vein. A 2F balloon emboleetomy catheter was inserted into the left carotid artery and passed throug the left common carotid artery leading to the aortic arck Vascdar injury was induced by repeatedly (x3) inflating the catheter balloon and withdrawing it to the bifurcation. Following this procedure, two hours after, equlgravimetric amounts of various agents were administered via SC route to each of the individual groups. Group A was administered with Certoparin OD for 21 days. Group B received a lower low molecular weight heprin (Mr 3.4 kDa). Group C received unfraetionated heparin and Group D served as a control. On day 21, perfusion fixation was performed on all animals. The injured and contralateral carotid arteries were excised for histelogic examinatior~ In comparison to the saline control, varying degrees of the inhibition of myointimal proliferation following arterial injury was noted in all groups.

However, the degree of inhibition followed the order: heparin<Certopann<LLMWH_ In contrast to heparin treated group (C), the other groups A, B, and D did not exhibit any hemorrhagic lesions. However, degree of perivascular fibrosis, hemorrhage and inflammation were observed at the infusion site. In comparison to heparin, Certoparin and its lower low molecular weight derivatives exhibit better efficacy in reducing balloon angioplasty induced restenosis. The observed efficacy of these agents may be due to better bioavailability and sustained duration of action. In the present paper the binding of heparin to granulocytes of human and animal species was analyzed and compared with the binding to monocytes and lymphocytes. A low-molecularmass heparin (LMMH) was covalently bound to tyramine (tyr) and subsequently tagged with fluorescein-5-isothiocyanate (fitc) by endpoint attachmenC. Binding to leukocytes was quantified using flow cytometry analysis. The leukocyte populations were identified by their light scatter properties in man by CD4, CD13, and CD14 antibodies, in mice by CD 4 and GR 1 antibodies, and in rat by CD 45, ED 9, and RP 3 antibodies. Granulocytes, monocytes and lymphocytes of all species bound dose-dependently LMM-heparin. The binding of LMMH-tyr-fitc ~to granulocytes was higher in human, rat, rabbit, dog, and pig as compared to monocytes and lymphocytes. Mouse and sheep granulocytes did not bind more heparin than monocytes or lymphocytes. Binding of LMMH-tyr-fitc was reversible in the presence of unlabeled heparin or LMMH. More than 99% of human, rat, rabbit, dog and sheep granulocyte populations could be'distinguished from monocytes and lymphocytes by their fluorescence intensity after incubation with LMMH-tyr-fitc. This separation was not obtained for mouse and pig granulocytes. The data present evidence of a specific heparin binding to granulocytes of many species and indicate the significance of fluorescent labeled LMM-heparin for biological investigations. BACKGROUND AND PURPOSE: PTCA is an effective treatment for restoring coronary artery pat~cy; however, acute thrombotic rencclusion is a drawback of the procedure. Currem prevention of rencclusion relies on antiplatelet agmts (aspirin) and antithrombin agents 0aeparin). In the present study we examined the time course of changes in blood coagulation and fibrinolytic function following PTCA in patients with ischaemic heart disease. 20 patients were randomly assigned to receive either standard therapy (250 nag aspirin p.o.; 5.000 IU hepafin i.e. bolus, and 5-10.000 IU heparin intra-proeadurally)(n=10), or adjunctive therapy (aspirin, hepann; 100 mg sodium pmtosan polysulphate, SPP intra-cortmarily and subcutaneously) (n=10). The sensitive markers of thrombin activation (thrombin-amithrombin III complex ('rAT), prothrombin fragmem F 1+2 (pl+2), D-dimer) and of fibrinolytic activation (plasmin-antiplasmin complex (PAP), tissue type plasminogan activator (t-PA) and plasminogen activator inhibitor (PAl)-I activities) were measured before, immediately after, and 6 hs after PTCA. RESULTS: We found elevated mean levels of TAT and Fl+2 in both groups during the procedure, while plasma coneentrafiuns of D-dimer and PAP remained in normal range. Activity of PAI-I was not elevated compared to normal comrol. The t-PA activity obtained immediately post-PTCA showed significant increase in patients given SPP. Primary angiologic success was achieved in 80% of both groups. One patient in each group had recurrent ischaemia and emergency interventions (angioplasty, stent, bypass surgery) were performed. One patient receiving adjunctive therapy died in cardiogenic shock within 24hs aRer PTCA. Major bleeding was observed in one patient given SPP. CONCLUSION: No significant intracoronary haemostatic activation occurred in the majority of patients during and up to 6hs after PTCA. Treatment with SPP was associated with enhanced fibrinolytic activity. The benefits and risks of adding SPP to heparm and aspirin should be evaluated by further studies, 

In July 19,¢1 the Paul Ehdich Institute became responsible for quality and safety of bt~d and blood products. Since ~his time, batch m~se of PPSB and Factor IX prapamtions were carded out regularly, whereas Ihe obligatoP i testlng of all other products derived frem pooled plasma will be starling in Janualy 1996.

The European Pharmacopoeia does .or contain directions for the quality control of PPSB preparations. Therefore, Ifua ~ coneenning Factor IX clotting concentrates are applied, Ircludmg the non activated PTT and the classical tkrombln test These m~ods are not su'rlable for reliable exclusion of Ituombogenk~b/ ,of PP,Sfl, because Vlla actlvRy is not d~.

A hllhedo unsolved problem is the high sensitivity Of file classical thrombin test often not correlating with ~ data of thrombogenlci~. In ordm tD es~abl~ an effective quality conlrd protocol, different methods for the qual~ative detennlna~on of Ila, Vlla. IXa, and Xa (chrornogenic, fluorege~c clolfing reactions) wera instaUed. In agreement with the [deralure, considerable differences were found among the PPSB pr~ons registered in Germany.

Cedain amounts of activated do~ng racers may be well tole~ted in redp/ents. ~no are in a steady state condition. However, in severely ill patienls, eg. post operatively, Tissue Factor may be expressed which pofenllatesthe dsk of ~aclor Vlta. In the case of a certain PPSB preparation hlgh ¢onceatratio~ of acthrated cto~ng factors, especially ~vated factor VII, cotmlaled with fatal COmlffcal~ns, Evaluating ~ expe~ concenlralbn limits for adJvated prothrombin co~np~.x factors as prerequisite for ~e batch r~ease have to be applied. Aprotinin is used as a hemostatic agent in cardiopulmonary bypass surgery and other indications where hemostatic compromise results in hemorrhagic manifestations. The mechanism by which this agent produces hemostatic effects is not known, however, inhibition of serine proteasas such as plasmin, kallikrein and dastase and modulataion of platelet receptors have been considered as possible target sites. To determine the effect of aprotinin on tissue factor mediated activation of human platelets, whole blood samples were drawn at baseline and 24 hours aider aspirin (650 mg PO) ingestion (n=5). Blood samples were supplemented with saline and aprotinin (40 ug/ml) and tissue factor activation was studied at 200 pg/ral. Platelet activation was investigated using gtycoprotein ffla and GMP 140 specific antibodies on a flow cytometer (Epic Coulter). Tissue factor was found to produce the activation of both the baseline and asp' "mnized platelets. Aprotinirt augmented the tissue factor activation of platelets in both the control and aspirin supplemented groups. However, the relative increase in the aspifinized group was much monger (25% of the baseline). These results suggest that aprotinin augments the tissue factor mediated activation of platelets in both the normal and aspirinized individuals. While these effects may result in hemostatic restoration in aspirin compromised patients, in normal individuals the clinical implications of this effect remain to be clarified. , 10-20 (group 2) or >20 years (group 3) duration. Anticoagulated whole blood was incubated with fluorescent antibodies to GPIb and GMP-140 (two colour method) and analyzed with a flow cytometer. Thrombomodulin, F1+2, protein S, 13-thromboglobulin were measured according to standard procedures. RESULTS: Surface expression of GMP-140 was not different in groups 1 to 3, however, there was a tendency to higher acitvation in group 1 (<10 years IDDM). Results for thrombomodulin, F1+2, protein S, 13-thromboglobulin will also be presented. CONCLUSION: Though it did not reach statistical significance, platelet acitvation seems to be more important during early diabetes. This wilt be correlated with endothelial and plasmatic activation markers. In our clinic four patients with HIV-related thrombocytopenia were treated with a lot of Gammagard (93F21ABllF), which later turned out to be HCV contaminated. Before infusion all patients were negative for HCV antibodies and HCV RNA. 2 to 8 months after infusion 2/4 patients, who suffered from ARC at the time of HCV infection with CD4 counts >100/pL, seroconverted, whereas in the two other patients, who suffered from AIDS with CD4 counts below 100/pl, there was no seroconversion. In all cases HCV RNA was found. Genotyping with Inno-LIPA (Innogenetice) showed HCV genotype l(b) in all patients. Liver enzymes and HCV RNA copies were measured repeatedly over a period of one year after infection. The 2 patients with ARC showed a strong increase of HCV RNA titre during the first 3 to 4 months after infection, followed by a rapid decrease within the next months. In the patients with AIDS HCV RNA copies increased moderately within the first 4 to 6 months, followed by a slow decrease. Elevation of liver enzymes was mild in the AIDS patients and seems to be independent from the HCV RNA titre. In the ARC patients liver enzymes changed parallel to HCV RNA titers with a delay of 2 to 3 months. The course of HIV infection was only slightly influenced by the acute hepatitis C as measured by CD4 counts, i%2microglobulin and HIV RNA copies. Introduction:Mechanisms underlying ischemia/reperfusion injury have been thoumughly investigated in experimental models. Leucocytes appear to play a main role through production of cytokines and overexpresssion of adhesion molecules. In experimental animals, administration of monocional antibodies (MAb) recognizing CD18 can reduce organ injury following ischemia/repedusion. No data, however, have been reported concerning clinical ischemia situations. Patients and Methods:We investigated expression of CDt8, CD1 la, CDf l b and CD1 lc in granulocytes, monocytes and lymphocytes from peripheral blood of five patients undergoing elective hand surgery. The tourniquet was applied on the upper arm and heparinized samples from cubital veins were obtained before and at the end of ischemia. Control samples were drawn from the nonischemic contralataral arm with the same timing, Duration ot ischemia ranged between sixty and one hundred minutes (80~16). Whole blood samples were incubated with specific, fluorochmme labelled antibodies and analyzed by fluorocytometry (FACScan, Becton Dickinson, San Jose, CA). Mean fluorescence intensity (MFI), quantitatively reflecting surface expression of the indicated markers was evaluated for the individual cell populations. Data were compared by the paired student's t-test, p<0,05 was evaluated as significant. Results:MFI for all markers was comparable in all cell populations in samples obtained before ischemia from both arms. In contrast, expression of CD18 was significantly enhanced in granulocytes (321_+50 vs. 189_+38), monocytes (653-+54 vs. 426+122) and lymphocytes (299_+45 vs. 228-+36) from samples derived from the ischamic arm, as compared with the nonischemic arm, as measured at end of ischemia. At the same time, an increase of CDf lb on granulocytes (500~_342 vs. 213+150) and monocytes (533+359 vs.237-+206) but not on lymphocytes was found, No modifications of CDlta and CDttc expression could be observed. There was no correlation between duration of ischemia and quantitative expression of these markers, Conclusions:Our data indicate that relatively short ischemia periods induce an increased expression of ~2" integrins adhesion molecules on leucocytes. These results suggest, at close similarity with findings from expodmental models, that overexpression of adhesion molecules might play an important role in the induction of ischemia/reperfusion injury, in humans. In patients suffering from chronic inflammatory bowel diseases, such as Morbus Crohn and Colitis ulcerosa, we observe massive, sometimes barely staunchable bleedings. Hereby, the deficiency of coagulation factors, especially of factor XIII in plasma is established. ttowever the influence of factor XIII on the pathomechanism of the underlying disease is still under discussion. Therefore we studied the F XIII content in the intestinal mucosa. An immunohistochemicat method was developed using commercially available antibodies against F XIII subunit-A, the detection of mucosal factor XIII depends on the amount of chromogen bound to the antibody-horseradish-peroxidase complex. With this method, it is possible to locate but not to quantify F XIlI in the intestinal tissue. Therefore we developed an ELISA-metbod in homogenized intestinal tissue, using commercially available antibodies. Its precision was validated using a standard curve with commercially available factor XIII preparations (Fibrogemmin®). The detection limit of this method is > 0.05 I.U. F XIII/ml of tissue solution. Freezed dried intestinal tissue (lmg) was homogenized in 1 ml buffer using a potter. Specimens of the large bowel revealed F XIII values of 0,21 + 0,0038 I.U. (x __+ SD), tissue solution. With this method it is possible to quantify tissue-bound faxtor XIII. Studies are in progress to elucidate the content of F XIII in the intestine of patient's suffering from infammatory bowel diseases in order to contribute data to the pathomechanisms of F XIII deficiency. In a previous double-blind, controlled trial we were able to show that aprotinin administration has significantly contributed to reduce periand postoperative bleeding complications without increasing the risk of thromboembotic complications. The question arises whether this beneficial effect may be associated with its effects on intraoperative fibrinolysis. Therefore, 20 patients were treated with or without aprotinin (2 million KIU loading dose over 15 minutes followed by 500,000 KIU per hour), and citrated blood samples were obtained at the following time points: Before operation, after induction of the anesthesia, at the beginning of operation, intraoperatively when the femur shaft was implanted, and 24 hours postoperatively. The determinations of plasmin/antiplasmin-complexes, D-Dimers, thrombin/Antithrombin III-complexes, and prothrombinfragments 1 +2 were performed by means of test kits from Behring, Germany (EnzygnostRpAP micro, Enzygnost R D-Dimer Testkit, Enzygnost R TAT micro and Enzygnost R F 1 +2 respectively). -All markers of activated fibrinolysis and blood coagulation were significantly increased in the groups with and without aprotinin treatment, the highest activities to be seen when the femur shaft was implanted. However, the values of PAP and D-Directs of the aprotinin group were below the values of the control group until the end of operation. The markers of activated coagulation showed the opposite effect, however the differences between the two groups were not significant. As expected, the aPTT was significantly prolonged in the aprotiningroup. The aprotinin treatment was also associated with a significantly lower blood loss in these patients. -Concluding it can be said it is not clear whether the blood saving effect of aprotinin may be exclusively attributed to its antiplasmin activity since the differences of the fibrinolysis parameters were not statistically significant. Further blood samples should be analysed between the implantation of the femur shaft and the end of operation. In our laboratory large amounts of human prothrombin are required (30-50 mg/week). As we try to produce meizothrombin and meizothrombin-des-fragment-1 from human prothrombin and to apply it as an antidote for hirudin, the classical adsorption to barium sulphate or aluminum hydroxide from human plasma cannot be used. Commercially available human prothrombin is expensive and of an unacceptable quality for our applications. In most of these batches we found small amounts of Factor X and prothrombin activation products. We now developed a procedure to isolate prothrombin from "Prothrombin Complex Concentrates" (PPSB-250-bulk, DRK-Blutspendedienst Nds.). The concentrate also contains fac-tor VII, factor IX, and factor X. The prothrombin had to be separated from these factors. The concentrate we used contained amounts of other proteins and activation products of prothrombin (e.g. prethrombin-1) as well.

For the preparation of prothrombin from PPSB we used anion exchangechromatography (Resource-Q ®) on an FPLC ®. We applied dissolved PPSB directly or after buffer exchange on Sephadex G-25 onto the column at room temperature. The prothrombin was eluted with an NaCI-gradient in trisodium citrate buffer, pH 7.0. The buffer conditions are similar to the conditions used in the preparation of PPSB. The quality of the prothrombin so obtained was sufficient for most of our experiments. A second purification step on ion-exchange resulted in a 99% pure product devoid of contaminating factor activities and activation intermediates as examined with coomassie and silver stained SDS-Page electrophoresis and assays for Factor X. This prothrombin contained full enzymatic activity and its activation by specific snake venom prothrombin activators showed the known activation products. We are now able to isolate the amounts of pure prothrombin required for preclinical investigations. Most of the commercially available LMWHs such as Enoxaparin, Fraxiparin, and Fragrnin are prepared by chemical methods which can result in desulfation and other chemical modifications of the internal structure leading to differences in the pharmacologic effects. On the other hand, tiactionated LMWHs retain their native characteristics and are structurally similar to heparin. In addition, the oligosaocharide sequence responsible for ATIII binding is not modified. Physical methods such as gamma irradiation (~Co) have been used to fi'agment sulfated glycosaminoglyeans yielding fragraents without chemical modifications (DeAmbrosi et at. In : Biomedical and Biotechnological Advances in Industrial Polysaccharides, pp. 45-53). Utilizing this technique, depolymerized heparius exhibiting different molecular weights can be obtained. This communication reports on the biochemical and pharmacologic effects of several such depolymerized heparins to demonstrate the molecular weight dependence on biologic activity. Fragments exhibiting molecular weights of 5, 7, 8, and 9 kDa were prepared by exposing concentrated heparin solutions to a rectilinear gamma ray beam at intermittent doses of 2.5 to 25 Mrad under controlled temperatures. Unlike the chemically depolymerized heparins, these fractions did not exhibit any decrease in charge density or ATIII affinity. In routine assays for heparin, a clear cut molecular weight dependance on the anticoagulant and antiprotease actions was observed. On a gravimetric basis, these agents produce superior antithrombotic actions in comparison to chemically depolymerized derivatives. These studies suggest that gamma irradiation can be used to prepare LMWHs which retain their molecular integrity and therefore may prove to exhibit a more comparable biologic profile to hepari~ Futthermore, LMWHs produced by gamma irradiation lack the usual double bond fommtion which requires the use of additives which can alter the product profile. University Hospital, Dept. of Angiology, Frankfurt a.M., Germany Introduction: Thromboembolic disease constitutes a major clinical problem and among others a defective fibrinolytic system has been suggested as a predisposing factor for the development of thrombosis. The plasma fibrinolytic system can be impaired by inherited deficiencies of plasminogen defective release from the wessel wall tissue plasminogen activator (t-P'A) or by high ptusma levels of regulatory proteins, such as plasmino-8en. activator inhibilors (PAl). The aim ....... of the present study w~s to eshmate the prevalence of decreased fibnnolyl~c actwlty m young pLs. with thrombophilia. Patients: A great population of 884 pts. (fenmle 478, male 406; age 21-61 ys median 39.8 ys) with venous thromtx~emolism before the age of 45 years were investigated in regard to their plasma fibrinolytie system. In none of them well established thrombophilia risk factors could be identified previously. Methods: Plasminogen ~Behdngwerke), PAI-1 activity (ehromogenic assay, Biopool), PAl-I anugen coneentration (ELISA, Biopool), t-PA activity (chromogenic assay, Biopool) and antigen concentration (ELISA, Biopool) were measured before and after venous oeclusion.VO was performed z 12 month after the last thromboembolic epi~xle. 24 healthy subjects (median age 24.7 ys) served as controls. Results." 24 pts.(2.7%) were classified as plasminogen deficiencies (activity and antigen). 142 pts.(16%) had significantly elevated levels of PAl activity (up to 120 U/ml) and PAl antigen (up to 90 ng/ml). None of the pts. with high PAl levels had laboratory signs of acute phase reaction. Low t-PA activity could be demonstrated and confirmed in 121 pts., aecordingto a prevalence of 13.6% (range: 0-2.7 U/ml; reference limils: 2.8 -21.8 U/ml). However, there was a significant negative correlation between t-PA activity and PAl values. In 67 pts. (55.4%) the low t-PA activity was associated with increased PAl levels whereas the t-PA antigen concentration was normal. A parallel reduction of t-PA activity and t-PA antigen (range: 0.35-3.5 ng/ml; reference limits: 3.6 -21.0 ng/ml) were determined repeatedly in 54 pts. (F 23, M 31, median age 39 ys). Thus, the prevalence of a defective t-PA release was 6.1% in our study group. Conclusion." In comparison to the frequency of inherited deficiencies of other well established thrombophila risk factors we have observed a relativel~ high prevalence of diminished t-PA activity, elevation of PAl respectively in our study group. Our data strongly indicate that besides t-PA and PAl acuvity, antigen concentration for both parameters should be determined in pts. with thrombophilia. The antithrombotic and anticoagulant effect of the supersulfated low molecular weight heparin SSH 14 was studied after i.v. and s.c. administration in rats. Thrombus formation in the jugular vein was induced by i.v. injection of activated human serum and following stasis for 20 rain and was assessed by a thrombus score ranging from 0 (no thrombus formation) until 3 (complete thrombus formation). SSH t4 injected either 10 min (i.v.) or 30 rain (s.c.) before thrombus induction caused a dose-dependent antithrombotic effect in a range from 0.25 to 2 mg/kg i.v. and 1 to 4 mg/kg s.c. There were clear differences in the antithromboric effectiveness between female and male animals, i.e, in female rats antithrombotically effective doses were lower than in male rats (EDh0 after i.v. injection in females 0.35 mg/kg, in males 0.9 mg/kg). The sex differences were confirmed in studies on the time course of the antithrombotic effect. After i.v. injection of fully effective doses (2 mg/kg i.v. and 4 mg/kg s.c., resp.) the antithrombotic effect disappeared after 8 h in female or after 4 h in male rats. For studies on the anticoagulant action blood was drawn from the femoral artery and after centrifugation global clotting assays were performed in plasma. Similar to its antithrombotic action SSH 14 also caused doseand sex-dependent anticoagulant effects. The most sensitive assays were the APTT and the heptest; thrombin time and prothrombin time were less or not influenced by SSH 14.

In conclusion, SSH 14 was found to be an effective anticoagulant and antithrombotic agent in experimental studies in rats. At present there is no explanation for the clear sex differences found in this species. Venous thromboembolic disease is the most frequent complication in patients undergoing total knee replacement therapy. Patients and methods: After informed consent 3x30 patients were included in an open randomized clinical study and the incidence of venous thromboembolisrn was examined using different regimes for heparin prophylaxis (30 patients received fraxiparin 36 rag once daily, 30 patients clexane 40 once daily and 30 patients 7500 U Calciparin twice daily). There were no differences between the groups concerning age, sex, body weight, risk factors, surgeons, decrease in hemoglobin~ and requirements for blood products. Pre surgery, day 1, day 5-7 phIebograms were performed and also TAT, dimers, Fl+2 prothrombin fragments were examined. Results: 1., DVT in 26 patients (28.9%). DVT in 5/30 patients under calciparm prophylaxis, 8/30 patients under fraxiparin and 13/30 patients under clexane treatment. 2., Low speciflty (3.4%) of dimers and TAT (24%) for detecting a DVT in these special patients undergoing knee replacement therapy, elevated FI+2 fragments in the DVT group at TI and T2 vs the patients without DVT (T1 DVT: 3.24+-1.8 vs. 1.6+-0.3 -p= 0.0042). 3, Only 8/26 patients (31%) with DVT had clinical signs of thrombosis. Conclusions: 1., There is an increase of thrombin gneeration measured by TAT and dimers after knee replacement therapy. There are further studies with more patients necessary to confirm that Fl+2 prothrombin fragments can discriminate between patients with and without DVT from a clinician's point of view. 2., PhlebographicaUy confimled DVT in almost 30% of our patients demonstrate the high thromboembolic risk in these patients. Von Willebrand's disease (VWD) type 2 is characterized by absence of high molecular weight muitimers. Qualitative changes in the structure of the molecule might be associated with enhanced binding of von Willebrand factor (VWF) to platelet glycoprotein lb. Therefore in some patients VWD type 2 is associated with severe thrombocytopenia. Here, we report on a 9 year old boy who presented with severe purpura and platelet counts about 20000/gl at the age of 2 years. Thrombocytopenia did not respond to corticosteroids. A normalized platelet count of short duration was observed after high-dose immunoglobulins. In addition, increase of platelets was seen after anti-D treatment. Thus, although platelet associated antibodies were not detected, thrombocytopenia seemed to be caused by an autoimmune mechanism. Despite platelet counts above 50000/gl, the patient experienced severe bleedings with a significant decrease of hemoglobin levels. Therefore, he needed several transfusions. Coagulation analysis revealed VWD. Application of DDAVP lead to a normalization of partial thromboplastin time (PTT) and an increase of factor VIII with subsequent cessation of bleeding symptoms. Recently, VWD was typed 2 by lack of high molecular weight multimers. In conclusion, we report a case with VWD type 2 responding to DDAVP. However it is unclear, whether thrombocytopenia is part of the VWD type 2 or of autoimmune origin. Since autoimmune antibodies have not been detected, the effect of immunoglobulin treatment might be explained by blockade of enhanced binding of VWF to glycoprotein lb. Von Willebrad disease (vWd) with a prevalence of 0,8% (Ruggeri 1994, Rodeignere 1987) seems to be the most frequent inherited hemostatic disorder. • The diagnostic criteria for vWd are clinical picture, family hostory, laboratory findings: bleeding time, partial tromboplastine time (PTT), level of factor VIII:e, vWF, vWF:Ag, ristocetin induced platelets aggregation (RIPA) and multim~-analysis.The diagnosis ofvWd is occasionally difficult, especially in early childhood because the laboratory data may vary due to time of investigation, as well as abnormalities may not be present in all sub-types The aim of this study was the evaluation of diagnostic approach to vWd in childhood and diagnostic reliability of all available laboratory tests. All previously mentioned laboratory tests have been done on our own material (51 child who satisfied all criteria for vWd, 23 boys and 28 girls, 1-9 years old) except mulfimer analysis which was unavailable in some cases. Majority of laboratory tests proved to be highly specific and necessary for diagnosis. However, the diagnostic reliability of FVIII:c and adhesion of platelets is much lower in mild cases in comparison to total sample, while PTT is an unvaiied test. The most specific screening test for vWd is vWF which diagnostic reliability is almost 1,00. The optimal strategy to establish general diagnosis of mild forms ofvWd is use of vWF and vWF:Ag plus RIPA if necessary and multimer analysis to classify variant types. We report on a new multimeric structural defect of vWF detected in a German family (two sisters and their three children):

All members of the family who presented to our outpatient clinic had an increased spontanous bleeding tendency (moderate or strong hematoma, epistaxis, menorrhagia). Prolonged bleeding could be observed after surgical procedures (adenotomia, tooth extraction) and after trauma (laceration). Wound heeling was impaired in two cases.

Clotting assays showed slightly prolonged aPTI" and a mild decrease of F VIII:C, vWF:AG and vWF:RCof levels. Collagen binding activity was within normal ranges. Bleeding time (Simplate I) was slightly prolonged. The analysis of the multimeric structure in plasma showed quantitative and qualitative abnormalities: All multimers were detectable; the structure of vWF was reproducably abnormal in all family members so that the defect must be caused genetically.

The thmmbocytic vWF showed neither qualitative nor quantitative alterations.

Minirin@ (DDAVP) was administered as a test dose of 0,3 ~tg/kg BW in 100 ml 0,9% NaCL-solution i.v. to evaluate efficacy and tolerance: Clotting assays showed normalization of a_VrT, F VIII:C, vWF:AG, vWF:RCof in plasma and shortening of bleeding time in three cases. An insufficient rise of vWF:AG and vWF:RCof levels could be observed in one case. One patient had no rise of F Vm:C but a corrected bleeding time.

Multimeric analysis showed no structural change.

The administration of DDAVP was well tolcrated in all cases.

The existance of all multimers in plasma and the normal collagen binding activity suggest that the structural abnormalities of vWF in this family does not cause functional defects so that the defect could be classified as a type I vWD.

The response to DDAVP was only partially effective. Mild von Willebrand disease (vWd) is far the most frequent congenital bleeding tendency. Its diagnosis is very helpful in pre-operative check-up in order to avoid bleeding complications during surgery. Following post-operative periods or monitoring the management of haemorrhagic episodes in vWd patients is also strongly recommended. Current methods involve complex technologies, are time consuming and require large series. These assays lack the expected flexibility for rapid individual testing in patients. A new and flexible assay which works on the fully automatic walk-away coagulation instrument, STA, has been developed for these applications (Liatest vWF). The technology is an immuno-turbidimetric method using mierolatex particles coated with rabbit polyelonal antibodies specific for vWF. The assay has a dynamic range from 2 to 420% yon Willebrand factor (vWF) concentration, it works with a 2 fold dilution of tested plasma (50 td) and it offers a calibration established with the NIBSC international standard. The total assay time is of less than 10 minutes and the detection threshold is of 2% There is no prozone effect up to concentrations higher than 1,000% vWF. Intra-assay reproducibility is < 4% and inter-assay one < 5%. In dilution studies a mean recovery of 98% was obtained. In a study on 55 plasma samples from norma~ individuals, patients with high vWF concentrations, and vWd, comparison with the ELISA technique demonstrated a correlation coefficient of 0.997 with a slope of 0.978 and an intercept of 3.30. In the low assay range too, a good agreement was obtained with the ELISA. We conclude that Liatest vWF is a reliable, flexible, sensitive, and rapid automated assay which fits well the vW'F assay applications in coagulation laboratories. Fibrinolysis, the process during which the active enzyme plasmin is generated in a regulated and localised way, is -in a classical understanding -responsible for the dissolution of blood clots formed in a vessel. For this activity, t-PA is generally assumed to be the most important plasminogen activator and its activity, is regulated by enzyme kinetic mechanisms dependent on the presence of fibrin. With this background t-PA is used for thrembolytic therapy with great success. However, data from t-PA knockout mice indicate that t-PA might not be responsible for inhibiting the spontaneous development of intravsacular thrombi but only for dissolution of fibrin formed upon a coagulation challenge. In contrast, u-PA, generally assumed to be important for extravascular proteolytie activity on activated or tumour cells, seems to lead to the development of spontaneous fibrin formation in a mouse knockout model. On the other hand, the major plasminogea activator inhibitor PAl-I seems not only to regulate intravascular fibrinolysis but seems to also be important for the progression of vascular diseases (neointima formation is e.g. increased in a PAI-1 knockout model, but increased levels of PAI-1 seem to predict reocclusion after angioplasty). In addition to their functioning as enzymes and inhibitors, components of the fibrinolytic system seem also to be involved in signalling processes in tumour and other cells. The u-PA/u-PA-receptor system could be shown to function as a chemotactic system and to elicit a migratory and mitogenle response in monoeytes and tumour ceils as well as in vascular cells. For such a response activation of tyrosine kinases of the sre-family might be responsible in some cell lines, but other signal transduction pathways e.g. involving caveolae and the starprotein can not be excluded. There seems to be a further important role of components of the fibrinolytic system which involves serine protease inhibitors (SERPINs): SERPINs have homologies to hormone binding proteins and cleavage of SERPINs by their target enzymes not only leads to inactivation of the enzyme but also to a possible release of bound hormones from the SERPINs. From these data clearly the relevance of any regulation of the fibrinolytie, system depends on the specific function of the system to be dealt with. In addition to "fibrin binding", "receptor mediated" and "genetic control" (e.g. 4G vs. 5G in the PAI-I promotor) also "signal transduction" and "hormone delivery" are distinct functions of the system with specific regulation.

PLASMATIC For both, healthy persons as well as for patients with angina pectoris it could be shown that increased values of plasma fibrinogen, factor VIIc and vWF:ag are significantly associated with the risk to suffer an acute myocardial infarction or cardiac sudden death. The same holds for tPA:ag. However, a group analysis in quintiles reveals that particularly low tPA:ag values are connected with a particularly low coronary risk. Unexpectedly also the acute phase protein CRP is positively associated with increased coronary risk. For clinical purposes these factors have already been included into coronary risk scores in order to improve the individual risk prediction in combination with lipids and other risk factors. The assessment of the pathophysiological significance of these observations remains at dispute. 4 pathways are discussed: 1. The assumption that increased plasma values of those factors indicate increased coagulation activity could so far not be established in prospective studies. 2. Both vWF:ag and tPA:ag are produced in endothelial cells. An increase of their plasma level could therefore indicate increased endothelial cell functions which accompanies progressive atheromatosis. The risk association of the two acute phase proteins CRP and fibrinogen could be interpreted analogously. 3. First prospective studies favour the assumption of a genetic determination to an increased production of coagulation proteins in persons at particular coronary risk. It could also be shown that there is a certain dependance of the gene-polymorphism for co-and 13-fibrinogen chains from the coronary risk. 4. Even slightly elevated concentrations of fibrinogen and/or vWF:ag may influence the quality of a coronary thrombus both by increased physical stability and by reduced fibrinolytic lysibility. This could mean that an early coronary clot under these conditions could more readily develop to a stable, Occlusive thrombus. A newborn with pronounced bleeding tendency had a prothrombin (prth) deficiency below 2.8% in a clotting assay. Both parents had activities of 71% and 69%, respectively. However, the immunological determination ofprth by ELISA revealed normal concentrations in all family members (93%-101%). Furthermore, thrombin generation as investigated by a chromogenic assay using ecarin for activation of prth was normal as well. Activation of prth by FXa was investigated by reealcificafion of the plasma samples and further analyzed for prth and its derivatives produced. Although clotting times still were different, finally, normal levels of Fl+2 and TAT were generated as determined by ELISA. Western blot analysis using polyclonal (rabbit) antibodies to prth and a monclonal antibody specific to human thrombin, revealed different patterns of prth degradation products. TAT was only weakly visible in the serum of the mother and nearly absent in the child.The mobility of prothrombin and thrombin was different compared to normals indicating a lower molecular weight. After reduction of disulfide bridges a higher molecular weight of thrombin was observed compared to normals indicating an insufficient cleavage ofprth and formation ofprethrombin 2. These observations let suggest that prothrombin Marburg is a deletion mutant lacking the cleavage region Arg 320-Ile321. Upon cleavage by factor Xa only prethrombin 2 is formed under liberation of Fl+2. This prethrombin 2 is able to cleave chromogenic substrates in the ecarin assay. Probably, prethrombin 2 forms a complex with ATIII which is detected by ELISA, but unstable under denaturing conditions as in the Western blot. As a major complication of haemophilia A treatment, up to 30% of the severely affected patients develop antibodies to substituted factor VIII. Investigating 133 patients and considering the data of further 231 patients of the haemophilia database, we could show, that risk of inhibitor developement depends on the patient's mutation type. Patients with more severe gene defects, like intron 22 inversions, stop mutations or large deletions had a risk of about 35% for inhibitor developement, which was about 7 times higher than for missense mutations or small deletions. Besides an influence of mutation type, we investigated other parameters e. g. immune response genes (I-ILA-genotype) and clinical aspects (treatment onset and frequency, type of concentrate) that might also affect inhibitor formation. To exclude any effect of mutation type, we focussed on 72 patients with an intron 22 inversion. HLA-typing showed that some t-ILA-alleles (DQB0602, BT) occurred more otten and others (DQA103, DQB603, DR13, C2) less frequent in inhibitor patients. Treatment onset, frequency and type of concentrate apparently do not affect inhibitor incidence. The results presented here, prove that inhibitor development is considerably influenced by the mutation type. This supports the hypothesis that patients with severe molecular defects have no endogenous factor VIII protein and that substituted factor VIII represents a foreign protein, leading to an immune response, e. g. the production of alloantibodies. In addition, the immune response seems to be modified by the HLA-genotype. However oar findings (in terms of genotype and treatment parameters) can only explain part of the inhibitor pathogenesis. It is still unsolved why substituted factor VIII does not lead to a recognizable immune response in 2/3 of the patients with severe molecular factor VIII gene defects. Consequently other factors, probably concerning the antenatal phase, must be involved. ViIa IN THE TREATMENT  OF PATIENTS WITH INHIBITORS AGAINST FACTOR VIII OR IX:  A GERMAN/SWISS/AUSTRIAN MULTI~CENTER TRIAL  D. Ellbriiek*, I. Scharrer**, J. Dethling***, and the rFVIIa Study Group *Section H~mostaseology, University Ulm **Dept. of Angiology, JWG-University Hospital Frankfurt a.M. ***Novo Nordisk, Mainz Administration of activated recombinant Factor VII (rFVIIa) can by-pass the FVnlWlX pathway and offers an alternative treatment for patients with antibodies (inhibitors) against these factors. From November 1994 to October 1995, a total of 25 bleeding episodes and 10 surgical interventions in 18 patients were treated with rFVIIa in a phase IIIb multicenter trial. Diagnosis was hemophilia A (n = 15) or B (n=l) with inhibitor, and acquired inhibitor against Factor VIII (n=2). Various serious bleeds, from complicated joint and gingival bleeds to lifethreatening psoas bleeds, have been treated. Operations have been tooth extractions, radiosynovectomy, implantation and explantadon of porth-acaths and one adenotomy. Dose regimen was 90-120/zg/kg BW every two to three hours until clinical improvement, with subsequent dose reduction. Results: For bleeding episodes, response to rFVIIa after 24 hours was effective in 72%, partially effective in 12 9"0, ineffective in 129"o and not evaluable in 1 (4%) of the patients. Two of the three treatment failures were associated with very long dosage intervals of rFVIIa. The third patient was in a critical situation with artificial high pressure respiration and polytransfusion because of a hematothorax, and suffered a terminal intracerebral bleed. The efficacy of rFVIIa for surgery was very good. Response to treatment was independent of antibody titer. No signs of DIC or activation of coagulation were noted. Conduslon: In our experience, rFVIIa is an efficient and safe treatment for inhibitor patients with acute bleeding episodes. It should be investigated, whether rFVIIa can be an alternative treatment also for the hometreatment situation.

SUCCESSFUL IMMUNETOLERANCE THERAPY OF F VIH-INHIBITOR IN CHILDREN AFTER CHANGING FROM HIGH TO INTERMEDIATE PURITY F VIH CONCENTRATE W. Kreuz, J. Joseph-$teiner, D. Mentzer, G. Auerswald*, T. Beeg, S. Becker Zentrum der Kinderheilkunde, J. W. Goethe-Universit~itj Frankfurt am Main *Professor Hess Kinderklinik Bremen Introduction: Inhibitor to F VIII is the most severe complication in treatment of patients with haemophilia A. The incidence of F VIII inhibitors is estimated to range between 15-33%. Several authors reported that the immunetolerance therapy (ITr) of F VIII-inhibitors can be induced with high dose F VIII concentrate. Objective: This presentation will show data of four children with haemophilia A and F VIII inhibitor (high responder), who had an unsuccessful lit with high dose F VIII concentrate (high purity) in the first step. F VIII concentrate was changed to an intermediate purity product (Haemate HS®) in the subsequent course of H'T. All patients received bleeding prophylaxis with an activated-prothrombin-complex-concentrate (FEIBA®). Results: Median age was 13 (9-18) months, when the inhibitor was first detected. In all four patients the F VIII inhibitor titre increased under immunetolerance treatment with F VIII concentrate (high purity) in the first step of therapy. After changing the F VIII concentrate (intermediate purity) the inhibitor titres decreased continuously after a rebooster effect to 0BE within months. Median duration of F VIII inhibitor elimination time (until first testing of 0 BE) was 3 (2-5) months. In all patients the F VIII inhibitor was successfully eliminated. Until now all patients are under prophylactic treatment with F VIII concentrate and had no positive inhibitor testing since. Median observation time since the first testing of 0 BE is 14 (4-60) months. Conclusion: Different studies concerning immunetolerance treatment have been successful with F VIII concentrates of different purity. According to our experience in these four presented patients, we assume that probably not the purity of the F VIII concentrate is important for the induction of immunetoleranee, rather than the type of F VIII presentation in the used concentrate. The used preparation (Haemate HS®) is a F VIII concentrate with high concentration of vWF, which is known to be important for the protection of F VIII against degradation by proteases. This may be a mechanism for a prolonged antigen presentation to the immunesystem and thus may have a positive impact on the outcome ot ITr. Long scale trials are needed to prove the above assumptions. Thrombasthenia Glanzmann is a disease affecting platelet function because of a partial or total lack of glycoprotein (GP) Ilbllla expression or a modification of this complex. Since the receptor dysfunction goes along with reduced or absent platelet aggregation and adhesion, it causes bleeding complications in case of injury.

Here we report about a 60 years old women, who suffered since early childhood from a severe bleeding disorder. Life threating bleeding complications occured after tooth extraction and after abdominal surgery. Analysis of the patients platelets revealed normal values for the platelet count, whereas their volume showed to be increased (11 fl). Clot retraction was diminished to 17%. Platelet adhesion to siliconised glass and human subendothelial matrix was reduced, as was the spreading of the platelets. ADP (I#M) induced platelet aggregation was inhibited, while collagen-, ristocetin-and thrombin-induced aggregation showed to be normal. Cross immunelectrophoresis resulted in an atypical peak of GPIIbllla with reduced electrophoretic mobility. In the electroimmunoassay according to Laurell 14% of GPIIbllla was detected. Moreover we observed a markedly diminished 125j-fibrinogen binding. Sequence analysis of the GPIIb and GPIIla cDNA after PCR amplification unraveled a G2508 --, A transition in GPIIb, substituting Gly805 --* Glu. The structure/function relationship of this mutation has still to be investigated. We report two new abnormal fibrinogen variants, denoted as Bem IV and Milano XI, both having an exchange of arginine to histidine in position 16 of the Ac~-chain. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times, low plasma fibrinogen concentrations determined by a functional assay but normal fibrinogen levels measured by the immunological assay. The onset of turbidity increase following addition ofthrombin to purified fibrinogen was markedly delayed in both variants. Release of fibrinopeptide B by thrombin, measured by reversed phase HPLC, was normal whereas only one half amount of normal fibrinopeptide A was released. In addition to normal fibrinopeptide A, an abnormal fibrinopeptide A* was cleaved from both dysfunctional fibrinogens. The structural defect was determined by asymmetric PCR and direct sequencing of a gene fragment coding for the NH2-terminus of the Aachain. Both variants were found to be heterozygous for the transition G to A at nucleotide position 1203, leading to the substitution Actl6 Arg-->His, resulting in a delayed fibrin polymerization. The simple assay permits detection of the most common amino acid substitutions occuring in the NH2-terminus of the Ac~-chain of the functionally abnormal fibrinogen variants. Protein C inhibitor (PCI) a member of the serpin family is also known as plasminogen activator-3 (PAl-3). PCI was first described as a component of human plasma, regulating the activity of activated protein C and other sedne proteases of the human coagulation and fibdnolysis system. Since then PCI was found to be present in extra-plasmatic systems also. High concentrations of PCI were detected in human seminal plasma suggesting a role for PCI in human fertility. Significant concentrations of PCI mRNA and antigen were located in lysosomes of proximal tubular kidney cells suggesting an intracellular function for PCI in this environment. In this study we present evidence that PCI is also present in human pancreas. RNA from human pancreas was reverse transcribed and PCR amplified. The resulting PCI cDNA was identical with PCI cDNA from human liver. ~P labeled antisense RNA probes used in in situ hybridization experiments with human pancreas tissue sections showed that PCI RNA was located in the acinar ceils. Pancreatic fluid was analyzed by SDS-PAGE and immunoblotting. Using monospecific antibodies directed against human plasma PCI, a 57 000 MW protein band was observed which comigrated with purified human plasma PCI. Our results show that pancreas cells contain a significant concentration of PCI mRNA. This message is localized in the secretory acinar cells. Therefore we conclude that PCI antigen found in pancreatic fluid is likely to originate in the pancreas. The role of pancreatic PCI is unknown at present. However, since thrombosis and systemic hypercoagulable states are known complications of pancreatic diseases our results and in vitro experiments by others showing that PCI can inhibit pancreatic enzymes such as chymotrypsin and trypsin indicate that PCI may be part of the inhibitor potential which protects pancreatic tissue from auto degradation. These inhibitors normally prevent the release of active pancreatic proteases into the vasculature or microcirculation where destabilization of the coagulation balance and subsequent thrombus formation could occur. Institute for Clinical Chemistry and Laboratory Diagnostics and *Clinic for Cardiology, Universi W of Duesseldorf P-selectin (CD 62p, the former granule membrane protein 140 or GMP 140) is an integrated membrane protein of platelets and endothelial cells. Under inactivated conditions it is stored in the alpha granules of platelets and in the WeibeI-Palade bodies of endothelial cells. Endothelial cells covering atherosclerotic plaques show an increased expression of P-selectin. 13-thromboglobulin (13-TG), which is also expressed from the alpha granules of platelets during adhesion or aggregation, is regarded as a marker of platelet activation in vivo. Coronary thrombosis plays a central role in the pathogenesis of acute coronary syndromes. We therefore analysed CD 62p and 13-TG in acute coronary syndromes, healthy subjects (HS, n=l I), patients with stable angina pectoris (sAP, n=20), unstable angina pectoris (uAP, n=l 2) and acute myocardial infarction (AMI, n= 12). Plasma samples were obtained by using CTAD Vacutainer tubes (0.109 M Na~-citrate, theophylline, adenosine dipyridamole).

Patients with CAD showed significantly increased plasma concentrations of CD 62p (HS: 98+20 versus sAP: 133+38ng/ml, p<0.05; versus uAP: 128+28 ng/ml, p<0.01; versus AMI: 144+72 ng/ml, p<0.05) independent of the severity of clinical symptoms. In comparison only patients with AMI showed significant higher 8-TG concentrations compared with HS (HS: 30+20 versus AMI: 39+14ng/ml, p<0.05). Although the CD 62p plasma concentrations showed no relationship to the clinical severity, hence there was a positive correlation between CD 62p (r=0.47; p< 0.001; n=55) to the severity of CAD classified as I, 2, 3 vessel disease.

It is concluded that elevated CD 62p concentrations are correlated with the severity of cardiovascular disease. CD 62p is not suitable for differential diagnosis of acute coronary syndromes, because it is elevated independently of the clinical status of the patients. The involvement of platelets in the pathogenesis of acute myocardial infarction may be indicated by the increased 13-TG concentrations. IKlinik Nr Herz-, Thorax-und herznahe Gef&Schirurgie und 2Institut x~tr Klinische Chemie und Laberatodumsmedizin der UniversiNt Regensburg

An increased blood loss following surgery with extracorporeal circulation (ECC) contributes to the morbidity and mortality. Postoperative haemorrhage following ECC has been related to a platelet function defect and the activation of the blood dotting and fibrinulytic system. We investigated platelet surface antigen expression and parameters indicating activation of the clotting and fibrinolytic cascade to assess the predictive potential of these variables for increased blood loss after ECC. g0 patients referred for coronary bypass gra~ing with no history of a bleeding disorder and normal routine clotting tests were included. On the day prior to surge~ and immediately upon arrival on the intensive care unit blood samples were drawn. The surface expression of glycoprotein (GP) lib-Ilia, GP lb, and P-selectin was meamred with and without in vitro stimulation with adenosine diphosphate (ADP) using whole blood flow cytomet~y. Platelet counts and platelet factor 4 (PF4), as well as, routine clotting tests were performed. Activation of the clotting and fibrinolytic system were judged from thrombin-antithrombin-III complex fiAT), fibrinogen fiG), D-dimers (DD), cc2-antiplasmin (tz2A), prothrombin fragment 1+2 (Fl+2),and tissue plasm~ activator (t-PA). Blood loss fxom chest tubes was measured hourly until removal of drains. Following ECC the levels of PF4, TAT, DD, o~2A, Fl+2, and DD were sigulticnatly increased (p<0.0001) compared to baseline values. GP IIb-IIla, GP Ib, P-selectin, platelet count, and FG were significantly reduced (p<0.0001). Analysis of variance (ANOVA) revealed that postoperative values of GP Ib (p<0.0001), DD (p<O.05), FG (p<0.01),and platelet count (10<0.05) had a significant influence on blood loss. None of the preoperative data was significantly associated with increased blood loss. A subgroup of patients who bled more than the mean + 1 SD (14 cases) was formed_ In logistic regression analysis GP Ib expression (p=0.01) and DD (p=0.007) were of value in identifying bleeders. The sensitivity of the equation was 56%, the stx~ificity 97% and the positive predictive value 82%. Although the reasons for an increased blood loss following ECC are multifactorial, we were able to identify a small number parameters with a significant infl~nco. The combination of the expression of yon Willebrand receptor on the platelet su_rfaco and D-dimer levels in plasma was successful in predicling an increased blood loss following heart surgery. This may be helpful to define indications for platelet transfusion and flesh frozen plasma.

The most widely distributed receptor for the Fc-region of IgG is the Fcreceptor IIa (FcyRIIa). For this receptor a functionally relevant polymorphism of amino acids ArgmHis is known. We previously showed HIT-antibodies to bind to platelets via FeyRIIa and now whether clinical manifestation of HIT is associated with the Fe'/RIIa polymorphism. Therefore, we developed a rapid two step allelle-specific PCR (ASP) method for genotyping the FoyRIIa. With control samples the ASP was compared with the time consuming and complicated allele-specific oligonucleofide hybridization technique, the phenotype expression of FcTRIIa (using the moabs IV.3 and 41H16) and the monocyte stimulation assay. Complete concordance between the assays was found. We used the ASP to determine the allelic distribution of the Inst. Physiol. Chem., Marburg; *)Univ. of Virginia, Charlottesville, The functional heterogeneity of blood platelets is clinically significant and may contribute to the pathogenesis of cardiovascular and atherosclerotic diseases l). In order to understand the biochemical basis of the functional heterogeneity of platelets, we have investigated the signal transducing pathway of platelet activation a) in terms of guanylate cyclase and phosphodiesterase activities and b) at the level of protein phosphorylation and the receptor-effector coupling via G i-and Gs-proteins in functional heterogeneous blood platelet subpopulations separated according to their densities. Furthermore, we analyzed the ADP-ribosylation of RhoA, which is a lowmolecular weight GTP-binding protein and is thought to be involved in the platelet "shape change" reaction and aggregation. Aggregation was enhanced in low-density platelets (SP I) and less inhibited when cGMP was increased by NO, compared to high-density platelets (SPIII). SP I possessed a lower basal level of cGMP and exhibited a smaller increase in cGMP after stimulation with NO. Cyclic AMP-dependent phosphodiesterase activity was higher in SP I compared to SPIII, whereas cGMP-dependent phosphodiesterase activity was similar in SP I and SP III. SP I, when compared to SP III platelets, showed a higher degree of prephosphorylation of proteins in the range of 47, 32-34, and 20-22 kDa. After thrombin stimulation, the phosphorylation of these proteins was similar in the subpopulations. Pertussis toxin,induced ADP-ribosylation of the 40-41 kDa Gi-protein was highest in SP I, whereas Cholera toxin induced ADPribosylation of the Gs-protein and botulinum C3 exoenzyme-induced ADPfibosylation of RhoA were highest in SP III. The findings suggest that a) the higher aggregability in SP I compared to SP III may be caused by a poorer ability of guanylate cyclase to form cGMP and a higher activitiy of cAMPdependent phosphodiesterase and that b) the ADP-ribosylation of G-protein subunits and RheA may contribute to the functional heterogeneity in the subpopulation leading to the greater ability of SP I for aggregation. 1) Opper et al., Atherosclerosis, 113, 211-217, 1995. Causes of accelerated atherogenesis in non-insulin-dependent diabetes mellitus (NIDDM), known to be associated with increased plasma activity of plasminogen activator inhibitor type 1 (PAl-l) as well as with increased plasma concentrations of proinsulin and insulin, remain enigmatic. This study was designed to determine whether proinsulin and insulin increase PAl-1 in vivo thereby potentially accelerating atherogenesis. To simulate hyper(pro)insulinemia seen with NIDDM, we infused equimolar concentrations of insulin (1 IU/kg), proinsulin, C-peptide, or placebo, all endotoxin-free, i.v. over 1 h in 53 conscious rabbits maintained euglycemic. Plasma PAl-1 did not increase with placebo (n=16) or C-peptide (n=4), but did with proinsulin and insulin peaking in 3 h at 3.5-and 3.7-fold (n=16 and n=17, p<0.O02 for each) in parallel with PAl-1 protein quantified by reverse fibrin autography. The increases were specific as reflected by unchanged activities of o~-antiplasmin and t-PA. PAt-1 gene expression (PAl-1 mRNA) increased with proinsulin and insulin by 2.1-and 2.1-fold in 3 h in aorta, 1.9-and 2.4-fold in liver (p<0.05 for each), but not in heart, lung, spleen, or kidney and returned to baseline in 24 h (n=4 each). In situ hybridization localized the increased PAl-1 mRNA in aorta to endothelium (n=3 each). Thus, both proinsulin and insulin directly augment gene expression of PAl-1 in arterial endothelium in vivo, thereby increasing plasma PAl-1 activity, attenuating endogenous fibrinolysis, and potentially accelerating atherogenesis. The sarco-endoplasmic-retikulum Ca+÷ATPases (SERCA) that sequester calcium into intracellular stores, represent the most important mechanism to maintain cytosolic calcium levels low and to return to a resting level after activation. Recent studies in platelets and rat endothelial cells have identified two forms belonging to the SERCA 2b and SERCA 3 types. The monoclonal antibody PL/IM 430 raised to human platelet intracellular membranes has been shown to recognise the SERCA 3 ÷+ isoform and to inhibit Ca sequestration in human platelets.

The aim of this study was to identify Ca ATPase distribution in human umbilical vein endothelial cells (HUVEC) and human polymorphonuclear leukocytes (PMNL). Polyclonal antibodies with known specificity towards SERCA 2b (R 2/88) and SERCA 3 (89/3) in addition to PL/IM 430 were used in histochemical studies of permeabilised cells and in Western blotting experiments using cell lysates and mixed membranes. Using the alkaline phosphatase anti-alkaline phosphatase (APAAP) method R 2/88 and 89/3 reacted strongly with proteins in permeabilised HUVEC and PMNL, however PL/IM 430 showed good staining only with PMNL. In Western blotting experiments R 2/88 recognised proteins with molecular sizes of 100 kDa (known to be the parent Ca++ATPase) and 70 kDa (which may represent antibody recognised degradation product), in both HUVEC and PMNL. 89/3 ÷+ recognised bands at 100 kDa (parent Ca ATPase), 74 kDa and 46 kDa in both HUVEC and PMNL membranes. In agreement with histochemical results PL/IM 430 recognised proteins (100, 74 and 46 45 + + kDa) only in PMNL membranes. In Ca transport studies PL/IM 430 ÷+ inhibited Ca uptake in membranes prepared from PMNL but not in HUVEC. The distinct reaction of PL/IM 430 with PMNL and HUVEC suggests the possible presence of distinct sub-isoforms of SERCA 3 with the PMNL and platelet proteins being similar and different to the SERCA 3 isoform present in HUVEC. Microvascular endothelial cell (EC) lines with a limited life span of 2-3 months in vitro were established from human bone marrow, skin or solid tumor tissue. Homogeneity of the lines was verified by flow cytometric analysis and cytokine expression patterns. The cultured EC constitutively expressed HLA-class I antigens, CD44, procollagen type I, CD62E, CD34, and receptors for granulocyte colony stimulating factor (G-CSF). Endothelial cells constitutively produced IL-6 and IL-8 as well as TPA and PAI-I as determined by ELISA. In the presence of 5 IU/ml of exogeneous IL-la, G-CSF was secreted at high amounts (lng/ml) and GM-CSF at low amounts (15pg/ml) in confluent monolayers and during over night incubation. These EC potently bound to isolated neutrophilic granulocytes. When granulocytes were stimulated by either 10~ 4 or 10 .7 M formyl-methionyl-leucyl-phenylanaline (fMLP) to produce oxygen radicals, the preincubation with a single cell suspension of EC caused oxygen radical scavenging. A ratio of 1 : 100 EC to neutrophilic granulocytes resulted in a 50 __+ 10% inhibition of fMLP-induced and luminol-enhanced chemiluminescence in vitro. This effect was probably due to the constitutive nitrogen oxide (NO) production by EC. Unexpectedly, preincubation of EC with 50 IU/ml of interferon-y (IFN-y) to increase NO-secretion did not alter the extent of the oxygen scavenging effect measured at different ratios of EC to neutrophilic granulocytes. According to the surface antigen expression pattern, identical IFN-F treatment upregulated adhesion receptors on EC. Concomitantly, binding of neutrophils to EC was significantly increased. Thus, the net oxygen-scavenging effect by EC appears to be stable under the condition of IFN-y stimulation which indicates maintenance of EC function and effective neutralizition of non-specific oxygen radical damage. Moreover, the established test system can be useful to investigate the selective contribution of other inflammatory cytokines, adhesion molecules, macrophages and platelets on EC function in vitro. Atherosclerosis is characterized by intimal migration and proliferation of smooth muscle cells (SMCs), by macrophage infiltration and extracellular matrix remodeling. Circumstantial evidence suggests that urokinasetype plasminogen activator (UPA) may be relevant in these processes. This study examines UPA expression and UPA antigen localization in human coronary arteries with ,carious degrees of atherosclerotic lesions. Representative segments of macroscopically normal areas (MNAs), early lesions (ELs) and fibrous plaques (FPs) were processed in parallel for in situ hybddization and immunohistochemical analysis.

In MNAs, UPA was detected both in the luminal endothelium and in celocalization with ~-actin-positive SMCs throughout the tunica media. In ELs, UPA mRNA and antigen were substantially reduced in the luminal endothelium. However, cellular UPA expression was detected in the subendothelial area in colocalization with tissue-infiltrating CD68positive macrophages. In the deeper intimal and in the medial layers strong UPA expression was observed in association with ~-actinpositive SMCs, with most of the UPA-positive cells being located in areas with a high proliferative activity as detected by PCNA staining. In FPs, UPA mRNA was widely expressed in macrophage-containing areas within the fibrous cap and at the periphery of the necrotic core of the lesions. In addition, UPA antigen was identified in acellular areas of the necrotic core regions. Compared to the diseased intima of these lesions, the adjacent media stained only weakly for UPA. In conclusion, this study demonstrates an enhanced expression and synthesis of UPA by intimal SMCs and macrophages in advanced atherosclerotic lesions of human coronary artedes These findings suggest a role for UPA in the progression of the coronary atherosclerosis. Adenosine produced by vascular cells, platelets and other tissues has been proposed to be a vasodilator, inhibits platelet aggregation, stimulates proliferation and migration of endothelial cells as well as exhibits antiinflammatory effects. Adenosine of endothelial origin intra-and extracellularly formed and highly concentrated at the luminal surface may constitute an important antiaggregatory mechanism, which is part of the well-known antithrombogenicity of an intact endothelial lining. It was the aim of this study to investigate whether adenosine could influence the fibfinolytic potential of endothelial cells. When confluent human umbilical vein endothelial cells were incubated with adenosine (0.01-10mM) a significant dose dependent decrease in plasminogen activator inhibitor type-1 (PAI-I) antigen production by this cells was seen as compared to control. This effect by adenosine was also time dependent and seen after 2 hours of incubation with adenosine. As determined by ELISA, PAI-I antigen in conditioned media of endothelial cells incubated with 10mM adenosine decreased to 57% as compared to control. PAI-I antigen in extracelullar matrix of these cells was also reduced by incubation with adenosine. These results were also reflected on the level of specific mRNA as determined by Northern blotting. PAI-I mRNA decreased in endothelial cells after incubation for 4 hours with adenosine (10mM) to 77% (2.2Kb) and 85% (3.2Kb) of control. In conclusion our data demonstrate that adenosine can downregulate the expression of PAI-1 in cultured endothelial cells thereby increasing the profibrinolytic capacity of these cells. This effect of adenosine might contribute to its antithrombotic potential in addtion to its known antiaggregatory effect. Sowohl die Effizienz als such die Komplikationen einer Antikoagulantien-Dauerbehandlung sind in erster Linie yon der Gore und Engmaschigkeit der Gednnungskontrellen abh~ngig Seit 1986 wird die Schulung zur Gerinnungs-Selbstkontrolte in der Herz-Kreistauf-Klinik Bad Bedebarg in Gruppen eingeObt. Die theoretische und praktische Anleitung erfolgt in 8 Stunden durch eine in der Patientenschuiung erfahrene Arzthelfedn (oder Krankenschwested-pfleger) und einen Arzt. Die praktische Schulung wird durch einen patientennahen theoretischen Tell erg~nzt. Verglichen mit konventionell ~berwachten oral antikoagulierten Patienten (ca. 50% der TPZ-Werte liegen im theapeutischen Bereich) erreichten 216 Quickwert-Selbstbestimmer, daf$ 83,1% yon 14 812 TPZ-Werten im therapeutischen Bereich lagen (Tabelle 1 ). Schwerwiegende thromboernbolische Ereignisse oder Blutungskomplikatienen wurden nicht beobachtet. Durchschnittlich kontrolliert der Quickwert-Selbstbestimmer seine Gerineungswerte w6chentlich, Itmgstens im Zweiwochen-Abstand. Ob sich Blutungs-und Thremboembolierisiko langfristig senken lassen bei gleichze~iger Verbesserung yon Prognose und Lebensqualit~it, bedarf des Nachweises im Rahmen einer randomisierten, kontrollierten und prospektiven Studie. Tabelle I Verteilun~l der selbstbestimrnten Gednnur ~swerte Zeitraum 1986 Zeitraum -1988 Zeitraum 1986 Zeitraum -1990 Zeitraum 1986 Zeitraum -1992 To evaluate the potential benefit of an optimized oral anticoagulation management with INR measurements performed by the patients themselves, two prospective randomized studies have been completed. The first study (n=60) was performed to evaluate the accuracy of INR self-testing (INR-ST) compared with standard laboratory controls as well as the improvement of anticoagutation monitoring by INR-ST, e.g. the percentage of measurements found inside the target therapeutic INR-range. The 231 parallel measurements revealed a high accuracy of INR-ST with a 4.5% median difference between parallel controls of single measurements. In addition, INR-ST resulted in a significant increase of measurements inside the target therapeutic range (from 54 to 80%) in the entire group. However, INR-ST performed every 4 weeks did not result in a significant improvement, while selfcontrols every week (from 44 to 84%) and every 2 days (from 51 to 84%) did. In a second study (150 patients) the incidence of bleeding and thromboembolic complications were evaluated in patients who either had standard anticoagulation measurements o[ performed INR-ST. During a median follow-up of 1.5 years in the group with standard oral anticoagulation management an incidence for bleedings of 10.9% per year and for thromboembolic complications of 3.6% per year was found, while in the group of patients with INR-ST bteedings occured with an incidence of 4.5% per year and thromboembolisms with 0.9% per year. It can be concluded that INR-ST can be performed with high accuracy by the patients themselves. INR-ST results in more frequent controls of the INR and consequently in significantly longer time periods where the patients are inside the optimal target therapeutic INR range. The more accurate anticoagulation management resulted in our study group in a significantly lower incidence of thromboembolic and hemorrhagic complications.

The Cell Biology of Tissue Factor (Thromboplastin) H. prydz, The Biotechnology Centre of Oslo, Norway Tissue Factor (TF,Thromboplastin) is the most potent trigger of blood clotting knuwn.lts presence in tissue extracts has been known since before the turn of the century.Erwin Chargaff (of DNA fame) first described that TF consisted of two parts (a protein and a lipid moiety), a fact that was independently rediscovered in the early sixties by Deutsch and Lechner in Vienna and by myself in Oslo. In 1973 TF was first purified and shown to be an integral membrane protein of Mr about 50 kDa (this was a slight overestimate,recent studies indicate 46-48 kDa). In 1987 four groupes independently cloned TF eDNA, later also genomic DNA. The structure of TF suggested that it was a membrane spanning receptor, with an extracellular part of about 219 amino acids folding into two domains remotely like the domains of the Ig-superfamiliy, and more specifically the eytokine receptors.

TF is synthesized to a varying degree in many tissues, but not normally in tissues in contact with blood. However, during acute inflammatory situations and under the influence of a number of agents (immune cnmplexcs, lectins, cytuklnes,bacterial components, complement components etc) monucytes and vascular endothelial cells are induced to synthesize TF. Another possible way tu get TF into contact with blood is thruugh trauma, surgical ur otherwise.

The fact that TF is inducible has created a lot uf interest in its cell biology. We reported recently that binding of Factor VIIa tu TF un the cell surface elicited a marked increase in intracenular Ca spikes, thus rendering the final proof that TF not only luoks like a receptor but really is one, since all the three essential criteria are fulfilled: TF has a transmembrane segment and a cytoplasmic tail TF has a high specificity/high affinity ligaud (Factor VII and Factor X) TF induces an intracellular signal upon ligand binding Recent results regarding TF as a receptor and regarding its intracellular traffic will be reported. Both F.IX and F.X are activated by the TF/F.VIIa complex. We have used a TF-initiated model system to examine the roles played by F.IXa and F.Xa in initiating coagulation. The in vitro model contained a cellular TF source; plasma concentrations of F.II, V, VIII, IX and X, tissue factor pathway inhibitor and antithrombin III. We tested the ability of exogenously added F.IXa or F.Xa to substitute for TF/VIIa activity in initiating platelet activation and thrombin generation. F.Xa was 10 times more potent than F.IXa in initiating platelet activation, but F.IXa was more potent in promoting IIa generation. In the presence of TF/VIIa, zymogen F.X was required both for platelet activation and IIa generation, while F.IX was only required for IIa generation. Thus, TF/VIIa-activated F.IXa and F.Xa have distinct physiological roles. The main role of F.Xa is to activate platelets by producing an initial small amount of IIa. The process of platelet activation is much more efficient if this initial IIa is produced on the TF-bearing surface. F.IXa, by contrast, enhances IIa generation on platelets by providing F.Xa for platelet surface prothrombinase assembly. We conclude that activation of both F.IX and F.X by TF/VIIa is essential for efficient initiation of coagulation. Furthermore, F.X activated on the surface of a TF-bearing cell isnot functionally equi.valent to F.X activated on the platelet surface. Therefore, F.X activation by TF/VIIa does not compensate for a lack of F.IX or VIII in hemophilia because the F.Xa activated by TF/VIIa is located on the wrong surface for efficient thrombin generation. Our results emphasize that the location, of an activated coagulation factor is a critical determinant of its role in hemostasis. The X-ray crystallographic structure of the FVIIa-TF complex located the areas within FVIIa in contact with TF to the first EGFlike and the protease domains. High-affinity binding of FVIIa to TF depends on Ca2+ and previous studies suggest that Ca2+ binding to a site in the protease domain is required. However, using surface plasrnon resonance we have shown that removal of the N-terminal Gla domain and hydrophobic stack (HS) from FVIIa results in a decreased affinity for TF. In search for the mechanism by which regions in FVUa not in direct contact with TF affect the affinity, various techniques have been employed. A plausible model is one in which the Gla domain and HS optimize the interaction with TF by affecting regions that mediate the association with TF. Using gel filtration we demonstrated that the hydrodynamic radii of des(1-38)-FVIIa and, especially, FVIIa decreased upon Ca2+ binding, whereas des(1-44)-FVIIa was virtually unaffected. This suggested that the Gla domain and the HS interact in a Ca2+-dependent manner with other regions in FVIIa, presumably the protease domain, making FVIIa more globular. Using CD spectroscopy, we could detect Ca2+-induced changes in the secondary structure of FVIIa, all assignable to an increased helical content of the Gla domain. The amidolytic activity of FVIIa in the absence of TF has a Ca2+ dependency that suggests involvement of the Gla domain in obtaining full activity. This activity was very sensitive to guanidine hydrochloride (C50<0.5 M) and its quenching corresponded to unfolding of the Ca2+-loaded Gla domain as measured by changes in the Trp fluorescence of FVIIa. Based on these results it is conceivable that the Gla domain and perhaps the HS of FVIIa interacts with the protease domain and that this globular form mediates the initial contact with "IT. Several studies have indicated a profound role for FVII(a) in arterial thrombogenesis. In the present study we quantified the inhibitory effect of dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone recombinant FVIIa (DEGR-rFVIIa) on acute thrombus formation at medium-sized artery blood flow conditions characterized by a wall shear rate of 650 s -1. Native human blood was drawn directly from an antecubital vein by a pump into a heparin coated mixing device where DEGR-rFVIIa (0.9 -880 nmol/L final plasma concentration) or buffer were mixed homogeneously with the flowing blood. Subsequently, the blood entered a parallel-plate perfusion chamber in which a thrombogenic surface consisting of immobilized TF and phospholipids was positioned. Fibrin deposition, platelet-fibrin adhesion and platetet thrombus volume triggered by this surface were measured by morphometry. DEGR-rFVIIa inhibited these thrombus parameters in a dose dependent manner with IC50 values between 0.5 and 0.7 nmol/L The plasma levels of thrombin-antithrombin m complexes were inhibited at an IC50 of 0.6 nmot/L Since the concentration of FVII and FVIIa in normal plasma is about 10 and 0.1 nmol/L, respectively, theIC50 values indicate that DEGR-rFVIIa is a very efficient inhibitor of thrombus formation in this model. Thus, inhibition of the extrinsic pathway of coagulation may represent and entirely new and effective arterial anti-thrombotic approach.

The anticoagulant agent from medicinal leeches, hirudin, is presently gaining popularity as an antithrombotic drug. The design of recombinant forms of the minipretein and their availability led to the clinical use of this naturally occurring thrombin

Inhibitor. Extensive studies in experimental animals have shown that due to its pronounced and specific antithrombin effect hirudin is an antithrombotic agent of high quality. Apart from its anticoagulant effect, hirudin is pharmacodynamically inert and is well tolerated in vivo. 

Brain abnormalities in male children and adoleescents with hemophilia: detection with MR Imaging

MRI of sickle cell cerebral infarction

In addition, portions of the preparations were incubated with 1.4nm-gold labelled fibrinegen molecules (Fg-Au; final concentration 4gp_g/ml) for 10 see at 37°C. The expenments were stopped al~er 3 or 10 rain by glutaraldehyde fixation. Serial sections were prepared and examined after silver-enhancement using Danscher's (1981) method to visualize the Fg-Au. HWdid not alter the resting GFP. After .3,rain, activated HW-platelets changed their shape moderately but in contrast to the controls did not aggregate. Moreover, the conatdcting cytoskeleton that centralizes platelet organelles was not formed. Fg-Au was found on the surface of the control platelets, in high density in the contact spaces between the aggregated platelets and internalized in the surface connected system. Only a very small number of HWtreated cells had Fg-Au on their surface and showed internalization. 10 rain after ADP most of the platelets in both preparations had retumod to a discospherecytic shape

It is confirmed that HW inhibits the platetet aggregation and in addition, 1) the formation of the contractile cytnskeleton as well as 2) the binding and interalization of the used ligand fibrinogen

Santoso 2 l.Institute for Immunology and Transfusion Medicine, Emst-Moritz-Arndt-University Greifswald; 2. Institute for

Transplant vasculopathy in cardiac allografts is the leading limitation to long-term survival of cardiac transplant recipients. Activation of the coagulation mechanism by adherent monocytes expressing tissue factor (TF) is expected to be involved in the development of transplant atherosclerosis. In this in vitro study the effect of the immunosuppressant Cyclosporine A (CsA) on monocyte TF expression and the regulation of the TF gene transcription was analysed. CsA was shown to inhibit the induction of monocyte procoagulant activity (PCA) by decreasing lipopolysaccharide (LPS)-induced tissue factor (TF) expression in monocytes. CsA exerted a dosedependent inhibitory effect on monocyte TF expression at concentrations (1,5 txmol/L CsA: 50 % inhibition) not decreasing the cellular protein synthetic rate. Addition of CsA during LPSstimulation prevented activation of the nuclear factor (NF) ~B as demonstrated by electrophoretic mobility shift assay. Western blot analysis confirmed reduced nuclear translocation of NF-~B in the presence of CsA. Inhibition of the LPS-induced NF-~B activation by CsA resulted in a reduced TF gene transcription as demonstrated by RT-PCR-analysis. Thus, Cyclosporine A prevents TF expression by inhibiting the induction of TF gene transcription in activated monocytes. CsA may therefore provide a therapeutical tool acting not only as a direct immunomodulating agent, but also by influencing local coagulation activation. The N-glyean pattern of recombinant human coagulation factors II (rF-I~ and IX (rF-IX), derived from both transfected Chinese hamster ovary (CHO) cells and African green monkey (Vero) cells, and produced at industrial scale were analyzed by binding to carbohydrate specific lectins and were compared with the glycan structure of human plasma-derived coagulation factors. Human plasma-derived coagulation factors 1I (hpF-I1) and IX (hpF-ix) exhibited complex-type glycan structures with carbohydrate chains capped with c~(2-6)sialic acid. Terminal galactose-B(1-4)-N-acetylglucosamine units were detected in hpF-IX. Both CHO cell-derived rF-]I and rF-IX exhibited complot-type glycosylafion and contained ~2-3)-sialio acid in addition to terminal galactose-B(1-4)-N-acetylglucosamine. Vero cell-derived if-IX exhibited a complex-type glycan structure sinailar to that of CHO cell derived rF-IX. In contrast, rF-lI produced by Vero cells exhibited a glycan microheterogeneity composed of hybrid-type glycosylation containing "highmarmose" structures and complex-type glycosylation containing et(2-3)-sialie acid. Galaetose-B(1-4)-N-acetylglucosamine structures and a low concentration of ct(2-6)-sialic acid were detected in both micro-heterogeneity fractions of Veto cell-derived rF-lI. Although different in the'tr carbohydrate structures, coagulation factors II and IX obtained recombinantly from both transformed CHO-cells and Vero-cells exhibited coagulation activities comparable to the plasma-derived proteins. Exposure of tissue factor (TF) to flowing blood, as a consequence of vascular injury, leads to TF/F.VIIa-initiated coagulation which may play a key role in triggering intravascular thrombosis. In order to evaluate the potential advantages of TF pathway blockade over existing therapy, the antithrombotic and the anticoagulant .effects of a monoclonal anti-rabbit TF antibody (AP-1) and heparin were compared in a rabbit arterial thrombosis model. Thrombosis was induced by a local injury to the carotid artery and the recurrence of thrombus formation was followed by monitoring the cyclic flow variations (CFV) of the arterial blood flow. Rabbits received intravenously either AP-1, heparin or vehicle, and CFV were monitored on line for 120 min. Activated partial thromboplastin time (aPTT), and activated clotting time (ACT) were measured at the end of the experiment control AP-I heparin <lmg/kg* 0.2mg/kg/hr 0.8mg/kg/hr intraabdominallretroperitoneal, CNS) on a named-patient basis. Later also mild/moderate bleeds, predominantly joint bleeds were included.In 80-90% of the bleeds NovoSeven was found to be hemostatically efficacious. Also in major surgery (hip-and knee-replacement including a bilateral total condylar arthroplasty, herniotomy) hemostatic efficacy has been found to be 94% Since rFVlla is proteotytically active only in complex with TF the risk for systemic activation of the coagulation system should be minimal. Also very few side-effects have been seen.Doses between 75-120 tJg/kg have been used, which initially have to be given at 2 hours intervals. To achieve satisfactory hemostasis both an initial dose high enough to induce immediate hemostasis AND an appropriate dose interval ensuring a plasma level of FVlI:C well above the hemostatic lower level for a period of time, the length of which is dependent on the type of bleeding, are important. Accordingly, the highel the initial dose the more robust with regard to dose interval will the treatment be. Acute myocardial ischemia is usually triggered by coronary thrombosis. Heparin (hep), along with aspirin, is commonly used for the treatment of unstable angina (UA) and myocardial infarction (MI). Hirudin (hiQ is a potent antitbrombin agent which may offer therapeutic advantages over standard hep for the treatment UA and MI. The OASIS phase I pilot study conducted in 24 Canadian centres, has evaluated the safety, feasibility and efficacy of hir (HBW 023, Behringwerke AG, Marburg) compared to hep in 601 patients with MI or suspected non Q-wave MI. Patients (95 % received aspirin) were randomized to a 72 hr infusion of either hep (5000 u bolus followed by 1200 u/hr; n=253), or low dose hir (0.2 mg/kg bolus then 0.10 mg/kg/hr; n=175) or medium dose lair (0.4 ms/ks bolus then 0.15 mg/kg/hr; n=173). At seven days of follow-up the incidence of major bleeds was 0.4 % in the heparin group, 0.6 % low dose hit, 1.2 % medium dose lair (p=NS), minor bleeds were 10.3 %, 17.1% and 19.1% respectively (p---0.008 hep vs med dose); there were no hemorrhagic strokes. The incidence of the primary outcome of cardiovascular death, new MI and severe recurrent ischemia (refractory or severe angina) was 17.0 %, 15.4 % and 8.7 % respectively (p---0.014 hep vs reed dose). There was also a trend towards lower rates of coronary artery bypass surgery in reed dose hir compared to hep. In a 200 patient substudy, levels of thrombin antithrombin complex, fibfinopeptide A and D-dimer were all suppressed to a greater extent by Mr compared to hep. These results indicate that hirudin may be a more effective agent for the treatment of acute coronary syndromes, but more information on safety and efficacy is needed from larger randomized trials. Full clinical and coagulation resuks will be presented. HAT is caused by an immunologic response to heparin. Affected patients need effective parenteral anticoagulation. We treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory confirmed HAT (HIPA-Test) with r-hirudin (Behringwcrke AG, Marburg). Treatment regimens were: AI acute HAT: 0.4 mg/kg b.w., continuous infusion 0A5 mg/kg b.w/h (51 patients); A2 acute HAT with thrombolysis: bolus 0.2 mg/kg b.w., continuous infusion 0.1 mg/kg b.w./h (5 patients); B: proph, treatment: continuous infusion 0. I mg/kg b.w.Pa (18 patients); C: during cardio-pulmunary bypass. Primary objective was: increase in platelet counts or maintenance of normal baseline values during effective anticoagulation. Secondary objectives were the incidence of new thromboembolic complications (TECs), major hemorrhage, limb amputations and deaths. Results were compared with a historical control of 120 IX-AT patients, all confirmed by the HIPA test, but treated before rhirudin was available (Danaparoid, Marcumar, no anticoagulation). Primary efficacy analysis: respunders AI: 74%o, A2: 60%, B: 44%, C: 50%, total: 65%. All adverse events beside bleeding complications had been judged to be caused by the underlying disease and not directly related to r-hirndin. Proportion of patients with event in the obse~wation period; death: A1 5.9%, A2 0%, B 16.774, C 0%, total: 7.3%, during rhirudin treatment: 1.2%; new TEC: AI 5.9%, A2 0%, B 27.8%, C 0%, total 9.8%, during rhirudin: 2.4%; limb amputation: AI 3.9%, A2 0%, B 5.6°/0, C 0%, total: 3.7%, during rbirudin: 3.7% Eaapoiat We conclude, that r-hirndin is an effective anticoagulant in HAT typa II patients allowing rapid re, coveD" of platelet counts. Compared to a historical control the r-hirudin group had a strongly reduced incidence of Combined endpuint of mortality, limb amputation and new TECs. Major bleedings occurred slightly more frequent than in the historical control. However, the dosage used in regimen B might have been too low, as flds group revealed an effective anticoagulatiun and increase in platelet counts in only 43% and concomitantly had the higl)est incidence of new TECs.
