Stief et al.indd Inhibition of Intrinsic Thrombin Generation Thomas W. Stief Department of Clinical Chemistry, University Hospital Giessen & Marburg, Germany. Abstract Background: The contact phase of coagulation is of physiologic/pathophysiologic importance, whenever unphysiologic polynegative substances such as cell fragments (microparticles) get in contact with blood. There are several clinically used inhibitors of intrinsic thrombin generation. Here the inhibitory concentrations 50% (IC50) of these anticoagulants are mea- sured by the highly specifi c thrombin generation assay INCA. Methods: Unfrozen pooled normal citrated plasma in polystyrole tubes was supplemented at 23°C in duplicate with 0–2 IU/ml low molecular weight heparin (dalteparin), 0–2 IU/ml unfractionated heparin, 0–500 KIU/ml aprotinin, or 0–40 mM arginine. 50 µl plasma or 1 IU/ml thrombin standard were pipetted into a polystyrole microtiter plate with fl at bottom. 5 µl SiO2/ CaCl2 - reagent (INCA activator) were added and after 0–30 min incubation at 37°C 100 µl 2.5 M arginine, pH 8.6, were added; arginine inhibits hemostasis activation and depolymerizes generated fi brin within 20 min at 23°C. The in the physiologic 37°C incubation phase generated thrombin was then chromogenically detected. The intra-assay CV values were < 5%. Results and Discussion: The approximate IC50 were 0.01 IU/ml dalteparin, 0.02 IU/ml heparin, 25 KIU/ml aprotinin, and 12 mM arginine. The effi ciency of any anticoagulant on intrinsic thrombin generation should be measured for each individual patient. Abbreviations: IIa, thrombin; ∆A, increase in absorbance; APTT, activated partial thromboplastin time; CRT, coagulation reaction time (at 37°C in water-bath); F-wells, polystyrole microtiter plates with fl at bottom; IC50, inhibitory concentra- tion 50%; INCA, intrinsic coagulation activity assay; IU, international units; KIU, kallikrein inhibiting unis; LMWH, low molecular weight heparin; mA, milli-absorbance units; PSL, pathromtin SL®; RT, room temperature (23°C); U-wells, polystyrole microtiter plates with round bottom. Keywords: INCA, thrombin, LMW-heparin, dalteparin, heparin, aprotinin, arginine Introduction Low molecular weight heparins (LMWH) are essential drugs for patients inside and outside the hospital; unfortunately, the global hemostasis assay APTT is not sensitive to LMWH [Fareed et al. 2004]. There is clinical need for a simple physiologic global hemostasis test that monitors the anticoagulant power of LMWH. The only LMWH - routine assay currently available is the anti-Xa assay, that is available in a clotting [Denson and Bonnar, 1973] and in a chromogenic version [Teien et al. 1976]. However, the target therapeutic dose range in the anti-Xa assay is 0.4–0.7 IU/ml heparin in the anti-Xa assay but only 0.2–0.4 IU/ml heparin in the APTT [Kitchen, 2000]. This discrepancy indicates that the Xa activity added in the anti-Xa assays might be supra-physiological and that the other pharmacologic actions of the heparins than just the inhibition of activated factor X are not refl ected by the anti-Xa assay. Recently, a new test for thrombin activity in plasma was developed [Stief, 2006; Stief et al. 2006]. This assay uses (I) a chromogenic thrombin substrate at fi nal concentrations < 0.6 mM and (II) arginine at fi nal concentrations > 1 M, resulting in highly specifi c thrombin determination. Of diagnostic importance are the following chromogenic thrombin tests that all base on this new technique: 1. basal thrombin activity (IIa) 2. recalcifi ed coagulation activity assay (RECA) 3. intrinsic coagulation activity assay (INCA) 4. extrinsic coagulation activity assay (EXCA). As equipment only a normal microtiterplate reader is required; these only 1 or 2 measuring point - based tests are easy to handle with high reproducibility (all have intra-assay CV values less than 5%), Correspondence: T.W. Stief, MD, Priv.-Doz. Department of Clinical Chemistry, University Hospital of Giessen & Marburg, D-35033 Marburg, Germany. Fax: +49-6421-286 5594; Email: thstief@med.uni-marburg.de Please note that this article may not be used for commercial purposes. For further information please refer to the copyright statement at http://www.la-press.com/copyright.htm PERSPECTIVES Drug Target Insights 2006:1 5-10 5 Inhibition of Intrinsic Thrombin Generation they are fast and economical, and can thus be used in routine diagnostic. Material and Methods INCA coagulation reaction time The INCA is a new simple global chromogenic hemostasis test that requires only a two-point deter- mination of thrombin generation in the important ascending part of the thrombin activity curve, i.e. the ratio between thrombin at a second time point divided by thrombin at a fi rst time point should be > 1. 50 µl unfrozen plasma (1 part 106 mM citrate + 9 parts of venous blood; centrifuged at 2800 g (4000 rotations per minute at 23°C) are pipetted into flat bottom polystyrole microtiter plate wells (F-wells, Polysorp®, NUNC, Wiesbaden, Germany; article nr. 446140). The INCA is started by addition of 5 µl SiO2/CaCl2 reagent (freshly with 278 mM CaCl2 1:10 diluted Pathromtin SL®, DadeBehring, Marburg, Germany). Always H2O-rinsed completely emptied new disposable polypropylene tips for the Eppendorf-multipette® are used: if this disposable tip of the multipette for addition of the INCA-activator to the reaction well is not rinsed, the intrinsic thrombin generation is strongly inhibited; some releasable plastic material in the new tip seems to act like a heparinoid; if the untreated polystyrole F-well plate (Polysorp®) is replaced by an irradiated one (Maxisorp®) or a U-well plate (for sample volumes < 25 µl U-wells result in higher precision than F-wells), then this plastic material inhibits the intrinsic coagulation to some extent. INCA inhibition by heparins Unfrozen pooled normal citrated plasma in 5 ml polystyrole tubes was supplemented with 0–2 IU/ ml dalteparin (a LMWH; Fragmin P®, Pharmacia, Erlangen, Germany) or with 0–2 IU/ml unfraction- ated heparin (Roche, Basel, Switzerland) and kept at 23°C. 50 µl samples were pipetted into F-wells, and the INCA was started by addition of 5 µl SiO2/ CaCl2 reagent. After 3 min coagulation reaction time (a CRT of 3 min is the initial time point of thrombin generation) at 37°C in the water-bath, 100 µl 2.5 M arginine, pH 8.6 (Sigma, Deisenhofen, Germany) were added. The plate was withdrawn from the water-bath. After 20 min (23°C = RT) 10 µl 3.85 mM chromogenic thrombin substrate HD-CHG-Ala-Arg-pNA (Pentapharm, Basel, Switzerland) in H2O were added and ∆A/t was determined by a microtiterplate reader with a 1 mA resolution (Milenia-DPC, Los Angeles, USA). The result was standardized against 1 IU/ml IIa in 6.7 % human albumin (Kabi, Stockholm, Sweden) replacing the plasma sample. INCA kinetic in plasma supplemented with heparins 20 µl unfrozen pooled normal plasma, supple- mented with 0 IU/ml, 0.063 IU/ml, 0.13 IU/ml, 0.25 IU/ml dalteparin or 0 IU/ml, 0.063 IU/ml, 0.13 IU/ml, 0.25 IU/ml, or 0.5 IU/ml unfractionated heparin were incubated in U-wells (NUNC) with 2 µl SiO2/CaCl2 reagent. After 0–30 min (37°C), 50 µl 2.5 M arginine, pH 8.6, were added. The plate was withdrawn from the water-bath. After 20 min (23°C) 20 µl 0.77 mM chromogenic thrombin substrate in 2 M arginine was added and the linear ∆A/t (RT) was determined. INCA inhibition by aprotinin Unfrozen pooled normal plasma in polystyrole tubes was supplemented with 0–400 KIU/ml aprotinin (Bayer, Leverkusen, Germany). The INCA was performed with 20 µl plasma samples in U-wells and 2 µl INCA-activator. After 0–30 min (37°C), 50 µl 2.5 M arginine, pH 8.6, were added. After 20 min (RT) 20 µl 0.77 mM chro- mogenic substrate in 2 M arginine was added and the linear ∆A/t (RT) was determined. Additionly, the APTT of the aprotinin-supplemented plasma samples was measured by a Behring Coagulation Timer®. INCA inhibition by arginine Unfrozen pooled normal plasma in polystyrole tubes was supplemented with 0–40 mM arginine (Braun, Melsungen, Germany) or alkaline arginine (pH = 8.7; Sigma). 50 µl samples were tested in the INCA (F-wells). Infl uence of fi brin on anticoagulants in INCA Unfrozen pooled normal plasma with 2.8 g/l fi brin- ogen was unsupplemented and supplemented with purifi ed human fi brinogen (Haemocomplettan, Aventis, Frankfurt, Germany; the preparation Drug Target Insights 2006:16 Thomas W. Stief contained about 1% soluble fi brin [Stief, 2000] that might act as antithrombin I [Mosesson, 2005]) to a fi nal active fi brinogen concentration of 4.0 g/l. The plasmas were then unsupplemented or supplemented with 0.01 IU/ml dalteparin, 0.02 IU/ml heparin, 25 KIU/ml aprotinin, or 12 mM arginine. The INCA was performed as described above. Addition of 0.1% Triton X 100® (Sigma) fi nal test concentration accelerates the decrease of the fi brin-related turbidity about 7fold, i.e. instead of 20 min arginine reaction time 3 min is suffi - cient before starting the IIa detection phase of the assay. A turbidity increase in plasma occurs within minutes, if the fi nal thrombin activity exceeds 0.02 IU/ml [Stief, 2007 in press]. Results The action of heparins on INCA is shown in Figure 1. The 50% inhibitory concentrations (IC50) were about 0.01 IU/ml for LMWH and 0.02 IU/ml for unfractionated heparin (Fig. 1a). In therapeu- tical heparin concentrations (about 0.2 IU/ml), 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0 0.05 0.1 0.1 5 0.2 0.25 0.3 Heparin conc. [IU/ml] II a A ct iv it y 3 m in C R T [ IU /m l] Figure 1. Inhibition of intrinsic thrombin generation by heparins. Figure 1a. IC50 determination. Unfrozen pooled normal plasma, supplemented with dalteparin (●) or unfractionated heparin (■) was analyzed in the INCA (50 µl sample into F-wells). The INCA was performed with a thrombin generation time = coagulation reaction time of 3 min (initial phase of thrombin generation). 10 µl 3.85 mM CHG-Ala-Arg-pNA in H 2O were added and ∆A/t was measured. 1 IU/ml IIa had 12.2 mA/min RT. 0 1 2 3 4 5 6 7 0 5 10 5 CRT [min] IIa A ct iv ity [I U /m l] 1 20 25 30 35 Figure 1b. Reaction kinetic of dalteparin. 20 µl unfrozen pooled normal plasma, supplemented with 0 IU/ml (O), 0.063 IU/ml (■), 0.13 IU/ml (♦), or 0.25 IU/ml (▲) dalteparin was incubated with 2 µl SiO2/CaCl2 reagent. After 0–30 min CRT at 37°C in the water-bath 50 µl 2.5 M arginine, pH 8.6, was added. After 20 min (RT) 20 µl 0.77 mM chromogenic thrombin substrate in 2 M arginine was added and the linear ∆A/t (RT) was determined. Drug Target Insights 2006:1 7 Inhibition of Intrinsic Thrombin Generation 0 1 2 3 4 5 6 7 0 5 10 15 20 25 30 35 IIa A ct iv ity [I U /m l] CRT [min] the thrombin generation in the INCA at CRT < 20 min does not exceed 0.5 IU thrombin/ml sample (Fig. 1b,c). The INCA curves are evaluated in their important pre-maximum phase. The IC50 of aprotinin on the INCA is about 25 KIU/ml (Fig. 2). This reflects the extreme sensitivity of the INCA: in the usual APTT the aprotinin concentration that prolongs the normal APTT 1.5 fold is 300 KIU/ml. Arginine dose-dependently inhibits INCA (Fig. 3). The IC50 values were about 12 mM for commercially available arginine for i.v. infusion and about 8 mM for alkaline arginine (at pH 8.7). Normal plasma supplemented with 1.2 g/l purifi ed fi brinogen containing 1% soluble fi brin resulted in a 50% decrease of IIa–generation: plasmatic fi brin entraps generated thrombin, that is why fi brin can be considered as antithrombin I (8). Figure 1c. Reaction kinetic of unfractionated heparin. 20 µl unfrozen pooled normal plasma, supplemented with 0 IU/ml (O), 0.063 IU/ml (■), 0.13 IU/ml (♦), 0.25 IU/ml (▲), or 0.5 IU/ml (●) heparin was incubated with 2 µl SiO2/CaCl2 reagent. After 0–30 min CRT at 37°C in the water-bath 50 µl 2.5 M arginine, pH 8.6, was added. After 20 min (RT) 20 µl 0.77 mM chromogenic thrombin substrate in 2 M arginine was added and the linear ∆A/t (RT) was determined. 0 0.5 1 1.5 2 2.5 3 0 5 10 15 20 25 30 35 IIa A ct iv it y [I U /m l] CRT [min] Figure 2. Inhibition of intrinsic thrombin generation by aprotinin. Figure 2a. 20 µl unfrozen pooled normal plasma, supplemented with 0 KIU/ml (O), 25 KIU/ml (∆), 50 KIU/ml (□), 100 KIU/ml (■), 200 KIU/ml (▲), or 400 KIU/ml (●) aprotinin, were tested in the INCA (U-wells). Drug Target Insights 2006:18 Thomas W. Stief This decrease in IIa-generation was independent of added anticoagulant. Discussion The APTT does not refl ect the effi ciency of some important clinically used anticoagulants, such as e.g. the LMWH [Fareed et al. 2004]. The SiO2 amount used to trigger the INCA is about 100fold below the contact activator amount used for the APTT, and in contrast to the usual global coagula- tion test APTT the plasma matrix in the INCA is not signifi cantly changed (only 1 part of reagent to 10 parts of citrate plasma). The INCA monitors the anticoagulant potency of anti-factor Xa or anti-thrombin drugs [Tobu et al. 2004]: LMW-heparins are powerful inhibi- tors of IIa generation, especially via inhibition of factor Xa. In an INCA test version with an assay incubation at 37°C prolonged to 12 min (INCA-12) patient APTT values of about 40 s correspond to about 1 IU/ml IIa generation, APTT values of 50–60 s to about 0.2 IU/ml IIa (< 36 s = 100% of norm APTT; 4.3 ± 1.4 IU/ml IIa = 100% of norm IIa generation) [Stief et al. 2006]. The usual plasmatic unfractionated heparin concentrations in therapeutic anticoagulation are about 10fold higher than the IC50 values observed here in the INCA. The superior anticoagulant capacity of LMWH compared to unfractionated heparin might be due to some contact activating 0 0.5 1 1.5 2 2.5 3 0 50 100 150 200 250 300 350 400 450 IIa A ct iv ity [I U /m l] Aprotinin activity [KlU/ml] Figure 2b. CRT = 10 min (●), CRT = 15 min (■). 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 2 4 6 8 1 4 16 IIa A ct iv ity [I U /m l] 0 112 CRT [min] Figure 3. Inhibition of intrinsic thrombin generation by arginine. Figure 3a. Unfrozen pooled normal plasma was supplemented with 0–40 mM arginine. 50 µl samples in F-wells were tested in the INCA: 0 mM (O), 5 mM (▲), 10 mM (●), 20 mM (♦), or 40 mM (■) arginine. 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