ujms109_2.pdf Upsala J Med Sci 109: 71–122, 2004 Coagulation, inflammation and myocardial dysfunction in unstable coronary artery disease and the influence of glycoprotein IIb/IIIa inhibition and low molecular weight heparin Stefan James Department of Medical Sciences, Cardiology, Uppsala University Hospital, Uppsala, Sweden ABSTRACT A free full-text copy of this article can be found at the web page of Upsala J Med Sci: http://www.ujms.se Patients with unstable coronary artery disease (CAD) have an increased risk of sub- sequent myocardial infarction and death. This study evaluated the safety and effica- cy of treatment with glycoprotein IIb/IIIa inhibition in addition to aspirin, low mol- ecular-weight heparin and its influence on coagulation and inflammation. Also, ear- ly and differentiated risk assessment utilising markers of inflammation, myocardial damage and dysfunction were evaluated. The Global Utilisation of Strategies To open Occluded arteries-IV (GUSTO-IV) trial randomised 7800 patients with unstable CAD to 24 or 48 hours infusion of abciximab or placebo in addition to routine treatment with aspirin and heparin or dalteparin. Baseline levels of creatinine, C-reactive protein (CRP), troponin T (TnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP) were analysed. At select- ed sites, all patients received subcutaneous dalteparin (n=974), in stead of heparin infusion (n=6826). In a sub-population of dalteparin treated patients (n=404), serial measurements of markers of coagulation, fibrinolysis and inflammation were also performed. Addition of abciximab to dalteparin as the primary treatment of unstable CAD was not associated with any significant reduction in cardiac events but a doubled risk of bleedings. The combination of abciximab with dalteparin seemed as safe when 71 Received 12 November 2003 Accepted 3 December 2003 Abbreviations: Acute coronary syndrome (ACS), Brain natriuretic peptide (BNP), Coefficient of vari- ance (CV), Coronary artery disease (CAD), Coronary artery bypass grafting (CABG), Creatine kinase- MB (CK-MB), C-reactive protein (CRP), Electrocardiogram (ECG), Glycoprotein IIb/IIIa (GP IIb/IIIa), Global Utilisation of Strategies To Open Occluded Arteries IV (GUSTO-IV), Interleukin-6 (Il-6), Low-molecular weight heparin (LMWH), N-terminal pro-Brain natriuretic peptide (NT-proB- NP), Myocardial infarction (MI), Percutaneous coronary intervention (PCI), Plasminogen activator (tPA), Plasminogen activator inhibitor (PAI-1), Soluble fibrin (SF), Thrombin-antithrombin complex (TAT), Troponin T (TnT), Unfractionated heparin (UFH). used with heparin. Despite full dose dalteparin and aspirin there was a simultaneous activation of the inflammation, coagulation and fibrinolysis systems without any influence of the abciximab treatment. Elevated levels of CRP, TnT, and NT-proBNP and reduced creatinine clearance were independently related to short and long-term mortality. The best prediction of high and low risk was provided by a combination of NT-proBNP and creatinine clearance. Any detectable elevation of TnT and reduced creatinine clearance, but neither elevation of CRP nor NT-proBNP, were also inde- pendently associated to a raised risk of subsequent myocardial infarction. INTRODUCTION Cardiovascular disease is the most common cause of death in the industrialised world, comprising 46 % of all deaths in Sweden (1). In spite of the steady decline in the incidence of myocardial infarction in recent years, the incidence of unstable coronary artery disease (CAD) has increased considerably(1). Chest pain, or other symptoms suggestive of unstable CAD, is furthermore one of the most common reasons for admission to the hospitals emergency rooms. These patients constitute a very heterogeneous population regarding the clinical history and the underlying cause of the symptoms. Subsequently, also the prognosis varies considerably. Early risk prediction is essential for identification of patients at high risk and selection of the most appropriate treatment strategy. Moreover, with early risk-prediction, patients at low risk can be identified and costly and potentially hazardous treat- ments and prolonged hospital stays can be avoided. Unstable CAD is caused by a complex interaction between the endothelium, platelets and cascades systems of inflammation, coagulation and fibrinolysis, lead- ing to thrombus formation and compromised coronary blood flow. Subsequent myocardial damage, caused by downstream embolization of thrombotic material, as well as inflammation and myocardial dysfunction, contributes to an increased risk of serious complications. Treatment of patients with unstable CAD aims to symp- tom relief and to limitation of myocardial damage and prevention of recurrent ischemic events at a tolerable level of side effects. Despite substantial improve- ments in the treatment there is still an approximately 7–10% risk of death or myocardial infarction the initial month after the index episode(2–4). Therefore, fur- ther improvements by new anti-thrombotic and anti-inflammatory agents and by better and more differentiated risk assessment to tailor the treatment to individual patients, is highly warranted. In the present study, the safety and clinical efficacy of the glycoprotein IIb/IIIa inhibitor abciximab in combination with either the low molecular-weight heparin, dalteparin or unfractionated heparin in patients with unstable CAD was investigated. Also, the influence of treatment with abciximab on the activation of systems of inflammation, coagulation and fibrinolysis was elucidated. Finally, the utility of bio- chemical markers of inflammation, myocardial damage and myocardial dysfunction for prognostication of the risk for death and myocardial infarction was elucidated. 72 BACKGROUND Clinical manifestations and definition of coronary syndromes The coronary arteries continuously supply blood for the myocardium’s demands of oxygen and nutrients. Interruption of the blood flow creates a state of ischemia with accumulation of acid waste products and disintegration of cell membranes resulting in ischemic chest pain. The induced changes in electric potentials can be detected with an electrocardiogram (ECG) and biochemical substances released from the injured myocardial cells can be detected in the peripheral blood. The classification of coronary syndromes is based on the acuteness by which they develop; stable coronary artery disease (CAD), acute coronary syndromes (ACS) and sudden cardiac death. Stable CAD is often a chronic disease with a deciduous onset and associated with a relatively low risk (figure 1). In contrast, ACS starts acutely and has a considerably worse prognosis. ACS is further divided into myocardial infarction (MI) and unstable angina (UA). Typically, MI is characterised by a sudden onset of severe chest pain in combination with a shortness of breath, 73 Fig. 1. Atherosclerosis, plaque rupture and thrombus formation. Definitions of various types of coro- nary heart disease. MI = Myocardial infarction. nausea and circulatory instability and defined as symptoms or signs of myocardial ischemia associated with a certain degree of elevation of biochemical markers(5). Unstable angina on the other hand is defined as a condition of coronary ischemia without elevation of biochemical markers and is clinically characterised by; a) recent onset (<4 weeks) of angina pectoris on minimal exertion, b) worsening of a stable angina with prolonged, more severe or more frequent episodes of chest-pain c) angina at rest or d) angina after a myocardial infarction (6). Transient ischemic ECG changes such as ST-depression or T-wave inversion are not obligatory but strongly support the diagnosis. Based on the patients ECG findings on arrival, MI are further classified as ST- elevation or non-ST elevation MI. ST-elevation MI is a special entity associated with the most dramatic symptoms and the strongest need for urgent treatment in order to restore the obstructed blood flow in the diseased coronary artery. Patients with a complete bundle branch block on ECG belong to the same high-risk catego- ry. Considering both the underlying pathophysiology and symptom evolution, non ST-elevation MI and unstable angina are parts of a continuum of unstable CAD, only separated from each other by the detection of biochemical markers of myocar- dial damage, above a somewhat arbitrary and variable limit. The focus of interest in the present study is non ST-elevation MI and UA. Atherosclerosis and plaque formation The predominating underlying cause of ACS is atherosclerosis, which starts in early adulthood in the innermost layer of the vessel wall, the intima. Several factors accelerate this process, among them smoking, hereditary factors, hypertension, ele- vated levels of serum lipids and diabetes mellitus. The first visible signs of athero- sclerosis are the so called “fatty streaks” consisting of pools of enlarged macrophages containing engulfed oxidised LDL particles that usually develop in response to mechanical stress (7). Over time, smooth muscle cells proliferate and tissue matrix is synthesised in the intima driven by a chronic inflammatory process creating a fibromuscular atherosclerotic plaque (8)(Figure 1). Late in the process, collagen and calcium is often deposited in the plaque as suggested by the term “ath- erosclerosis”(9). As the coronary plaque grows larger, the blood flow will be obstructed and may not be able to meet to current metabolic demands of the myocardium distal to the coronary stenosis. The latter situation is the pathophysio- logical background to the clinical entity called “chronic stable angina pectoris”. Plaque disruption and thrombus initiation The atheromatous core of a plaque is a soft, gruel-like hypo-cellular material mainly consisting of lipids. The overlying fibrous cap separates the plaque content from the blood. Disruption of a plaque is not an uncommon event but still a complex patho- logical process that is central to the initiation of the acute coronary syndrome (10). Importantly, not only the large plaques or tight stenoses create the acute coronary 74 syndrome. Two thirds of arteries with a plaque-rupture and an occlusive thrombus have stenoses of 50 percent or less (11). The major determinants of plaque rupture are the size and consistency of the atheromatous core, the thickness of the fibrous cap, the inflammation and repair within the plaque and the phenomenon of plaque fatigue (12). However, also sudden changes in blood pressure and increased shear stress are important contributors. The plaque rupture leads to exposition of highly thrombogenic factors to the coagulation system and platelets, initiating the throm- bus formation, which further exaggerate the narrowing of the coronary artery. Platelet activation Platelets are small cells found in large numbers in the blood and play a pivotal role in the thrombotic process. At the site of arterial injury, subendothelial tissue is 75 Fig. 2. Platelet activation. exposed to the blood constituents (figure 2). Platelets adhere to the exposed colla- gen, von Willebrand factor (13) and fibrinogen by specific cell receptors i.e. glyco- protein (GP) Ia/IIa and Ib-IX-V complex (14). Platelet adhesion results in outside-in signalling resulting in a rapid change of shape. Thereby, the platelets increase their surface in order to cover the lesion. In addition to adhesion, several independent mechanisms are important platelet activators, such as thromboxane, serotonin, epi- nephrine, adenosine diphosphate and thrombin (15). Simultaneously, they also release vasoconstrictors, chemotactic factors, clotting factors and receptors (i.e. p- selectin and CD40 ligand) from their granules, promoting vasospasm, additional platelet aggregation and thrombin generation. Release of adenosine diphosphate and thromboxane from �-granules amplifies the process by autocrine feedback loops. Activated platelets undergo structural changes for activation of the most numer- ous receptor found on the platelet surface, the GP IIb/IIIa receptor which exists at a number of 40.000–80.000 per platelet (16). The activated GP IIb/IIIa receptor crosslinks platelets by binding to fibrinogen, creating a platelet- fibrinogen net over the site of tissue injury. Coagulation cascade (Proteins indicated with bold text were evaluated in the study) At the site of a plaque rupture, the glycoprotein tissue factor (TF) will be exposed to the blood. TF is the initiator of the coagulation cascade in vivo and is expressed on smooth muscle cells, fibroblasts and macrophages in the vicinity of the vessel wall(17) (Figure 3). The coagulation cascade (18) (19) is complex and includes a series of interactions between a large number of pro-coagulant and anti-coagulant proteins interacting with platelets and endothelial cells. TF binds to small amounts of factor VII, which become activated by proteolytic cleavage into its active form, VIIa. An auto-amplifying system called the “extrinsic system” thereby becomes activated, ending up with the conversion of prothrombin to thrombin. Thrombin is considered a key protein in the cascade by its ability to activate other coagulation factors i.e. factor V, VIII and XI, and by controlling the final step in the clot forma- tion; conversion of fibrinogen to soluble fibrin. Soluble fibrin (SF) is formed when fibrinopeptide A and B are cleaved from fib- rinogen by the action of thrombin. SF circulates in plasma with a half-life of 4–6 hours. The level of SF reflects thrombin activity as the basis for fibrin formation and is therefore a sensitive marker of activation if the coagulation cascade (20). Monomers of fibrin rapidly polymerise into cross-linked strands which are finally stabilised by factor VIII (activated by thrombin) to form the skeleton in the arterial clot together with the platelets. Several anti coagulant proteins are involved in the counter regulation of the coag- ulation system. One of the most important inhibitors is antithrombin, which acts by forming inactivated complexes with thrombin, Thrombin- antithrombin complex (TAT). The inhibition of antithrombin is markedly accelerated in the presence of heparin. Thus, TAT can be detected in plasma after thrombus formation and is con- 76 sidered to reflect thrombin production and activity. The half-life of the complexes is thought to be approximately 5 minutes (20). In the present study fibrinogen, TAT and SF were used as markers of coagulation activity for the investigation of influences of abciximab on the coagulation system. Fibrinolysis (Proteins indicated with bold text were used in the study) The fibrinolytic system has a very important role in balancing the coagulation cas- cade by inhibiting formation of thrombi and dissolving existing clots (21) (figure 3). The key protein, plasmin, acts by degrading fibrin into its soluble degradation com- ponents, one of which is D-Dimer. Plasmin is produced on the fibrin surface in the blood, by cleavage of the inactive pro-enzyme plasminogen. Plasminogen, in turn, is activated by plasminogen-activators. The most important plasminogen activator in the blood is tissue plasminogen activator (tPA), which is produced and released into the blood by endothelial cells as a response to various stimuli, for example ischemia. tPA has short half-life of about 5 minutes due a rapid removal from plas- ma by the liver and by complex formation with plasminogen activator inhibitor 1 (PAI-1). tPA antigen concentration therefore reflect the free tPA level as well as the tPA/ PAI-1 complex concentration (22). Free plasmin in plasma is extremely rapidly inactivated by �2-antiplasmin in order to reduce the potent effects of plasmin in the circulation. Thus, PAI-1 activity in plasma attenuates endogenous fibrinolysis and 77 Fig. 3. Coagulation system. increase fibrin concentration (22). PAI-1 is produced by endothelial cells and smooth muscle cells in the vessel wall and by macrophages in the liver and spleen as well as by adipose tissue. While the biosynthesis rate of PAI-1 is high, its short biological half-life (8–10 minutes) causes low plasma concentrations (23). Since PAI-1 belongs to a group of acute phase reactants, its activity in plasma can be increased by inflammatory stimuli as well as by high fibrinolytic activity. In the pre- sent study tPA and PAI-1 were used as markers of fibrinolytic activity. Inflammation Inflammation plays an essential role in the pathogenesis of atherosclerosis (7). The earliest evidence of the influence of inflammatory mediators are the “fatty streaks”, which are pure inflammatory lesions, containing monocyte-derived macrophages and T-lymphocytes. Continued inflammation at every stage of the disease is indicat- ed by an increased number of macrophages and lymphocytes, (24) secreting cytokines and hydrolytic enzymes. Sero-epidemiological and pathophysiological studies suggest that infectious organisms such as herpesvirus, Chlamydia pneumo- niae and Helicobacter pylori may play a role in the initiation and progression of ath- erosclerosis (25). The exact mechanisms for the regulation of the atheroma progres- sion are still however, largely unknown but contains a complex interaction between different cell-types and inflammatory mediators. Furthermore, the rupture of a fibrous cap and thus the initiation of the acute coro- nary syndrome involves inflammatory action (26) as reflected by an increased con- centration of activated macrophages and T-lymphocytes promoting degradation of extracellular matrix in the unstable plaque (27, 28). Increased concentrations of monocytes (29), lymphocytes (30), inflammatory mediators such as C-reactive pro- tein (CRP), (31) interleukin-6 (IL-6), serum amyloid protein A (32) as well as cytokines (33, 34) have been reported in patients with UA and myocardial infarc- tions at the time of hospital admission. Interleukin-6 (Il-6) is a cytokine with pro-inflammatory and pro-coagulant prop- erties affecting many different cell-types. It also affects endothelial function and platelet production and is the only cytokine known to induce the synthesis of all the acute phase proteins by the liver. The production of Il-6 is stimulated by tumour necrosis factor and interleukin-1 (35, 36). C-reactive protein (CRP) is an acute phase protein synthesised by, and released from, the liver in response to circulating interleukin-6. CRP is a sensitive marker of infection, tissue damage and inflamma- tion (37). Its plasma half-life is rapid (approximately 19 hours) and identical under all circumstances. Thus, in contrast to other cytokines the concentration of CRP in plasma is mainly dependent on its production. The increased inflammatory activity in patients with unstable CAD may also, at least partly, reflect an acute phase reaction resulting from myocardial necrosis (38). In unstable CAD there is also evidence of a widespread inflammation in the coro- nary arteries but the actual source of inflammatory mediators is still unknown (39). However, ischemic episodes themselves, as reflected by ST-depressions, do not 78 seem to illicit CRP elevation in patients with unstable angina (40). Furthermore, recent evidence suggests that CRP itself might be involved in the pathogenic mech- anisms of the myocardial damage (41). CRP has been shown to display pro-inflam- matory properties (42) by co-localisation with and ligand binding to, complement in the myocardium (43). Activated complement may in turn mediate further vascular and myocardial destruction and induce arrythmias (41). In addition, CRP induces monocytes to express tissue factor, which may contribute to the development of dis- seminated intravascular coagulation and thrombosis in inflammatory states (44). Fibrinogen is an acute phase protein produced in the liver and exists in the circu- lation in increased concentrations in various inflammatory conditions. There is a high degree of correlation between levels of fibrinogen and several well-established clinical risk factors for cardiovascular disease (45). Since fibrinogen is both an inflammatory and coagulation mediator it is an unspecific marker reflecting increased coagulation as well as inflammatory activity. In the present study the above mentioned inflammatory markers were used for evaluation of a potential influence of abciximab on the inflammation system as well as regarding CRP, also for prediction of coronary events. Myocardial damage The troponin complex is formed by three different forms of structural proteins (tro- ponin C, I and T) located in the thin filament of the contractile apparatus of both skeletal and myocardial myocytes regulating the calcium dependent interaction between actin and myosin. Cardiac isoforms of troponin I and T are expressed sole- ly on myocardial cells and released from the cytoplasma after disintegration of the cell membrane caused by myocardial necrosis. Accordingly, measurable levels of troponin I or T are highly specific for myocardial damage indicating even micro- scopic areas of necrosis irrespective of the cause (46). The initial rise of troponin concentration occurs 3 to 4 hours after the ischemic injury with a persistent eleva- tion up to two weeks after the event. The MB isoform of Creatine kinase (CK-MB) is a cardiac specific enzyme also useful for detection and exclusion of myocardial damage. However, the specificity as well as the sensitivity is lower than for tro- ponins. Thus, CK-MB is regarded as the second best alternative for diagnostic and prognostic purposes. Myocardial dysfunction Brain natriuretic peptide (BNP) is a neurohormone synthesised and released from the cardiac ventricles in response to increased wall tension (47). By affecting the nephrons resulting in natriuresis, BNP and other natriuretic peptides are involved in the tight regulation of extracellular volume. The plasma level of BNP is increased in patients with heart failure and increases in proportion to the degree of left ventricu- lar dysfunction (48). BNP levels also increase after MI and in unstable angina pec- toris (49). BNP is produced as a pro-hormone, pro-BNP, which is enzymatically cleaved into BNP and the amino terminal portion of the pro-hormone, N-terminal 79 proBNP (NT-proBNP) (50). It has been shown that mean levels of NT-proBNP are similar to mean levels of BNP in healthy people whereas in response to cardiac impairment the absolute and relative increase of NT-proBNP exceeds that of BNP up to fourfold (50). RISK ASSESSMENT IN PATIENTS WITH ACUTE CORONARY SYNDROMES Clinical factors Patients with ACS are heterogeneous both regarding the clinical background, extent and severity of the underlying coronary disease. Several clinical (51) as well as bio- chemical and ECG indicators have been described by multivariable analyses (52). Age and male gender are among the most important clinical predictors associated with an unfavourable outcome. Various indicators of a history of cardiovascular dis- ease, such as diabetes mellitus, heart failure, hypertension and renal dysfunction are also important factors for risk assessment (52). Due to the heterogeneity of ACS- patients and the variable risk and alternative treatment strategies, a better and a more individualised risk assessment than clinical factors can offer, is needed. Electrocardiogram ST segment depression > 0.1 mV on the ECG on admission has consistently been found to indicate an increased risk of subsequent death or myocardial infarction (53) with a further increased risk in relation to the magnitude of ST-depression (54). Even ST-depressions of 0.05–0.1 mV provide prognostic information regarding future coronary events (55). T-wave inversion is a less specific ECG finding, con- cerning the diagnosis as well as the prognosis (56, 57). Continuous ST monitoring with 12-lead ECG or vectorcardiography better reflects the dynamic nature of myocardial ischemia than the occasional ECG recordings and adds to risk assessment(58) and identification of patients who bene- fit from extended anti-thrombotic treatment (59). Coronary angiography Coronary angiography provides an anatomic outlining of the coronary arteries and is considered the golden standard for the assessment of the existence, location and severity of the CAD. The number of diseased vessels (>50% stenosis) as well as the complexity of the lesions (60) and existence of visible thrombi (61) contribute to an increased risk. Furthermore, coronary angiography is a prerequisite for the decision on interventional procedures. However, still coronary angiography is an invasive procedure with an inherent, although low, risk of adverse events with a not negligi- ble cost. Therefore, mainly patients with a high likelihood of unstable CAD and a moderate-high risk of subsequent events, based on other predictors, are suitable for an invasive assessment. 80 Markers of coagulation and fibrinolysis activity Activation of the coagulation and fibrinolytic systems, as demonstrated by elevated markers of thrombin generation, thrombin activity and fibrin turnover, have been demonstrated in the acute phase of unstable CAD and are associated with an adverse outcome (62–64). Also reduced fibrinolytic capacity has been associated with an increased risk of coronary events in community based populations (65) as well as in unstable angina (66). In addition, increased levels of PAI have been asso- ciated with an increased rate of events in survivors of MI (67). None of these hemo- static markers are however recommended for use as risk predictors in a clinical set- ting (68). Inflammatory markers Elevated markers of inflammatory activity are associated with an increased risk for future cardiovascular events in healthy individuals (69,70), as well as in patients with stable (71), and unstable CAD (72–75). In patients with unstable CAD the ery- throcyte sedimentation rate as well as CRP and fibrinogen levels were found to be higher in patients developing refractory unstable angina than in patients with an uneventful clinical course (76). Elevation of IL-6 on admission for ACS seems to be associated with an increased risk of long-term mortality (77, 78) and also identifies those who derive the greatest benefit from an early invasive treatment (78). In large epidemiological studies, an increased fibrinogen concentration has been shown to predict future coronary events (79). Also in unstable CAD, fibrinogen lev- els have been shown to contribute to prediction of future risk of death and/or MI in short and long term, independent of troponin elevation and ECG changes (72). In unstable CAD, CRP elevation on hospital admission has been shown to be an independent predictor of mortality (72) (73–75). However, the association between the CRP level and the early risk of MI in unstable CAD has not been established, as most studies have presented small patient numbers and combined endpoints (table 3) (31, 71, 74, 80). Markers of myocardial damage Numerous previous studies have shown that troponin elevation is associated with an impaired outcome in patients with unstable CAD (81–83). It has been convincingly shown that elevation of troponin T raises the probability of significant coronary stenoses and advanced coronary artery disease i.e. three vessel disease and left main disease (84). Also, thrombus formation is more frequent in patients with troponin elevation (84). Recently it has been shown that even very low troponin levels just above the detection limit of the assay is associated with an increased risk of MI and death (84). However, few studies have had a sample size and event rate allowing the separate evaluation of the relations to death and MI separately. The raised mortality associated with troponin elevation is evident in short- as well as long-term (73) and independent of inflammatory activity, ECG changes and impaired renal function 81 82 Table 3. Published studies on the predictive value of CRP elevation in unstable coronary artery disease. In order of sample-size. SA denotes Stable angina pectoris; UA, Unstable angina pectoris; Atypical CP, Atypical chest pain; UCAD, Unstable coronary artery disease ie. unstable angina pectoris + non-ST elevation MI, Myocar- dial infarction; Emerg. revasc., Emergency revascularization; Refract. angina, Refractory angina. D.L, Detection Limit. (85). Identification of patients with troponin elevation is also useful for targeting therapy with low-molecular weight heparin (86), glycoprotein IIb/IIIa inhibitors (87, 88) and early revascularisation (89) as these patients derive a particular benefit from these treatment modalities. Markers of myocardial dysfunction It is well recognised that elevation of BNP indicates a worse prognosis in patients with heart failure and after MI (48). Recently it has been shown that elevation of BNP as well as NT-proBNP levels obtained after the acute phase (median time 40 hours) in patients with a broad range of ACS, independently predicts mortality (90, 91). On multivariate analyses, BNP levels have not been associated with subsequent MI (91). However, the relationship between levels of BNP or NT-proBNP and other risk-markers in the assessment of risk in ACS patients has not been fully elucidated. Renal dysfunction Renal insufficiency is associated with a worse prognosis in a wide spectrum of patients with cardiovascular disease, including ACS (92–94). A part of the increased risk associated with reduced renal function is attributable to a large number of co-existing conditions such as age, diabetes and hypertension. Still however, renal failure itself has consistently been shown to be associated with a worse prognosis(95). It has been proposed that renal dysfunction is a measure of the extent of vascular damage caused by a variety of insults on the endothelium. In many previous studies, patients with renal insufficiency have been excluded. Hence, the prognostic value of cardiac troponin and BNP in patients with and without renal dysfunction presenting with suspected acute coronary syndromes have not been defined. Risk scores and combinations of risk markers As several clinical, ECG and biochemical factors have been identified as indepen- dent markers of increased risk of subsequent cardiac events, the combinations of different markers have been evaluated. Several risk-scores have been developed from large clinical trials of ACS patients, by adding different indicators after weighing their relative importance (96). Thus, by combining BNP, CRP and tro- ponin I, there was a near doubling of the mortality for each additional bio-marker that was elevated in a recent study of patients with ACS (97). The higher value of the risk-score, the more benefit from early interventional treatment is derived for ACS-patients according to the recently presented score from the FRISC II trial (98). Before the present study was performed, neither the relations between levels of NT-proBNP and levels of markers of myocardial damage, inflammation and clinical risk factors nor the combination these markers for risk assessment had been eluci- dated. 83 TREATMENT (Therapies marked with bold text were evaluated in the study) The aims of treatment in patients with unstable CAD are symptom relief, limitation of myocardial damage and prevention of future coronary events i.e. myocardial infarction and death. Anti-ischemic drugs reduce oxygen utilisation by decreasing heart rate, contractility and blood pressure and inducing vasodilatation. �-blocking agents exert their effects by inhibiting �-receptors in the myocardium and thereby decreasing oxygen consumption. Treatment of ACS patients with �-blockers is associated with a 13 % reduction in the progression to acute myocardial infarction (99). Nitrates and calcium channel blockers are vasodilating agents relieving pain but not convincingly shown to reduce coronary events (100). Platelet inhibition As platelet aggregation plays a central role in the pathogenesis of ACS, antiplatelet regiments are essential to reduce thrombus formation and peripheral embolization of thrombotic material in the coronary arteries. Aspirin Aspirin irreversibly inhibits cyclooxygenase, impairing prostacyclin metabolism and thromboxane A 2 synthesis predominantly in platelets. As a result, platelet acti- vation and aggregation in response to collagen, ADP and thrombin is inhibited. However, at higher concentrations, thrombin and ADP still can activate platelets in the absence of thromboxane A 2 . A large number of trials have shown that aspirin reduces death and MI in patients in unstable angina, even in low doses (101). A loading dose of 300 mg is therefore recommended in all patients with ACS, followed by long term treatment (68). ADP receptor inhibitors Inhibition of the ADP receptor of the platelets results in decreased platelet aggrega- tion. In a large clinical trial including patients with unstable angina confirmed by ECG changes or elevation of biochemical markers, Clopidogrel on top of aspirin was found to reduce the incidence of cardiac events significantly during a median follow-up of 9 months (102). Particularly, the incidence of MI was reduced. Fur- thermore, treatment with Clopidogrel in combination with aspirin is considered a routine treatment at least one month after implantation of a coronary stent due to its superior effect in reducing acute stent-thromboses (103). Glycoprotein IIb/IIIa inhibitors Activation of the GP IIb/IIIa receptor is considered the final common pathway in platelet aggregation. Blocking this receptor almost completely abolishes aggrega- tion of platelets. Abciximab is the Fab fragment of a monoclonal antibody 7E3, binding with strong affinity to the GP IIb/IIIa receptor. In contrast to other GP 84 IIb/IIIa inhibitors, abciximab has a long half-life and binds to the vitronectin (� v � 3 ) and other receptors. By interaction with these receptors, abciximab has been sug- gested to influence also systems of coagulation and inflammation. Glycoprotein IIb/IIIa receptor inhibitors have consistently been shown to reduce the rate of procedure related myocardial infarctions in patients undergoing percuta- neous interventions in a large number of trials (104–108). Long term mortality is reduced in patients treated with abciximab and heparin in conjunction with coronary stenting as compared to patients on sole heparin treatment (109). Furthermore it has been shown that GP IIb/IIIa inhibitors, on top of aspirin and heparin, have reduced the rate of death and myocardial infarctions in special patient settings with unstable angina and non q-wave myocardial infarctions (110–113). The benefit has been shown to be most pronounced in high-risk patients with elevated troponin levels (87, 88) and in patients treated with early coronary interventions (52, 114). Before the performance of the GUSTO-IV trial, which constitutes the basis of the present study, no GP IIb/IIIa inhibitor had been evaluated in a pure non-interventional set- ting. However, in the CAPTURE trial the administration of abciximab to high-risk ACS patients for 18 to 24 hours before percutaneous intervention reduced the pre- intervention incidence of MI by 70% (relative risk reduction; 2.1% to 0.6 %, P=0.029) (105). Several oral GP IIb/IIIa inhibitors have been tested in large trials without any evi- dence of benefit. In fact, the therapies have been associated with an increased inci- dence of bleedings and a modest increase in mortality (115), illustrating the poten- tial risk with this type of treatment. Coagulation inhibitors Unfractionated heparin (UFH) is a heterogeneous mixture of sulphated polysac- carides of varying chain length increasing the effects of antithrombin, which most important way of action is inhibition of thrombin. UFH is effective in reducing the incidence of myocardial events in aspirin treated patients with unstable angina, although the number of clinical trials, and the number of patients included in those trials are relatively few (116). Nevertheless, in an international perspective, UFH treatment is regarded standard treatment in patients with unstable CAD. Low-molecular weight heparins (LMWH) are fragments of unfractionated heparin that possess a greater anti-Xa activity in relation to anti-IIa (anti-thrombin) than UFH. In addition, LMWH, have several potential advantages over UFH (117, 118). The dose response is more predictable and reliable, the immunogenicity is reduced with less frequent thrombocytopenia, and finally there is less rebound effect after discontinuation of therapy. Other advantages from a practical point of view are; longer biological half-life enabling easier administration with subcuta- neous injections and less need for monitoring the anticoagulant effect. There is con- vincing evidence that LMWH is more effective than placebo in reducing cardiac events (119). Furthermore, LMWH has been shown to be at least as effective as UFH in short-term. Dalteparin was shown to be similar to UFH (120), while 85 Enoxaparin was significantly more effective than UFH in the reduction of the car- diac events in large trials (121, 122). The direct thrombin inhibitor Hirudin has been evaluated in two large scale trials and found to be slightly more effective than heparin in the short-term, without long- lasting effects. In spite of treatment with a combination of aspirin and short term LMWH, there is still a 15–25% risk for recurrent ischemic events the first month after the ACS (119) (121, 122). Thus, there is a compelling need for improvement of long-lasting treatment effects. Complications due to antiplatelet and anticoagulation regimens Thrombocytopenia may occur as a result of treatment with UFH as well as GP IIb/IIIa inhibitors. However, the incidence is fairly low and the condition is reversible upon discontinuation of the drug infusion. Mild thrombocytopenia (<100.000/ml) has been reported in 2.5–5.6 % and severe (<50.000/ml) in 0.9–1.6% among abciximab and heparin treated patients (123). With “small molecule” GpIIb/IIIa inhibitors, somewhat lower incidence numbers have been reported (124) (125). Combining the anti-platelet properties of a GP IIb/IIIa inhibitor with the antico- agulant effect of a LMWH have theoretical advantages which may offer clinical improvements in the medical treatment of patients with ACS. The risk of bleedings may however outweigh the potential treatment benefits. Thus, close monitoring and reporting side effects of new pharmacological agents or combination of agents are therefore important. Acute coronary syndrome trials with GP IIb/IIIa inhibitors in addition to UFH have reported major bleeding levels around 1.4 to 10.8% and minor bleedings in up to 13.1% (126). The intervention rates and definitions for major and minor bleedings have however varied. The safety of abciximab and enoxaparin as well as tirofiban and enoxaparin has previously been evaluated in rel- atively small series and seemed to be well tolerated with low and similar bleeding rates (127, 128). Coronary revascularisation The difference between a treatment strategy including early coronary angiography and revascularisation if suitable, as compared to a conservative medical strategy and revascularisation only in case of recurrent ischemia or ischemia at a pre-dis- charge exercise test has been evaluated in a number of clinical trials. Since the plan- ning of the GUSTO-IV trial, convincing evidence has emerged that an early inva- sive strategy is superior for patients with non-ST elevation ACS. In the FRISC II trial the composite primary endpoint of death or MI at 6 months was reduced signif- icantly from 12.1% to 9.4% (2). At one-year follow-up there was a significant reduction in mortality in favour of the invasive strategy (129). The results were con- firmed in the Tactics trial, in which the GP IIb/IIIa inhibitor tirofiban was used as adjunctive treatment in the invasive group (130). Also with enoxaparin as adjunc- 86 tive treatment, the primary composite endpoint was reduced with an interventional strategy in the RITA-III trial (131). Depending on the extent and characteristics of the coronary lesions, as identified by coronary angiography, revascularisation may be carried out by either percuta- neous coronary intervention (PCI) or coronary artery bypass grafting (CABG). In patients with left main disease or three-vessel disease, particularly in combination with left ventricular dysfunction, CABG is the treatment of choice by virtue of the well-documented reduction in mortality (132, 133). Due to a rapid progression in technology, including an increased utilisation of coronary stents and potent platelet inhibitors, PCI is however becoming increasingly used as the first line treatment alternative. In patients with one or two vessel disease the choice of PCI is indis- putable but also in patients with three-vessel disease the results appear to be as good as with surgery (134). However, in the present study (GUSTO IV) a primary med- ical treatment was evaluated and early coronary angiography was discouraged. AIMS The aims were to investigate a large cohort of patients with non-ST elevation acute coronary syndrome regarding: � Safety and efficacy of abciximab combined with dalteparin or unfractionated heparin. � Activation of inflammation, coagulation and fibrinolysis and the influence of abciximab when added to dalteparin. � The activation of inflammation and signs of myocardial damage for the separate prediction of death and myocardial infarction. � Signs of myocardial dysfunction for the separate prediction of mortality and sub- sequent myocardial infarction. MATERIALS AND METHODS Patients The Global Utilisation of Strategies To open occluded arteries-IV (GUSTO-IV) trial included 7800 patients with acute coronary syndromes between 1999 and 2000 (135) (figure 4) for evaluation of abciximab treatment without early revascularisa- tion. The primary endpoint in the trial was the occurrence, of death (of any cause), or myocardial infarction (MI), within 30 days after randomisation. In Scandinavia, Switzerland and selected sites in the US., 72 centres recruited all their patients (n=974) to the dalteparin substudy, referred to as the dalteparin cohort (figure 4). The non-dalteparin patients recruited in all other centres of the GUSTO IV trial (n=6826) are referred to as the UFH cohort. The primary objective of this substudy was to evaluate the rate of side-effects, during the initial seven days in 87 relation to the randomised abciximab or placebo treatment within the dalteparin cohort (randomised comparison) as well as between the dalteparin and UFH cohorts (non-randomised comparison). A special blood-sampling program for repeated analyses of inflammation and coagulation factors performed at 28 selected Swedish sites, enrolling 404 patients, which constituted 74.3% of the patients recruited in Sweden and 42% of the patients in the dalteparin cohort. Study Design Inclusion Patients with an acute coronary syndrome without persistent ST segment elevation were recruited. Eligible patients were 21 years of age with one or more episodes of angina lasting 5 min within the last 24 hours if they had either a positive car- diac troponin T or I test or 0.5mm of transient or persistent ST-segment depres- 88 Fig. 4. Study design of the main GUSTO IV-ACS trial with the sub-studies included in the thesis indi- cated. sion. Patients with a history of acute myocardial infarction distinct from the qualify- ing event within 10 days prior to enrolment were required to have new ST-segment depression and CK-MB levels below the upper limit of normal. Exclusion Exclusion criteria were myocardial ischemia precipitated by a condition other than atherosclerotic coronary artery disease, persistent ST-segment elevation myocardial infarction or new left bundle branch block, percutaneous coronary intervention (PCI) within previous 14 days, planned PCI or coronary bypass surgery within 30 days after enrolment, confirmed hypertension (systolic >180 mmHg or diastolic >100 mmHg), coexistent condition associated with limited life expectancy and a number of factors associated with an increased bleeding risk (135). Randomised and concomitant treatment The study was conducted in a double-blind fashion with patients randomly assigned to; abciximab therapy for 24 h (0.25 mg/kg bolus followed by a 0.125 ug/kg/min infusion up to a maximum of 10 ug/min for 24 h) followed by 24 h of placebo infu- sion, or abciximab therapy for 48 h (same bolus and infusion for total duration of 48 h), or matching placebo (bolus and 48 h infusion). All patients received 150–325 mg of aspirin as soon as possible after randomisation for long term treatment. The patients received a 70 U/kg unfractionated heparin (UFH) bolus (to a maximum of 5,000 U) followed by a continuous infusion of 10 U/kg/hour (to a maximum of 800 U/hour) titrated to maintain the aPTT between 50 and 70 seconds for 48 hours or dalteparin (120 IU/kg to a maximum of 10,000 IU) subcutaneously every 12 hours for 5–7 d or until a revascularisation procedure or discharge. Concomitant therapy with beta-blockers was strongly recommended. Use of all other cardiac medications was left to the discretion of the investigator. Coronary angiography was discouraged during or within 12 hours after the comple- tion of study agent infusion unless the patient had recurrent or continuing ischemia at rest associated with ischemic ST-T changes that was unresponsive to intensive medical therapy. If percutaneous coronary intervention was required during study drug adminis- tration, blinded crossover to active therapy with abciximab was provided. Definitions of clinical endpoints All patients had ECG examinations at baseline, 48 hours and at 30 days, as well as blood samples collected at baseline and 8, 16, 24, 36 and 48h after randomisation to be analysed in a central laboratory for CK-MB. Additional samples were to be col- lected at 0, 8, 16, and 24h after any ischemic episode lasting >20 minutes. MI was defined as either a new significant Q-wave ( 0.04 s or at least a quarter of the R- wave amplitude in two or more contiguous leads) or CK-MB 3 times upper limit of normal. For patients with CK-MB elevation at entry, a new episode of chest-pain in combination with a new CK-MB elevation was required for MI diagnosis the ini- tial 7 days as presented in detail in the GUSTO IV-ACS publication (135). Follow- 89 ing coronary by pass surgery a new significant Q-wave was the only criterion. A clinical endpoint committee blinded to treatment assignment adjudicated all sus- pected cases of myocardial infarction. During 30-days of follow-up, mortality and rate of adjudicated myocardial infarctions were recorded. At 12 months only all- cause mortality was collected. Bleeding classification Bleeding was classified as major, minor or insignificant by the TIMI criteria (136). Major bleeding during baseline hospitalisation was defined as either 1) intracranial haemorrhage or 2) bleeding associated with a haemoglobin drop greater than 50 g/L. Minor bleeding was defined as any of the following: 1) spontaneous gross hematuria or hematemesis; 2) observed blood loss with decrease of haemoglobin >3 but 50 g/L. Insignificant bleeding was defined as any bleeding not meeting the cri- teria for major or minor. Statistical methods According to the protocol the outcome analyses were made with the abciximab 24 hours and 48 hours treatment groups combined. The safety data were analysed for the abciximab groups separately but since there was no major difference between the abciximab groups these results were also presented with the groups combined. Comparisons were then made between the dalteparin and UFH cohorts. Compar- isons between groups were made with Chi2-test. Fischer’s exact test was used for groups with small numbers. For continuous skewed variables a non-parametric, Mann-Whitney, test was performed. Differences between groups were presented as odds ratios O.R. with 95% confidence interval. Differences in levels of biochemical markers were evaluated with non-parametric tests given that they were not normally distributed. Bivariate correlations were cal- culated with Spearman rank correlation coefficients. Significance tests of between- group comparisons were made with Kruskal-Wallis or Mann-Whitney tests and of within-group comparisons between different time-points with Wilcoxon signed rank test. Also, the change in median levels from the previous sample for the randomised treatment groups was evaluated with Kruskal-Wallis test. The material was divided into quartiles on the basis of levels of biochemical markers. Means were expressed with one standard deviation for continuous vari- ables and medians were shown with 25–75 percentiles for skewed variables. Differ- ences in categorical base-line variables between quartiles were evaluated with Chi2 tests for trend. Differences between mean or median values for continuous variables were evaluated with one way ANOVA or Kruskal-Wallis tests as appropriate. Skewed variables were log transformed for calculation of independent associations between the variables in a multiple linear regression analysis. Kaplan-Meyer esti- mates were used for evaluation of the occurrence of the individual endpoints death and myocardial infarction after enrolment. Logistic regression analyses were per- 90 formed for evaluation of the significance of predictors of MI at 30 days and mortali- ty at 30 days and one-year. Included variables are specified in detail in the respec- tive paper. Laboratory methods Venous blood samples were drawn from direct punctures at randomisation, 24, 48 and 72 hours. The blood was collected into vacutainer tubes containing sodium cit- rate (0.13 M) or EDTA. Plasma was prepared within 30 minutes of collection by centrifugation at 2000 × g at room temperature for 20 minutes. After centrifugation serum was frozen at –20˚C in aliquots and sent for central laboratory analyses of CK-MB levels. One aliquot of the serum samples at baseline was stored at –70˚C and sent in batches of 500 to the Department of Clinical Chemistry, University Hos- pital, Uppsala, Sweden for analyses of troponin T, C-reactive protein (CRP) and NT-proBNP. One batch was unfortunately lost during transportation. At selected Swedish sites all patients were recruited to a special blood-sampling program for analyses of inflammation and coagulation factors from serum-samples obtained at baseline, 24 hours, 48 hours and 72 hours. Troponin T levels were determined by a third generation assay on an Elecsys (Roche diagnostics) with the detection limit 0.01ug/L and a total coefficient of vari- ance, CV of 8% at 0.05 ug/L and 4.1 to 6.0% between 0.1 to 11 ug/L. CRP concentrations were measured with a high-sensitive chemiluminescent enzyme-labelled immunometric assay (Immulite CRP, Diagnostic Products Corpo- ration) with a detection limit of 0.1 mg/L. and a total CV of 5.6% at 2 mg/L and 5% at 10 mg/L. Plasma NT-proBNP was determined with a sandwich immunoassay on an Elec- sys 2010. The analytical range extends from 20 to 35.000 ng/L. At the central lab, the total CV was 3.3% (n=21) at a level of 209 ng/L and 3.0% (n=21) at a level of 7431 ng/L. In a healthy population (n=407) matched to the FRISC II population (2, 3) for age (range 40–75 years) and gender (32% females) the 97.5 percentile was 290 ng/L. Serum creatinine at baseline was analysed at the local laboratories for 7706 patients. The creatinine clearance rate was calculated with the Cockcroft and Gault equation with correction for gender: {(140-age) × (weight (kg)} / Serum creatinine (umol/L) (137). Measurements of plasma levels of Il-6, thrombin-antithrombin (TAT) com- plexes and tissue Plasminogen-activator (tPA) antigen were performed using com- mercial enzyme-linked immunosorbent assay (Elisa) kits (Quantikine from R&D systems, Abingdon, UK, Enzygnost TAT, Behring Diagnostics, Marburg, Germany and Imulyse tPA, Biopool, Umeå, Sweden). Soluble fibrin (SF) was analysed using a quantitative spectrophotometric assay (Berichrom FM from Behring Diagnostics, Marburg, Germany). Plasminogen-activator-inhibitor (PAI-1) activity was deter- mined by Chromolize PAI-1 (Biopool, Umeå, Sweden). 91 RESULTS The main GUSTO-IV trial Baseline characteristics As planned, GUSTO-IV-ACS enrolled 7800 patients between July 21, 1998 and April 21, 2000, in 458 hospitals in 24 countries. Western European sites enrolled 48% of the patients, Eastern Europe 31% and North America 14%. The mean age was 65 years (SD 11) and mean weight 77 kg (SD 14) and 62% were males. Most patients had a history of coronary artery disease, including myocardial infarction (31%), and previous revascularisation (16%). All baseline characteristics were bal- anced among the three treatment groups. At enrolment, 59% tested positive on a qualitative or quantitative assay for cardiac troponin T or I, 80% had ST-segment depression and 32% had both ST depression and an abnormal cardiac troponin. Primary and secondary endpoints The combined primary endpoint of death and MI at 30 days’ follow-up was similar among the three treatment groups: 8.0% with placebo, 8.2% with abciximab 24 hours and 9.1% with abciximab 48 hours (figure 5). Moreover, no significant differ- ences were apparent among the three treatment groups at any time point, 48 hours, 7 days and 30 days. The components of the combined endpoint were also evenly distributed among the three treatment groups. Within 30 days after enrolment coro- 92 Fig. 5. Kaplan-Meyer analysis regarding the primary outcome (death or myocardial infarction) in the main GUSTO-IV trial during the initial 30-days. nary revascularisation was performed in 30% of patients: percutaneous coronary intervention (PCI) 19% and bypass surgery 11%. Yet, only few patients (2,5%) underwent revascularisation within 48 hours, while on study treatment (PCI 1.6%, bypass surgery 0.9%). 93 Fig. 6. Primary endpoint events (death or myocardial infarction at 30-days) in pre-specified subgroups of the main GUSTO-IV trial. As might be expected, events (death and myocardial infarction) were more fre- quent among patients with high-risk characteristics, such as advanced age, ST-seg- ment depression, elevated cardiac troponin or diabetes mellitus than among those without such characteristics. However, no significant treatment effect of abciximab was apparent in any of these subgroups (figure 6). The dalteparin substudy of GUSTO-IV Baseline characteristics The randomised treatment groups were well matched regarding all baseline charac- teristics. However, as compared to the 6826 patients in the UFH treated cohort, the 974 patients in the dalteparin cohort constituted a higher risk population as indicat- ed by higher age and weight, more prior by-pass surgery, more often an evolving myocardial infarction and a positive troponin test at baseline. Safety Major bleedings (not related to coronary artery by-pass surgery) were rare in all groups, although numerically doubled by abciximab treatment in both the UFH and 94 Fig. 8. Result of the primary outcome analysis of the dalteparin substudy of the GUSTO-IV for sub- groups based on age. Major and minor bleedings presented for the abciximab and placebo groups in the unfractionated heparin and dalteparin cohorts. dalteparin cohorts (table 1). Minor bleedings occurred at similar low rates in the dalteparin and the UFH groups but were also doubled by abciximab treatment in both cohorts (table 1). In the dalteparin cohort, major and minor bleedings occurred in 5.0% in the abciximab group as compared to 1.8% in the placebo group (O.R. 2.71; 1.14–6.41). In the UFH cohort the difference between the abciximab and placebo groups were similar (3.8% vs. 1.8%: O.R. 2.17; 1.52–2.99). Particularly elderly (> 65 years) patients had an increased risk of major and minor bleedings with abciximab (figure 8). Stroke (any type) was an uncommon event in all groups in the trial (table 1). The highest stroke rate was found in the abciximab group (0.6%) of the dalteparin cohort, not statistically different from the placebo group (0%). The UFH treated patients experienced similar stroke rates (0.3% for both abciximab and placebo). Intracranial haemorrhage occurred only in one patient in the dalteparin cohort receiving abciximab 24 hours. Thrombocytopenia was more common with abciximab than placebo in the whole patient population. Severe thrombocytopenia (<100.000/ml) occurred only with abciximab, both in the dalteparin- (0.8% vs. 0.0%, n.s.) and in the UFH cohort (1.6% vs. 0.0%, n.s.). Moderate thrombocytopenia (<50.000/ml) was also more fre- quent with abciximab than placebo both in the dalteparin (3.1% vs. 1.5%, ns) and in the UFH cohort (6.3% vs. 0.9%, p<0.001). Thus, during abciximab treatment the risk of thrombocytopenia was halved in the dalteparin as compared to the UFH cohort (p<0.01). 95 Table 1. Safety at 7 days. Comparisons between placebo, abciximab (24 hours + 48 hours) and between dalteparin and heparin treated cohorts. Dalteparin Heparin Placebo Abciximab Placebo Abciximab N=328 N=646 N=2270 N=4556 %(N) %(N) %(N) %(N) Stroke 0 (0) 0.6 (4) 0.3 (7) 0.3 (14) Intracr. haemorrhage‡ 0 (0) 0.2 (1) 0 (0) 0.1 (4) Major bleeding§ 0.6 (2) 1.2 (8) 0.2 (5) 0.7 (34)** Minor bleeding§ 1.2 (4) 3.7 (24)* 1.5 (35) 3.0 (137)*** Insign. bleeding§ 24.7 (81)††† 46.4 (300)***, ††† 5.0 (113) 15.1 (690)*** Blood transfusion 4.9 (16)††† 4.3 (28) 1.9 (44) 3.0 (136)* Platelet count <100.000/ml 1.5 (5) 3.1 (20)††† 0.9 (21) 6.3 (285)*** <50.000/ml 0.0 (0) 0.8 (5) 0 (1) 1.6 (73)*** Platelet transfusion 0.6 (2) 1.7 (11) 0.1 (3) 1.2 (53)*** *=p<0.05, **=p<0.01, ***=p<0.001 Abciximab versus placebo. † p<0.05, ††† p<0.001. Dalteparin ver- sus heparin. ‡ Intracranial haemorrhage, § Bleedings according to TIMI-criteria, not related to coronary artery by-pass surgery. 96 Fig. 7. Results of the secondary endpoint, the outcome analysis, in the dalteparin substudy of the GUSTO-IV depicted in Kaplan-Meyer curves for the unfractionated heparin and dalteparin cohorts respectively. Efficacy The secondary endpoint, outcome at 30 days, did not differ significantly between placebo and abciximab in the dalteparin substudy. In the dalteparin cohort the rate of death or MI in the abciximab groups vs. placebo was 9.6% vs. 11.3% (O.R. 0.85; 0.58–1.25) while in the UFH cohort the corresponding event rates were 8.5 % vs. 7.6 % (O.R. 1.12; 0.95–1.34) (figure 7). Thus, there was a trend for a higher event rate in the dalteparin cohort than in the UFH cohort (O.R. 1.27; 1.01–1.58). Howev- er, when correcting for other known predictors of an adverse outcome in a forward stepwise multiple regression analysis the dalteparin treatment did not remain an independent predictor of death or MI at 30 days. Activation of coagulation, fibrinolysis and inflammation in relation to abciximab treatment Coagulation Levels of coagulation markers, TAT and SF, increased in the acute phase. Median TAT level increased significantly from baseline (3.1 ug/L) to 24 hours and stayed elevated at the same level at 48 hours (3.7 ug/L). At 72 hours the median level had decreased again and was not different from baseline. Median SF level also increased from baseline (20.0 ng/L) to 24 hours and remained elevated until 72 hours (23 nmol/L). At all time-points the median levels of the coagulation markers were similar in the three different randomised treatment groups. Neither the absolute nor the relative changes in median levels from previous sample differed between the groups at any time-point. Fibrinolysis The median tPA level showed a continuous rise from baseline (8.7 ug/L) until the last sample at 72 hours (17.5 ug/L). In contrast, the PAI-1 levels remained unchanged during the 72 hours sampling period. Neither the median levels at differ- ent time-points, nor the changes within the groups differed significantly between the randomised treatment groups. Inflammation The median level of Il-6 increased from baseline (5.4 ng/L) and reached its peak median level at 24 hours (7.8 ng/L). The median level decreased thereafter and was at 72 hours no longer statistically significant different from baseline. Median CRP level increased similarly from baseline (4.4 mg/L) to 24 hours (8.7 mg/L) but in contrast to Il-6, the median level remained significantly elevated until 72 hours. The median level of fibrinogen increased continuously from baseline (3.3 g/L) until the last sample at 72 hour (3.9 g/L). There were no differences in median levels or changes of levels of the inflammatory markers between the randomised treatment groups. 97 Biochemical markers for prediction of coronary events Markers of myocardial damage The median time from the onset of the qualifying episode of ischemic chest pain to randomisation was 9.5 (5.0–16.6) hours. Troponin T analyses were available from 7115 (91.2%) of the patients. The troponin T-levels ranged from 0 to 17.3 ug/l and the quartile limits were 0.01, 0.12 and 0.47 ug/L. The rate of the primary combined endpoint of the GUSTO IV-ACS study, death or MI, was increasing with higher tro- ponin T quartiles at all time-points of follow-up (p<0.001). Also mortality was markedly increasing with increased troponin T quartiles from 1.1% to 7.4% between the first to the fourth quartile at 30 days (figure 9 a). The rate of MI was 98 Fig. 9. Rate of a) death and b) MI at 48 h, 7 days and 30 days in relation to quartiles of Troponin T. The number of patients with events is noted under the bars. increasing from the first- to the second quartile and was at 30 days 2.5% vs. 6.7%. However, no difference was observed between the upper three quartiles (figure 9 b). There was even a trend to a lower rate of MI in the fourth quartile as compared to the third quartile (5.6% vs. 7.2%, p=0.055). In a multiple logistic regression analy- sis, increasing troponin T quartile was independently related to both death (O.R. 1.63; 1.43–1.87) and MI (O.R.1.23; 1.11–1.37) at 30 days (table 4). Any elevation of troponin T (> 0.01 ug/L vs. 0.01) however, provided a larger differentiation of high and low risk regarding both death and MI (O.R. 3.36;2.10–5.34 and 2.48; 1.79–3.42 respectively) Markers of inflammation CRP analyses obtained at baseline were available from 7108 (91.1%) patients. The range of CRP-levels was 0 to 489 mg/L with quartile limits 1.84, 3.96 and 9.62 mg/L. The rate of the primary combined endpoint, death or MI was significantly increasing with higher CRP quartiles at 30 days: 7.1%, 7.3%, 8.1%, 10.5%, p=0.001. This difference was entirely driven by the difference in mortality, which was observed already at 48 hours (figure 10a). At 30 days, mortality increased from 2% in the first quartile to 6.3% in the fourth quartile with increased rates also from the first to the second and from the third to the fourth quartiles. However, at no time-point was there any relationship between the rate of MI and the quartiles of CRP (figure 10 b). In the multiple logistic regression analysis increasing CRP quar- 99 Table 4. Multiple logistic regression analyses on Troponin T and CRP as predictors of death, MI and death or MI at 30 days. Death MI Death or MI O.R (95% C.I.) P O.R (95% C.I.) P O.R (95% C.I.) P Model 1 TnT quartiles* 1.63 (1.43–1.87) <0.001 1.23 (1.11–1.37) <0.001 1.39 (1.27–1.52) <0.001 CRP quartiles† 1.19 (1.05–1.35) 0.006 0.94 (0.85–1.04) 0.26 1.00 (0.92–1.09) 0.91 Model 2 TnT (ug/l) >0.01 vs. 0.01‡ 3.36 (2.10–5.34) <0.001 2.48 (1.79–3.42) <0.001 2.84 (2.14–3.76) <0.001 CRP (mg/l) >1.84 vs. 1.84‡ 1.72 (1.17–2.55) 0.009 0.76 (0.59–0.98) 0.033 0.93 (0.75–1.16) 0.53 Model 3 TnT (ug/l) >0.47 vs. 0.47‡ 2.47 (1.86–3.28) <0.001 1.22 (0.79–1.32) 0.89 1.51 (1.23–1.86) <0.001 CRP (mg/l) >9.62 vs. 9.62‡ 1.31 (0.98–1.74) 0.07 0.98 (0.76–1.25) 0.85 1.07 (0.87–1.31) 0.55 O.R = Odds ratio, C.I. = confidence interval, MI = myocardial infarction. All three models included: age, male gender, body-weight, smoking, previous angina, stroke, heart failure, diabetes mellitus, hypertension, hypercholesterolemia, previous revascularization, previous myocardial infarction, cur- rent treatment with �-blockers and ACE-inhibitors, aspirin treatment prior to inclusion, ST-depression 0.5 mm and randomized treatment (abciximab 24 hours, 48 hours or placebo). tiles independently predicted 30-day mortality (O.R. 1.19; 1.05–1.35), while there was no relationship to subsequent MI (table 4). Creatinine clearance Patients with renal dysfunction are at high risk partly because of the high preva- lence of multiple risk factors. In the GUSTO-IV population, a reduced creatinine clearance was significantly correlated with a large number of predictors of a worse outcome, such as diabetes, hypertension, age, heart failure, previous myocardial infarction (85) and elevation of CRP, troponin T and N-terminal proBrain Natriuret- ic Peptide (95). Still, a creatinine clearance below the 1st quartile (51 ml/min) was independently associated with mortality as well as subsequent myocardial infarction in multivariate analyses (95). 100 Fig. 10. Rate of a) death and b) MI respectively 48 h, 7 days and 30 days in relation to quartiles of CRP. The number of patients with events is noted under the bars. 101 Fig. 11. Kaplan-Meier survival curves regarding probability of (a) death during one-year and (b) myocardial infarction during 30-days of follow-up for patient strata based on quartiles of NT-proBNP. Hyperlipidemia (including elevated Lp(a) lipoprotein levels), the insulin resis- tance syndrome, and hyperhomocystinemia are other factors contributing to coro- nary artery disease in patients with renal dysfunction. The chronic anemia and vol- ume overload associated with severe renal dysfunction, may be important contribu- tors to an increased vascular stiffness, the development of heart failure and subse- quent mortality. Other specific cardiovascular risk factors contributing to the vascu- lopathy induced by the renal disease include secondary hyperparathyroidism, increased sympathetic-nerve activity caused by afferent renal reflexes, elevated lev- els of oxidized low-density lipoprotein, endothelial dysfunction and diminished vas- cular nitric oxide production (138). Moreover, a reduced secretion of erytropoetin and insulin-like growth factor in patients with renal dysfunction may also specifi- 102 Fig. 12. Multiple logistic regression analyses for the prediction of myocardial infarction at 30 days and death at 1-year follow-up. cally contribute to an increased risk of thrombotic cardiovascular events by an inhi- bition of vascular repair (139). Markers of myocardial dysfunction Determinations of NT-proBNP levels in serum samples obtained at randomisation were available from 6809 (87.3%) of the patients in the GUSTO-IV trial. The NT- proBNP levels ranged from 5.3 to 35000 ng/L with a median level of 669 (interquartile range 237–1869) ng/L. Increasing levels of NT-proBNP were independently positively associated with age, female gender, current smoking, diabetes mellitus, hypertension, previous myocardial infarction and heart failure but negatively with body-weight, hypercho- lesterolemia and the occurrence of ST-depression at baseline. NT-proBNP levels were also associated with time from symptom onset and the magnitude of myocar- dial necrosis i.e. troponin elevation. In addition, NT-proBNP levels were associated with renal dysfunction and inflammatory activity as reflected by levels of creatinine and CRP. There was an increased mortality among patients in increasing quartiles of NT- proBNP (figure 11). The Kaplan-Meier survival curves for the quartiles separated 103 Fig. 13. Mortality at 30-days follow-up among strata of patients based on quartiles of Troponin-T and quartiles of C-reactive protein. 104 a b early. Already at 48 hours after randomisation the difference in mortality between the quartiles was statistically significant (p=0.001), with a mortality of 0.2% (n=3), 0.4% (n=6), 0.4% (n=7) and 1.4% (n=23) respectively. The separation of the curves continued throughout the first year after the index event (p<0.001, log rank). Thus, at one-year follow-up the mortality was 1.8 % in31, 3.9% in 66, 7.7% in 131 and 19.2% in 327 in the respective quartile. At one year there was an exponentially increasing mortality in the entire spectrum of NT-proBNP levels with a mortality of 0.4% in 3 in the lowest decile (<98 ng/L) and 27.1% in 185 in the highest decile (>4634 ng/L). In a multivariable logistic regression analysis, adjusting for a large number of predictors of long-term mortality, increasing quartiles of NT-proBNP still independently contributed to the prediction of 1-year mortality (figure 12). The risk of subsequent MI after the index event was also increasing with increas- ing quartiles of NT-proBNP with an MI rate of 2.7% in 46, 5.4% in 91, 5.7% in 98 and 7.5% in 128 (p<0.001) for the respective quartile at 30-days follow-up (figure 11b). However, in a multivariable logistic regression analysis, age above 65 years, previous MI, creatinine clearance < 50.8 ml/min, angina pectoris, troponin T eleva- tion (>0.01ug/L) and ST-depression at baseline, but not the level of NT-proBNP constituted independent predictors of myocardial infarction at 30 days (figure 12). 105 c Fig. 14. Mortality at one-year follow-up among strata of patients based on quartiles of NT-proBNP and quartiles of creatinine clearance (a) Troponin-T (b) and C-reactive protein (c). Combinations of biochemical markers As both Troponin T and CRP were independent predictors of 30-day mortality (table 4), the prognostic value of combining these markers was also evaluated. The highest mortality at 30 days was found in the patients with both markers in the top quartiles, 9.1% and the lowest in the patients with both makers in the bottom quar- tiles, 0.3% (figure 13). Finally, the long-term prognostic value of different combinations of NT-proBNP, troponin T, CRP and creatinine clearance were evaluated as all these markers were independent predictors of one-year mortality. A very low mortality was found in patients with NT-proBNP in the bottom quartile in combination with creatinine clearance in the top quartile (0.3%) or in combination with troponin T or CRP in the bottom quartile (1.6%) (figure 14a, b, c). The highest one-year mortality, 25.7%, was found in patients with levels of NT-proBNP in the top and creatinine clearance in the bottom quartile. A similar high mortality was found in patients with NT-proB- NP in combination with troponin T or C-reactive protein levels in the top quartiles, 22.3 or 23.4 %. DISCUSSION Effect of abciximab and dalteparin treatment There was no benefit with abciximab administered intravenously during the first 24 or 48 hours after enrolment of patients with acute coronary syndromes who did not undergo early coronary revascularisation. These findings are in sharp contrast to earlier investigations with abciximab in patients with refractory angina (105), in patients undergoing percutaneous coronary intervention (PCI) (104, 106, 140), and in studies with other parenteral glycoprotein IIb/IIIa receptor blockers (110, 111, 141). The reasons for these differences are unclear, and several possible explana- tions have been discussed. The inclusion of patients was based on chest pain and either elevated cardiac tro- ponin levels and/or ST segment depression >0.5 mm aiming at patients at a relative- ly high risk. Yet, the observed event rate in the placebo group, and in the trial over- all was lower (8%) than expected (11%). It is possible that the minimal duration of chest pain required (5 minutes) and the low level of ST-segment depression required, allowed enrolment if relatively low risk patients. However, the event rate was in fact similar or even higher than in other ACS-trials when differences in end- point definitions are taken into consideration (111, 119, 141, 142). In several sub- groups of patients the event rate was clearly higher but there was still no effect of abciximab treatment in these groups (figure 2). Based on the concept of a dominating role of platelet aggregation and coagula- tion activation in ACS, a combination of a GP IIb/IIIa inhibitor and a LMWH in the treatment of patients with non-ST elevation ACS, has several theoretical, in addition to the practical, advantages. Still, no treatment benefit of abciximab could be 106 observed in the dalteparin substudy that included a patient population at somewhat higher risk, resulting in significantly higher event rates than in the UFH cohort. The GP IIb/IIIa inhibitor abciximab, in addition to its anti-thrombotic effect, also has been suggested to suppress the inflammatory response in unstable CAD. An anti-inflammatory effect might be related to abciximab cross-reacting with other integrin receptors (143). In the present study however, abciximab did not suppress the activation of inflammation in unstable CAD. This is in contrast to the findings in a substudy of EPIC (144) in patients undergoing percutaneous coronary interven- tion. In the latter the suppression of inflammatory activity may have been related to the prevention of myocardial damage at the time of the intervention by abciximab therapy. In GUSTO-IV no reduction in myocardial damage by abciximab was observed, and the rate of coronary interventions was very low, according to the trial design (135). On the contrary, a possible pro-inflammatory effect of abciximab has emerged as one plausible explanation for unexpected lack of clinical benefit with abciximab in the GUSTO-IV ACS (145), as there even was a trend to a raised long-term mortality in the 48-hour abciximab group. In particular, an unfavourable trend with an excess in mortality was observed in patients with signs of inflammation and without evi- dence of ongoing intracoronary thrombosis, as reflected by elevated CRP levels and the absence of elevated cardiac troponin levels at enrolment (145). However, the results of the current study do not support such an explanation, as abciximab did not influence the development of indicators of inflammatory activity. There is evidence that high dose dalteparin in placebo controlled ACS trials reduces thrombin generation and activity, as demonstrated by the reduction of the F1+2, TAT and SF levels (146). In the present study TAT and SF levels still increased during the initial 72 hours of dalteparin treatment, demonstrating an acti- vation of the coagulation system. Although there was an activation of the coagula- tion and fibrinolytic system, there was no attenuation of the elevation of these by abciximab treatment. Previously a number of in vivo and in vitro studies have indi- cated that abciximab, by its blockade of the glycoprotein IIb/IIIa receptor, might have an indirect inhibiting effect also on the coagulation system (147, 148). Howev- er, the present study does not support any effect of abciximab on the coagulation system. Abciximab, in contrast to clopidogrel, is ineffective in reducing platelet-leuko- cyte aggregates and p-selectin expression in vivo and even increases expression of p-selectin, presumably caused by “outside-in” signalling when abciximab binds to the GP IIb/IIIa receptor (149). Formation of platelet-leukocyte aggregates by platelet p-selectin binding to leukocytes at the site of vascular injury increases thrombin generation and might be an important mechanism that contributes to haemostasis and thrombosis (150). However, the present results also refute any pro- coagulative effect of abciximab, at least in patients treated with dalteparin and aspirin. Thus, in a patient population with non-ST elevation ACS treated with aspirin and 107 low molecular weight heparin, the addition of abciximab infusion does not prevent an initial activation of the inflammation, coagulation or the fibrinolysis systems. Safety of abciximab and dalteparin treatment The safety of abciximab in addition to LMWH and aspirin in the treatment of ACS has not been investigated previously in a large-scale trial. In the current trial the numbers of major and minor bleedings were overall low and comparable to other trials of anti-thrombotic treatments in acute coronary syndromes (136). There were no significant differences in bleedings in the non-randomised comparison between full dose of dalteparin and a reduced dose of UFH (table 2). However the addition of abciximab was associated with a doubling in major and minor bleedings, both within the UFH and dalteparin cohorts, in the present trial. Acute coronary syndrome trials with GP IIb/IIIa inhibitors in addition to UFH have reported major bleeding levels in up to 10.8% and minor bleedings in up to 13% (123, 126). The safety of abciximab and enoxaparin combined as conjunctive thera- py to intervention was tested in the open label NICE 4 trial that included 818 patients which did not show an increased incidence of major bleedings or transfu- sions (0.2% major and 6.8 % minor bleedings, 1.2% transfusions) (151). Tirofiban and enoxaparin vs. UFH was evaluated in the ACUTE-II trial that included 525 patients presenting with unstable angina or suspected myocardial infarctions with- out ST-segment elevation and permitted coronary angiography after 24 hours. The combination treatment was well tolerated with low and similar bleeding rates in the two groups, i.e. major 1.0% vs.0.3% and minor 2.5 %vs. 4.3% (125). After the pub- lication of the present study, a randomised comparison between enoxaparin and UFH in ACS-patients treated with eptifibatide has also been published. Major bleedings were less frequent (1.8% vs. 4.6%), while minor bleedings were more fre- quent for enoxaparin than UFH (30.3% vs. 20.8%) (152). Taking differences in def- initions into account, these results are in accordance with the present findings of a similar rate of major and minor bleedings when UFH or dalteparin was combined with abciximab. The increased numbers of insignificant bleedings (<30 g/L fall in haemoglobin) in the dalteparin substudy was to a large extent attributable to the 108 Table 2. Independent predictors of bleedings in the GUSTO-IV ACS study. 95% C.I for O.R Independent predictors for major and minor bleedings* O.R Lower Upper Age >65 years† 2.81 2.08 3.80 Abciximab treatment 1.62 1.37 1.91 PCI†† during initial 7 days 1.81 1.96 2.57 * Bleedings according to TIMI-criteria, not related to coronary artery by-pass surgery. † Prespecified cut-off for the outcome analyses and the mean age. †† Coronary intervention. Included variables: age, sex, weight, history of stroke or TIA, diabetes, hypertension, smoking, dalteparin cohort, randomised abciximab treatment, clopidogrel, aspirin, oral anticoagulant and other lmw-heparin usage. subcutaneous heamatomas caused by dalteparin injections, which commonly create. Insignificant bleedings were particularly common for the patient groups with aug- mented risk of bleedings, such as elderly (Figure 8) and females. As expected thrombocytopenia was more common with the addition of abcix- imab both to UFH and dalteparin. Severe thrombocytopenia was however rare. Therefore concerning the risk of thrombocytopenia at abciximab treatment LMWH seems preferable to abciximab. Differential risk assessment with biochemical markers Troponin and CRP In concordance with other studies the current study showed that both troponin T and CRP were significant predictors of an adverse outcome in the early phase after an episode of ACS (31, 73, 75). In contrast to previous studies, both CRP and troponin T levels were available from a sufficiently large number of patients to allow prospective evaluation of their separate relationships to mortality and risk of MI. Furthermore, the independent associations to the different outcome events could also be demonstrated in multivariable analyses. Increasing troponin T levels were associated with a continuous rise in mortality. In contrast, any detectable troponin T, i.e. above 0.01ug/L, was associated with a raised risk of MI without any further risk at higher troponin T levels. Increased CRP levels during the acute stage of unstable CAD were related to increased mortality in accordance with previous findings (31, 72, 73, 75). In this large patient cohort the relationship to increased mortality was evident early and further accentuated throughout the 30-day follow-up. There was however no association between the CRP levels and the risk of MI. No previous study on inflammatory markers in ACS has contained a sufficient number of patients enabling separation of the endpoints death and MI. What might be the reason for the relationship between CRP and subsequent mor- tality but not MI in the acute phase of unstable CAD, in contrast to the well-estab- lished relationship between CRP elevation and subsequent coronary events in the chronic phase of atherosclerotic disease (69, 153–155)? In the acute phase of unsta- ble CAD the elevation of CRP level is transient and to a large extent caused by an acute phase reaction (156). Some unstable CAD patients might have a hyperrespon- siveness of the inflammatory system, which might exaggerate the acute phase reac- tion and increase the immune system reaction (157). Such a mechanism is support- ed by the observations of co-localisation of CRP and activated complement in infarcted myocardium (43). CRP may in itself contribute to inflammation by activa- tion of complement that in turn may mediate myocardial damage, induce arrythmias and provoke contractile dysfunction (41). Such an interpretation is in accordance with the relationships between the CRP level and the occurrence of cardiac rupture, left ventricular aneurysm formation and mortality after acute MI (158). Thus, the CRP elevation in unstable CAD might indicate a different process than the low- grade CRP elevation that is associated to subsequent coronary events among healthy 109 individuals(69, 153, 159) and in the chronic phase of atherosclerosis after myocar- dial infarction (160). In unstable CAD as well as in chronic atherosclerotic disease there is a lasting elevation of the fibrinogen level (156) that might indicate an underlying chronic low-grade inflammatory condition that in both conditions is associated with a raised risk of later MI. NT-proBNP in relation to other markers In the present study, from a large cohort of patients of non-ST elevation ACS, we demonstrated that baseline levels of NT-proBNP are independently related not only to age and female gender (161) but also to low body-weight and renal dysfunction. Part of this relationship might be explained by an increased sensitivity to volume overload, as BNP levels have been shown to be secreted at a response to volume overload and to raised intra-cardiac pressure (162) irrespective of the cause. The present study also demonstrated that levels of NT-proBNP were independently relat- ed to clinical factors indicating any kind of cardiovascular damage or dysfunction supporting that elevation of BNP (or NT-proBNP) is a general indicator of reduced cardiac performance rather than a specific indicator of systolic dysfunction (163). Moreover, our study demonstrated that ongoing myocardial damage (i.e. minimal troponin elevation), time since start of myocardial ischemia and damage and the inflammatory response (i.e. CRP elevation) were related to the magnitude of eleva- tion of NT-proBNP, further supporting the concept of BNP being a sensitive and rapid marker of reduced cardiac performance. This is in accordance with the recent report that BNP levels increase as a result of temporary occlusion of a coronary artery in conjunction with a coronary intervention (164) even when intracardiac fill- ing pressures remained unchanged (165). In the present study NT-proBNP levels were negatively associated with the presence of ST-depression (> 0.5mm) at base- line which, however, might be attributable to the fact that ST-depression was part of the inclusion criteria and the low level of > 0.5 mm for the definition of ST-depres- sion on admission. Recently it has been shown that elevation of BNP as well as NT-proBNP levels obtained after the acute phase (median 40 to 72 hours after symptom onset) in patients with a broad range of ACS independently predict mortality (90, 91). In the present study we extended these results in a considerably larger population of non- ST elevation ACS, for NT-proBNP obtained already on admission, at a median 9.5 hours after symptom onset in accordance with a previous study from our group in an unselected chest pain population (166). Thereby, we could demonstrate that NT- proBNP predicted one-year mortality in patients with blood samples obtained with- in 5.0 hours (first quartile) as well as more than 16.6 hours (fourth quartile) after symptom onset. The present study also demonstrated that any elevation of NT- proBNP above the 97.5 percentile, 290 ng/L, in a healthy population matched for age and gender, seemed to be associated with an increased risk of death after the index event. Despite the fact that the level of NT-proBNP was independently related to sev- 110 eral riskfactors, the NT-proBNP level still was the strongest independent indicator of mortality in the multivariate analysis. Also elevation of troponin T and CRP as well as reduced creatinine clearance rate (85) independently predicted an increased mortality. Accordingly, the combination of several of these markers allowed an even better stratification of future risk of fatal events. The combina- tion of quartiles of increasing NT-proBNP levels and quartiles of decreasing crea- tinine clearance rates provided the best prediction of long-term mortality. Among patients in every quartile of creatinine clearance, mortality was increased with increasing quartiles of NT-proBNP. The combination of quartiles of NT-proBNP and either quartiles of CRP or troponin T provided a similar prediction of mortali- ty. Interestingly however, elevated levels of troponin T seemed to contribute to an increased mortality only in patients with NT-proBNP levels in the top quartile. Thus, ACS-patients without myocardial dysfunction seem to tolerate even moder- ately large myocardial infarctions without a lethal outcome. On the other hand, ACS-patients with renal dysfunction, myocardial damage or increased inflamma- tory activity in addition to any reduction in cardiac performance, as indicated by a release of NT-proBNP, have a high risk of fatal complications to their heart dis- ease. In contrast to ST-depression and troponin elevation at baseline, the risk of sub- sequent MI at 30-days follow-up was not independently predicted by increasing levels of NT-proBNP in accordance with previous studies (167). The reason for this finding might be that BNP is a regulatory myocardial hormone which is not involved in the processes related to the rupture of a coronary plaques or formation of coronary thrombi. In contrast, elevation of BNP has been shown to predict sud- den death in patients with heart failure (168). Thus, the release natriuretic pep- tides from ventricular myocytes, in response to increased wall-tension due to ischemia or volume overload, might indicate a propensity to develop ventricular arrythmias, ventricular rupture or terminal heart failure rather than myocardial infarction. Clinical implications What biomarkers should be recommended in the early evaluation of patients with unstable CAD (figure 15)? For prediction of MI troponin elevation is the strongest biomarker and also reduced creatinine clearance is independently associated with subsequent myocardial infarction. However, for prediction of mortality, several clinical as well as ECG and biochemical markers seem to be useful. On multivari- able analysis, NT pro-BNP seems to be the strongest biochemical riskmarker. By virtue of being an unspecific marker of reduced cardiac performance NT proBNP seems to be very useful for selection of low-risk patients. A level below the 97.5 percentile (i.e. 290 ng/L) of a healthy population is associated with a very low mor- tality. In fact it also seems very unlikely that the patient has any significant heart disease at these low levels. Patients with elevation of NT pro-BNP on the other hand have increased risk of a fatal complication in relation to the level, which may 111 merit further investigation and treatment. It has been shown that Carvedilol treat- ment is particularly effective in patients with heart failure and elevated levels of NT-proBNP (169). However, it remains to investigate whether patients with high levels of NT-proBNP might derive a particular benefit from ACE-inhibition, early coronary interventions, implantable cardioverter-defibrillators (ICD) or other thera- peutic modalities. Patients with any detectable troponin elevation have an increased risk of death and derive a particular benefit from early coronary intervention (89), GP IIb/IIIa inhibition (88) (87) and extended low molecular weigh heparin treat- ment (86) (170). Also a moderate reduction in creatinine clearance is independently associated with an increased mortality which should be increasingly recognised although not possible to treat. CRP elevation is particularly useful for prediction of long-term mortality. There is evidence that statin treatment reduce CRP levels among survivors of MI and that statin therapy is particularly beneficial for patients with CRP elevation, independent of lipid levels (171). Furthermore, ST-depression upon admission as well as several baseline factors are useful for prediction of mor- tality. 112 Fig. 15. Risk indicators in unstable coronary artery disease. CONCLUSIONS In patients with non ST-elevation ACS not scheduled for early revascularisation: � Abciximab infusion on top of dalteparin and aspirin treatment was generally well tolerated although abciximab increased the number of bleedings as compared to placebo. In patients treated with abciximab, bleeding-rates were similar if com- bined with dalteparin than if combined with UFH. � Addition of abciximab to standard treatment with dalteparin as the primary treat- ment of ACS, did not reduce the rate of cardiac events at 30-days follow-up. � Despite dalteparin treatment there was a simultaneous activation of inflamma- tion, coagulation and fibrinolytic systems not influenced by abciximab infusion. � Baseline levels of troponin T and C-reactive protein were independently related to 30-day mortality. Any detectable elevation of troponin T, but not of C-reactive protein was also associated to a raised risk of subsequent MI. Concerning mortal- ity, the combination of both markers provided a better risk stratification than either one alone. � Increasing quartiles of NT-proBNP were independently related to short and long term mortality. The combination of NT-proBNP and creatinine clearance provid- ed the best prediction of one-year mortality. � A multimarker strategy with creatinine clearance, troponin, CRP and NT-proBNP together with ischemic ECG changes and clinical background characteristics has the potential to make risk assessment and clinical decision-making individualized and substantially improved. SUMMARY Patients with ACS constitute a heterogeneous population with different clinical his- tory, extent and severity of the coronary artery disease. In the assessment of risk of cardiac events, several clinical as well as ECG and biochemical markers are useful. Different biochemical markers obtained on admission provide different and comple- mentary prognostic information. By using a combination of biochemical markers an individualised and differentiated risk prediction can be made. Thus, with increasing levels of NT proBNP, CRP or troponin T there is a commensurate rise in short and long-term mortality that is independent of other risk indicators. Elevated levels of NT proBNP provide the strongest prediction of long-term mortality with a continu- ous increase in mortality in relation to the levels. The combination of NT-proBNP with creatinine clearance rate, or with levels of troponin T or CRP, provides a better risk stratification concerning the long-term risk of death in ACS patients than either one of the markers alone. At any detectable troponin level, in contrast to elevated levels of CRP or NT-proBNP, there is also a raised risk of a later myocardial infarc- tion independent from other risk indicators. 113 114 Addition of abciximab to the standard treatment with UFH or LMWH and aspirin as primary treatment of ACS is not associated with any significant reduction in car- diac events but a doubled risk of bleedings. If abciximab is used the combination with dalteparin seems as safe as the combination with UFH, although nuisance bleedings are more common. On the other hand thrombocytopenia is more rare. Despite full dose low molecular weight heparin and aspirin treatment there is still a simultaneous activation of the inflammation, coagulation and fibrinolysis systems in non ST-elevation ACS. Prolonged treatment with abciximab has no influence on the activation of these systems. Therefore, more effective attenuation of the coagu- lation and inflammation systems as well as reduction of myocardial damage and dysfunction might be new objectives for pharmacological stabilisation of unstable CAD. REFERENCES 1. Swedish Board of Health and Welfare (2002). 2002 yearbook. Internet. 2. FRISC-II-study-group (1999). Invasiv compared with non- invasiv treatment in unstable coro- nary-artery disease: FRISC II prospective randomised multicenter study. Lancet. 354:708–15. 3. FRISC-II-study-group (1999). Long-term low-molecular-mass heparin in unstable coronary- artery disease: FRISC II prospective randomised multicenter study. Lancet. 701–07. 4. Cannon CP, Weintraub WS, Demopoulos LA, et al. (2001). Comparison of early invasive and conservative strategies in patients with unstable coronary syndromes treated with the glycopro- tein IIb/IIIa inhibitor tirofiban. N Engl J Med. 344:1879–87. 5. Myocardial infarction redefined–a consensus document of The Joint European Society of Cardi- ology/American College of Cardiology Committee for the redefinition of myocardial infarction (2000). Eur Heart J. 21:1502–13. 6. Braunwald E (1980). Unstable angina. Heart disease- A textbook of cardiovascular medicine 1331–1332. 7. Ross R (1999). Atherosclerosis--an inflammatory disease. N Engl J Med.340:115–26. 8. Libby P, Ridker PM, Maseri A (2002). Inflammation and atherosclerosis. Circulation. 105:1135– 43. 9. Davies M (1997). The composition of coronary artery plaques. New England J Med. 1997:1312. 10. Yeghiazarians Y, Braunstein JB, Askari A et al. (2000). Unstable angina pectoris. N Engl J Med. 342:101–14. 11. Little WC, Constantinescu M, Applegate RJ, et al (1998). Can coronary angiography predict the site of a subsequent myocardial infarction in patients with mild-to-moderate coronary artery dis- ease? Circulation. 78:1157–66. 12. E.Falk M (1995). Coronary plaque disruption. Circulation. 92:657–671. 13. Fitzgerald DJ (2001). Vascular biology of thrombosis: the role of platelet-vessel wall adhesion. Neurology. 57:S1–4. 14. Berndt MC, Shen Y, Dopheide SM et al. (2001). The vascular biology of the glycoprotein Ib-IX- V complex. Thromb Haemost. 86:178–88. 15. Clemetson KJ (1995). Platelet activation: signal transduction via membrane receptors. Thromb Haemost. 74:111–6. 16. Ferguson JJ, Waly HM, Wilson JM (1998). Fundamentals of coagulation and glycoprotein IIb/IIIa receptor inhibition. Eur Heart J. 19 Suppl D:D3–9. 17. Moreno PR, Bernardi VH, Lopez-Cuellar J, et al. (1996) Macrophages, smooth muscle cells, and tissue factor in unstable angina. Implications for cell-mediated thrombogenicity in acute coronary syndromes. Circulation. 94:3090–7. 18. Furie B, Furie BC (1992). Molecular and cellular biology of blood coagulation. N Engl J Med. 326:800–6. 115 19. Mann KG (1999). Biochemistry and physiology of blood coagulation. Thromb Haemost. 82: 165–74. 20. Ardissino D, Merlini PA, Eisenberg PR et al. (1998). Coagulation markers and outcomes in acute coronary syndromes. Am Heart J. 136:S7–18. 21. Collen D (1999). The plasminogen (fibrinolytic) system. Thromb Haemost. 82:259–70. 22. Ottani F, Galvani M (2001). Prognostic role of hemostatic markers in acute coronary syndromes patients. Clin Chim Acta. 311:33–9. 23. Huber K, Christ G, Wojta J et al. (2001). Plasminogen activator inhibitor type-1 in cardiovascu- lar disease. Status report 2001. Thromb Res. 103 Suppl 1:S7–19. 24. Jonasson L, Holm J, Skalli O et al. (1986). Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis. 6:131–8. 25. Libby P, Egan D, Skarlatos S (1997). Roles of infectious agents in atherosclerosis and restenosis: an assessment of the evidence and need for future research. Circulation. 96:4095–103. 26. Falk E, Shah PK, Fuster V (1995). Coronary plaque disruption. Circulation. 92:657–71. 27. Moreno PR, Falk E, Palacios IF et al. (1994). Macrophage infiltration in acute coronary syn- dromes. Implications for plaque rupture. Circulation. 90:775–8. 28. Libby P (1995). Molecular basis for the acute coronary syndromes. Circulation. 91:2844–2850. 29. Jude B, Agraou B, McFadden EP, et al. (1994). Evidence for time-dependent activation of mono- cytes in the systemic circulation in unstable angina but not in acute myocardial infarction or in stable angina. Circulation. 90:1662–8. 30. Neri Serneri GG, Prisco D, Martini F, et al. (1997) Acute T-cell activation is detectable in unsta- ble angina. Circulation. 95:1806–12. 31. Liuzzo G, Biasucci LM, Gallimore JR, et al. (1994) The prognostic value of C-reactive protein and serum amyloid a protein in severe unstable angina. N Engl J Med. 331:417–24. 32. Biasucci LM, Vitelli A, Liuzzo G, et al. (1996). Elevated levels of interleukin-6 in unstable angi- na. Circulation. 94:874–7. 33. Neumann FJ, Ott I, Gawaz M, et al (1995). Cardiac release of cytokines and inflammatory responses in acute myocardial infarction. Circulation. 92:748–55. 34. Zhang R, Brennan ML, Fu X, et al. (2001). Association between myeloperoxidase levels and risk of coronary artery disease. Jama. 286:2136–42. 35. Le JM, Vilcek J (1989). Interleukin 6: a multifunctional cytokine regulating immune reactions and the acute phase protein response. Lab Invest. 61:588–602. 36. Barton BE (1996). The biological effects of interleukin 6. Med Res Rev. 16:87–109. 37. Yu H, Rifai N (2000). High-sensitivity C-reactive protein and atherosclerosis: from theory to therapy. Clin Biochem. 33:601–10. 38. de Beer FC, Hind CR, Fox KM et al. (1982). Measurement of serum C-reactive protein concen- tration in myocardial ischaemia and infarction. Br Heart J.47:239–43. 39. Buffon A, Biasucci LM, Liuzzo G et al. (2002). Widespread coronary inflammation in unstable angina. N Engl J Med. 347:5–12. 40. Liuzzo G, Biasucci LM, Rebuzzi AG, et al. (1996). Plasma protein acute-phase response in unstable angina is not induced by ischemic injury. Circulation. 94:2373–80. 41. Lagrand WK, Visser CA, Hermens WT, et al. (1999). C-reactive protein as a cardiovascular risk factor: more than an epiphenomenon? Circulation. 100:96–102. 42. Volanakis JE (1982). Complement activation by C-reactive protein complexes. Ann N Y Acad Sci. 389:235–50. 43. Lagrand WK, Niessen HW, Wolbink GJ, et al. (1997). C-reactive protein colocalizes with com- plement in human hearts during acute myocardial infarction. Circulation. 95:97–103. 44. Cermak J, Key NS, Bach RR, et al. (1993). C-reactive protein induces human peripheral blood monocytes to synthesize tissue factor. Blood. 82:513–20. 45. Meade TW, Chakrabarti R, Haines AP, et al. (1979). Characteristics affecting fibrinolytic activi- ty and plasma fibrinogen concentrations. Br Med J. 1:153–6. 46. Collinson PO, Boa FG, Gaze DC (2001). Measurement of cardiac troponins. Ann Clin Biochem. 38:423–49. 47. Levin ER, Gardner DG, Samson WK (1998). Natriuretic peptides. N Engl J Med. 339:321–8. 48. Valli N, Gobinet A, Bordenave L (1999). Review of 10 years of the clinical use of brain natri- uretic peptide in cardiology. J Lab Clin Med. 134:437–44. 49. Kikuta K, Yasue H, Yoshimura M, et al. (1996). Increased plasma levels of B-type natriuretic peptide in patients with unstable angina. Am Heart J. 132:101–7. 50. Hunt PJ, Richards AM, Nicholls MG, et al. (1997). Immunoreactive amino-terminal pro-brain natriuretic peptide (NT- PROBNP): a new marker of cardiac impairment. Clin Endocrinol (Oxf). 47:287–96. 51. Calvin JE, Klein LW, VandenBerg BJ, et al. (1995). Risk stratification in unstable angina. Prospective validation of the Braunwald classification. Jama. 273:136–41. 52. Boersma E, Pieper KS, Steyerberg EW, et al. (2000). Predictors of outcome in patients with acute coronary syndromes without persistent ST-segment elevation. Results from an international trial of 9461 patients. The PURSUIT Investigators. Circulation. 101:2557–67. 53. Nyman I, Areskog M, Areskog NH, et al. (1993).Very early risk stratification by electrocardio- gram at rest in men with suspected unstable coronary heart disease. The RISC Study Group. J Intern Med. 234:293–301. 54. Holmvang L, Clemmensen P, Wagner G, et al. (1999). Admission standard electrocardiogram for early risk stratification in patients with unstable coronary artery disease not eligible for acute revascularization therapy: a TRIM substudy. ThRombin Inhibition in Myocardial Infarction. Am Heart J. 137:24–33. 55. Hyde TA, French JK, Wong CK, et al. (1999). Four-year survival of patients with acute coronary syndromes without ST- segment elevation and prognostic significance of 0.5-mm ST-segment depression. Am J Cardiol. 84:379–85. 56. Miller WL, Sgura FA, Kopecky SL, et al. (2001). Characteristics of presenting electrocardio- grams of acute myocardial infarction from a community-based population predict short- and long- term mortality. Am J Cardiol. 87:1045–50. 57. Jernberg T, Lindahl B (2002). A combination of troponin T and 12-lead electrocardiography: a valuable tool for early prediction of long-term mortality in patients with chest pain without ST- segment elevation. Am Heart J. 144:804–10. 58. Jernberg T, Lindahl B, Wallentin L (2000). The combination of a continuous 12-lead ECG and troponin T; a valuable tool for risk stratification during the first 6 hours in patients with chest pain and a non-diagnostic ECG. Eur Heart J. 21:1464–72. 59. Jernberg T, Abrahamsson P, Lindahl B, et al. (2002). Continuous multilead ST-monitoring iden- tifies patients with unstable coronary artery disease who benefit from extended antithrombotic treatment. Eur Heart J. 23:1093–101. 60. Bugiardini R, Pozzati A, Borghi A, et al. (1991). Angiographic morphology in unstable angina and its relation to transient myocardial ischemia and hospital outcome. Am J Cardiol. 67:460–4. 61. Zhao XQ, Theroux P, Snapinn SM et al. (1999). Intracoronary thrombus and platelet glycopro- tein IIb/IIIa receptor blockade with tirofiban in unstable angina or non-Q-wave myocardial infarction. Angiographic results from the PRISM-PLUS trial (Platelet receptor inhibition for ischemic syndrome management in patients limited by unstable signs and symptoms). PRISM- PLUS Investigators. Circulation. 100:1609–15. 62. Oldgren J, Linder R, Grip L, et al. (2001). Coagulation activity and clinical outcome in unstable coronary artery disease. Arterioscler Thromb Vasc Biol. 21:1059–64. 63. Ardissino D, Merlini PA, Gamba G, et al. (1996). Thrombin activity and early outcome in unsta- ble angina pectoris. Circulation. 93:1634–9. 64. Kruskal JB, Commerford PJ, Franks JJ, et al. (1987). Fibrin and fibrinogen-related antigens in patients with stable and unstable coronary artery disease. N Engl J Med. 317:1361–5. 65. Meade TW, Ruddock V, Stirling Y, et al. (1993). Fibrinolytic activity, clotting factors, and long- term incidence of ischaemic heart disease in the Northwick Park Heart Study. Lancet. 342: 1076–9. 66. Munkvad S, Gram J, Jespersen J (1990). A depression of active tissue plasminogen activator in plasma characterizes patients with unstable angina pectoris who develop myocardial infarction. Eur Heart J. 11:525–8. 67. Hamsten A, de Faire U, Walldius G, et al. (1987). Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet. 2:3–9. 68. Bertrand ME, Simoons ML, Fox KA, et al. (2002). Management of acute coronary syndromes in patients presenting without persistent ST-segment elevation. Eur Heart J 23:1809–40. 116 69. Ridker PM, Cushman M, Stampfer MJ, et al. (1997). Inflammation, aspirin, and the risk of car- diovascular disease in apparently healthy men. N Engl J Med. 336:973–9. 70. Ridker PM, Hennekens CH, Buring JE, et al. C-reactive protein and other markers of inflamma- tion in the prediction of cardiovascular disease in women. N Engl J Med. 342:836–43. 71. Haverkate F, Thompson SG, Pyke SD, et al. (1997). Production of C-reactive protein and risk of coronary events in stable and unstable angina. European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group. Lancet. 349:462–6. 72. Toss H, Lindahl B, Siegbahn A, et al.(1997). Prognostic influence of increased fibrinogen and C- reactive protein levels in unstable coronary artery disease. FRISC Study Group. Fragmin during Instability in Coronary Artery Disease. Circulation. 96:4204–10. 73. Lindahl B, Toss H, Siegbahn A, et al. (2000) Markers of myocardial damage and inflammation in relation to long-term mortality in unstable coronary artery disease. FRISC Study Group. Frag- min during Instability in Coronary Artery Disease. N Engl J Med. 343:1139–47. 74. Heeschen C, Hamm CW, Bruemmer J, et al. (2000). Predictive value of C-reactive protein and troponin T in patients with unstable angina: a comparative analysis. CAPTURE Investigators. Chimeric c7E3 AntiPlatelet Therapy in Unstable angina REfractory to standard treatment trial. J Am Coll Cardiol. 35:1535–42. 75. Morrow DA, Rifai N, Antman EM, et al. (1998). C-reactive protein is a potent predictor of mor- tality independently of and in combination with troponin T in acute coronary syndromes: a TIMI 11A substudy. Thrombolysis in Myocardial Infarction. J Am Coll Cardiol. 31:1460–5. 76. Verheggen PW, de Maat MP, Cats VM, et al. (1999). Inflammatory status as a main determinant of outcome in patients with unstable angina, independent of coagulation activation and endothe- lial cell function. Eur Heart J. 20:567–74. 77. Koukkunen H, Penttila K, Kemppainen A, et al. (2001). C-reactive protein, fibrinogen, inter- leukin-6 and tumour necrosis factor-alpha in the prognostic classification of unstable angina pec- toris. Ann Med. 33:37–47. 78. Lindmark E, Diderholm E, Wallentin L, et al. (2001). Relationship between interleukin 6 and mortality in patients with unstable coronary artery disease: effects of an early invasive or nonin- vasive strategy. Jama. 286:2107–13. 79. Meade TW, North WR, Chakrabarti R, et al. (1980). Haemostatic function and cardiovascular death: early results of a prospective study. Lancet. 1:1050–4. 80. Rebuzzi AG, Quaranta G, Liuzzo G, et al. (1998). Incremental prognostic value of serum levels of troponin T and C-reactive protein on admission in patients with unstable angina pectoris. Am J Cardiol. 82:715–9. 81. Lindahl B, Venge P, Wallentin L. (1996). Relation between troponin T and the risk of subse- quent cardiac events in unstable coronary artery disease. The FRISC study group. Circulation. 93:1651–7. 82. Antman EM, Tanasijevic MJ, Thompson B, et al. (1996). Cardiac-specific troponin I levels to predict the risk of mortality in patients with acute coronary syndromes. N Engl J Med. 335: 1342–9. 83. Hamm CW, Ravkilde J, Gerhardt W, et al. (1992). The prognostic value of serum troponin T in unstable angina. N Engl J Med. 327:146–50. 84. Lindahl B, Diderholm E, Lagerqvist B, et al. (2001). Mechanisms behind the prognostic value of troponin T in unstable coronary artery disease: a FRISC II substudy. J Am Coll Cardiol. 38:979–86. 85. Aviles RJ, Askari AT, Lindahl B, et al. (2002). Troponin T levels in patients with acute coronary syndromes, with or without renal dysfunction. N Engl J Med. 346:2047–52. 86. Lindahl B, Venge P, Wallentin L. (1997). Troponin T identifies patients with unstable coronary artery disease who benefit from long-term antithrombotic protection. Fragmin in Unstable Coro- nary Artery Disease (FRISC) Study Group. J Am Coll Cardiol. 29:43–8. 87. Hamm CW, Heeschen C, Goldmann B, et al. (1999). Benefit of abciximab in patients with refractory unstable angina in relation to serum troponin T levels. c7E3 Fab Antiplatelet Thera- py in Unstable Refractory Angina (CAPTURE) Study Investigators. N Engl J Med. 340: 1623–9. 88. Heeschen C, Hamm CW, Goldmann B, et al. (1999.) Troponin concentrations for stratification of patients with acute coronary syndromes in relation to therapeutic efficacy of tirofiban. PRISM 117 Study Investigators. Platelet Receptor Inhibition in Ischemic Syndrome Management. Lancet. 354:1757–62. 89. Morrow DA, Cannon CP, Rifai N, et al. (2001). Ability of minor elevations of troponins I and T to predict benefit from an early invasive strategy in patients with unstable angina and non-ST elevation myocardial infarction: results from a randomized trial. Jama. 286:2405–12. 90. de Lemos JA, Morrow DA, Bentley JH, et al. (2001). The prognostic value of B-type natriuretic peptide in patients with acute coronary syndromes. N Engl J Med. 345:1014–21. 91. Omland T, Persson A, Ng L, et al. (2002). N-terminal pro-B-type natriuretic peptide and long- term mortality in acute coronary syndromes. Circulation 2002;106:2913–8. 92. Szczech LA, Best PJ, Crowley E, et al. Outcomes of patients with chronic renal insufficiency in the bypass angioplasty revascularization investigation. Circulation. 105:2253–8. 93. Best PJ, Lennon R, Ting HH, et al. (2002). The impact of renal insufficiency on clinical out- comes in patients undergoing percutaneous coronary interventions. Journal of the American Col- lege of Cardiology. 39:1113–9. 94. Reis SE, Olson MB, Fried L, et al. (2002). Mild renal insufficiency is associated with angio- graphic coronary artery disease in women. Circulation. 105:2826–9. 95. James S.K., Lindahl B., Siegbahn A., et.al. (2003). N-Terminal Pro-Brain Natriuretic Peptide and Other Risk Markers for the separate Prediction of Mortality and Subsequent Myocardial Infarc- tion in Patients With Unstable Coronary Artery Disease. Circulation 108:275–281. 96. Antman EM, Cohen M, Bernink PJ, et al. (2000).The TIMI risk score for unstable angina/non- ST elevation MI: A method for prognostication and therapeutic decision making. Jama. 284: 835–42. 97. Sabatine MS, Morrow DA, de Lemos JA, et al. (2002). Multimarker approach to risk stratifica- tion in non-ST elevation acute coronary syndromes: simultaneous assessment of troponin I, C- reactive protein, and B-type natriuretic peptide. Circulation. 105:1760–3. 98. Diderholm E (2002). Early Invasive Strategy in Unstable Coronary Artery Disease : Outcome in Relation to Risk Stratification. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 2002. 99. Yusuf S, Wittes J, Friedman L. (1988). Overview of results of randomized clinical trials in heart disease. II. Unstable angina, heart failure, primary prevention with aspirin, and risk factor modi- fication. Jama. 260:2259–63. 100. Held PH, Yusuf S, Furberg CD. (1989). Calcium channel blockers in acute myocardial infarction and unstable angina: an overview. Bmj. 299:1187–92. 101. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. Bmj 2002;324:71–86. 102. Yusuf S, Zhao F, Mehta SR, et al. (2001). Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation. N Engl J Med 345:494–502. 103. Smith SC, Jr., Dove JT, Jacobs AK, et al. (2001). ACC/AHA guidelines for percutaneous coro- nary intervention (revision of the 1993 PTCA guidelines)-executive summary: a report of the American College of Cardiology/American Heart Association task force on practice guidelines (Committee to revise the 1993 guidelines for percutaneous transluminal coronary angioplasty) endorsed by the Society for Cardiac Angiography and Interventions. Circulation 103:3019–41. 104. EPIC-investigators (1994). Use of a monoclonal antibody directed against the platelet glycopro- tein IIb/IIIa receptor in high-risk coronary angioplasty. The EPIC Investigation. N Engl J Med. 330:956–61. 105. The-CAPTURE-investigators (1997). Randomized placebo controlled trial of abciximab before and during coronary intervention in refractory unstable angina. Lancet 349:1429–1435. 106. The-EPILOG-investigators.(1997). Platelet glycoprotein IIb/IIIa receptor blockade and low-dose heparin during percutaneous coronary revascularization. The EPILOG Investigators. N Engl J Med. 336:1689–96. 107. Adgey AA (1998). An overview of the results of clinical trials with glycoprotein IIb/IIIa inhibitors. Eur Heart J. 19 Suppl D:D10–21. 108. Boersma E, Harrington RA, Moliterno DJ, et al. (2002). Platelet glycoprotein IIb/IIIa inhibitors in acute coronary syndromes: a meta-analysis of all major randomised clinical trials. Lancet. 359:189–98. 109. Topol EJ, Mark DB, Lincoff AM, et al. (1999). Outcomes at 1 year and economic implications 118 of platelet glycoprotein IIb/IIIa blockade in patients undergoing coronary stenting: results from a multicentre randomised trial. EPISTENT Investigators. Evaluation of Platelet IIb/IIIa Inhibitor for Stenting. Lancet. 354:2019–24. 110. PARAGON-investigators (1998). International, randomized, controlled trial of lamifiban (a platelet glycoprotein IIb/IIIa inhibitor), heparin, or both in unstable angina. The PARAGON Investigators. Platelet IIb/IIIa Antagonism for the Reduction of Acute coronary syndrome events in a Global Organization Network. Circulation. 97:2386–95. 111. PRISM-PLUS-investigators (1998). Inhibition of the platelet glycoprotein IIb/IIIa receptor with tirofiban in unstable angina and non-Q-wave myocardial infarction. Platelet Receptor Inhibition in Ischemic Syndrome Management in Patients Limited by Unstable Signs and Symptoms (PRISM-PLUS) Study Investigators. N Engl J Med. 338:1488–97. 112. Theroux P, Kouz S, Roy L, et al. (1996). Platelet membrane receptor glycoprotein IIb/IIIa antag- onism in unstable angina. The Canadian Lamifiban Study. Circulation. 94:899–905. 113. Boersma E, Akkerhuis KM, Theroux P, et al. (1999). Platelet glycoprotein IIb/IIIa receptor inhi- bition in non-ST-elevation acute coronary syndromes: early benefit during medical treatment only, with additional protection during percutaneous coronary intervention. Circulation. 100: 2045–8. 114. Ronner E, Boersma E, Akkerhuis KM, et al. (2002). Patients with acute coronary syndromes without persistent ST elevation undergoing percutaneous coronary intervention benefit most from early intervention with protection by a glycoprotein IIb/IIIa receptor blocker. Eur Heart J. 23:239–46. 115. Leebeek FW, Boersma E, Cannon CP, et al. (2002). Oral glycoprotein IIb/IIIa receptor inhibitors in patients with cardiovascular disease: why were the results so unfavourable. Eur Heart J. 23:444–57. 116. Oler A, Whooley MA, Oler J, et al. (1996). Adding heparin to aspirin reduces the incidence of myocardial infarction and death in patients with unstable angina. A meta-analysis. Jama. 276:811–5. 117. Weitz JI (1997). Low-molecular-weight heparins. N Engl J Med. 337:688–98. 118. Wallentin L (1996). Low molecular weight heparins: a valuable tool in the treatment of acute coronary syndromes. Eur Heart J. 17:1470–6. 119. Frisc-study-group (1996). Fragmin during instabilty in coronary artery disease (FRISC) study group. Low molecular weight heparin during instability in coronary artery disease. Lancet. 347:561–68. 120. Klein W, Buchwald A, Hillis SE, et al. (1997). Comparison of low-molecular-weight heparin with unfractionated heparin acutely and with placebo for 6 weeks in the management of unstable coronary artery disease. Fragmin in unstable coronary artery disease study (FRIC). Circulation. 96:61–8. 121. Cohen M, Demers C, Gurfinkel EP, et al. (1997). A comparison of low-molecular-weight heparin with unfractionated heparin for unstable coronary artery disease. Efficacy and Safety of Subcutaneous Enoxaparin in Non-Q-Wave Coronary Events Study Group. N Engl J Med. 337:447–52. 122. Antman EM, al e. (1999). Enoxaprin prevents death and crdiac ichemic events in unstable angi- na/non-q-wave myocardial infarction. Resulats from the Thrombolysis in Myocardial Infarction (TIMI) 11B trial. Circulation. 100:1593–1601. 123. Ferguson JJ, Kereiakes DJ, Adgey AA, et al. (1998). Safe use of platelet GP IIb/IIIa inhibitors. Eur Heart J. 19 Suppl D:D40–51. 124. Madan M, Berkowitz SD (1999). Understanding thrombocytopenia and antigenicity with glyco- protein IIb- IIIa inhibitors. Am Heart J. 138:317–26. 125. Cohen M, Theroux P, Borzak S, et al. (2002). Randomized double-blind safety study of enoxa- parin versus unfractionated heparin in patients with non-ST-segment elevation acute coronary syndromes treated with tirofiban and aspirin: the ACUTE II study. The Antithrombotic Combi- nation Using Tirofiban and Enoxaparin. Am Heart J. 44:470–7. 126. Blankenship JC (1999). Bleeding complications of glycoprotein IIb-IIIa receptor inhibitors. Am Heart J. 138:287–96. 127. Cohen E (2000). ACUTE II – Anti-thrombotic combination using Tirofiban and Enoxaparin. Abstract AHA 2000. 119 128. Kereiakes DJ, Grines C, Fry E, et al. (2001). Enoxaparin and abciximab adjunctive pharma- cotherapy during percutaneous coronary intervention. J Invasive Cardiol. 13:272–8. 129. Lagerqvist B, Husted S, Kontny F, et al. (2002). A long-term perspective on the protective effects of an early invasive strategy in unstable coronary artery disease. Two-year follow-up of the FRISC-II invasive study. J Am Coll Cardiol. 40:1902–14. 130. Cannon CP, Weintraub WS, Demopoulos LA, et al. (1998). Invasive versus conservative strate- gies in unstable angina and non-Q-wave myocardial infarction following treatment with tirofiban: rationale and study design of the international TACTICS-TIMI 18 Trial. Treat Angina with Aggrastat and determine Cost of Therapy with an Invasive or Conservative Strategy. Thrombolysis In Myocardial Infarction. Am J Cardiol. 82:731–6. 131. Fox KA, Poole-Wilson PA, Henderson RA, et al. (2002). Interventional versus conservative treatment for patients with unstable angina or non-ST-elevation myocardial infarction: the British Heart Foundation RITA 3 randomised trial. Randomized Intervention Trial of unstable Angina. Lancet. 360:743–51. 132. Yusuf S, Zucker D, Chalmers TC (1994). Ten-year results of the randomized control trials of coronary artery bypass graft surgery: tabular data compiled by the collaborative effort of the original trial investigators. Part 1 of 2. Online J Curr Clin Trials 1994; Doc No 145. 133. Yusuf S, Zucker D, Chalmers TC. (1994). Ten-year results of the randomized control trials of coronary artery bypass graft surgery: tabular data compiled by the collaborative effort of the original trial investigators. Part 2 of 2. Online J Curr Clin Trials 1994; Doc No 144. 134. Serruys PW, Unger F, Sousa JE, et al. (2001). Comparison of coronary-artery bypass surgery and stenting for the treatment of multivessel disease. N Engl J Med. 344:1117–24. 135. GUSTO-IV-ACS-investigators. (2001). Effect of glycoprotein IIb/IIIa receptor blocker abcix- imab on outome in patients with acute coronary syndromes without early coronary revasculariza- tion: The GUSTO IV-ACS randomized trial. Lancet. 357:1915–24. 136. Rao AK, Pratt C, Berke A, et al. (1988). Thrombolysis in Myocardial Infarction (TIMI) Trial-- phase I: hemorrhagic manifestations and changes in plasma fibrinogen and the fibrinolytic sys- tem in patients treated with recombinant tissue plasminogen activator and streptokinase. J Am Coll Cardiol. 11:1–11. 137. Cockcroft DW, Gault MH. (1976). Prediction of creatinine clearance from serum creatinine. Nephron. 16:31–41. 138. Becker BN, Himmelfarb J, Henrich WL, et al. (1997). Reassessing the cardiac risk profile in chronic hemodialysis patients: a hypothesis on the role of oxidant stress and other non-traditional cardiac risk factors. Journal of the American Society of Nephrology. 8:475–86. 139. Juul A, Scheike T, Davidsen M, et al. (2002). Low serum insulin-like growth factor I is associat- ed with increased risk of ischemic heart disease: a population-based case-control study.[com- ment]. Circulation. 106:939–44. 140. EPISTENT-investigators (1998). Randomised placebo-controlled and balloon-angioplasty-con- trolled trial to assess safety of coronary stenting with use of platelet glycoprotein-IIb/IIIa block- ade. The EPISTENT Investigators. Evaluation of Platelet IIb/IIIa Inhibitor for Stenting. Lancet. 352:87–92. 141. PURSUIT-investigators (1998). Inhibition of platelet glycoprotein IIb/IIIa with eptifibatide in patients with acute coronary syndromes. The PURSUIT Trial Investigators. Platelet Glycoprotein IIb/IIIa in Unstable Angina: Receptor Suppression Using Integrilin Therapy. N Engl J Med. 339:436–43. 142. Antman EM, Cohen M, Radley D, et al. (1999). Assessment of the treatment effect of enoxaparin for unstable angina/non-Q-wave myocardial infarction. TIMI 11B-ESSENCE meta-analysis. Cir- culation. 100:1602–8. 143. Tam SH, Sassoli PM, Jordan RE, et al. (1998). Abciximab (ReoPro, chimeric 7E3 Fab) demon- strates equivalent affinity and functional blockade of glycoprotein IIb/IIIa and alpha(v)beta3 integrins. Circulation. 98:1085–91. 144. Lincoff AM, Kereiakes DJ, Mascelli MA, et al. (2001). Abciximab suppresses the rise in levels of circulating inflammatory markers after percutaneous coronary revascularization. Circulation. 104:163–7. 145. Ottervanger JP, Armstrong P, Barnathan ES, et al. (2003). Long-term results after the glycopro- tein IIb/IIIa inhibitor abciximab in unstable angina: one-year survival in the GUSTO IV-ACS 120 (Global Use of Strategies To Open Occluded Coronary Arteries IV–Acute Coronary Syndrome) Trial. Circulation. 107:437–42. 146. Ernofsson M, Strekerud F, Toss H, et al. (1998). Low-molecular weight heparin reduces the gen- eration and activity of thrombin in unstable coronary artery disease. Thromb Haemost. 79:491–4. 147. Moliterno DJ, Califf RM, Aguirre FV, et al. (1995). Effect of platelet glycoprotein IIb/IIIa inte- grin blockade on activated clotting time during percutaneous transluminal coronary angioplasty or directional atherectomy (the EPIC trial). Evaluation of c7E3 Fab in the Prevention of Ischemic Complications trial. Am J Cardiol. 75:559–62. 148. Ammar T, Scudder LE, Coller BS. (1997). In vitro effects of the platelet glycoprotein IIb/IIIa receptor antagonist c7E3 Fab on the activated clotting time. Circulation. 95:614–7. 149. Klinkhardt U, Graff J, Harder S (2002). Clopidogrel, but not abciximab, reduces platelet leuko- cyte conjugates and P-selectin expression in a human ex vivo in vitro model. Clin Pharmacol Ther. 71:176–85. 150. Lindmark E, Tenno T, Siegbahn A. (2000). Role of platelet P-selectin and CD40 ligand in the induction of monocytic tissue factor expression. Arterioscler Thromb Vasc Biol. 20:2322–8. 151. Kereiakes DJ, Fry E, Matthai W, et al. (2000). Combination enoxaparin and abciximab therapy during percutaneous coronary intervention: “NICE guys finish first”. J Invasive Cardiol. 12 Sup- pl A:1A–5A. 152. Goodman SG, Fitchett D, Armstrong PW, (2003). Randomized evaluation of the safety and effi- cacy of enoxaparin versus unfractionated heparin in high-risk patients with non-ST-segment ele- vation acute coronary syndromes receiving the glycoprotein IIb/IIIa inhibitor eptifibatide. Circu- lation. 107:238–44. 153. Ridker PM, Buring JE, Shih J, et al. (1998). Prospective study of C-reactive protein and the risk of future cardiovascular events among apparently healthy women. Circulation. 98: 731–3. 154. Danesh J, Collins R, Appleby P, et al. (1998). Association of fibrinogen, C-reactive protein, albumin, or leukocyte count with coronary heart disease: meta-analyses of prospective studies. Jama. 279:1477–82. 155. Ridker PM, Rifai N, Pfeffer MA, et al. (1998). Inflammation, pravastatin, and the risk of coro- nary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and Recurrent Events (CARE) Investigators. Circulation. 98:839–44. 156. Oldgren J, Siegbahn A, Wallentin L (2001). Reversal of C-reactive protein but not fibrinogen elevation in unstable coronary artery disease. Eur Heart J. 22:1301. 157. Caligiuri G, Liuzzo G, Biasucci LM, et al. (1998). Immune system activation follows inflamma- tion in unstable angina: pathogenetic implications. J Am Coll Cardiol. 32:1295–304. 158. Anzai T, Yoshikawa T, Shiraki H, et al. (1997). C-reactive protein as a predictor of infarct expansion and cardiac rupture after a first Q-wave acute myocardial infarction. Circulation. 96:778–84. 159. Danesh J, Whincup P, Walker M, et al. (2000). Low grade inflammation and coronary heart dis- ease: prospective study and updated meta-analyses. Bmj. 321:199–204. 160. Ridker PM, Rifai N, Clearfield M, et al. (2001). Measurement of C-reactive protein for the tar- geting of statin therapy in the primary prevention of acute coronary events. N Engl J Med. 344:1959–65. 161. Redfield MM, Rodeheffer RJ, Jacobsen SJ, et al. (2002). Plasma brain natriuretic peptide con- centration: impact of age and gender. J Am Coll Cardiol. 40:976–82. 162. Osajima A, Okazaki M, Kato H, et al. (2001). Clinical significance of natriuretic peptides and cyclic GMP in hemodialysis patients with coronary artery disease. Am J Nephrol. 21:112–9. 163. Struthers AD (2002). Introducing a new role for BNP: as a general indicator of cardiac structural disease rather than a specific indicator of systolic dysfunction only. Heart. 87:97–8. 164. Kyriakides ZS, Markianos M, Michalis L, et al. (2000). Brain natriuretic peptide increases acute- ly and much more prominently than atrial natriuretic peptide during coronary angioplasty. Clin Cardiol. 23:285–8. 165. Tateishi J, Masutani M, Ohyanagi M, et al. (2000). Transient increase in plasma brain (B-type) natriuretic peptide after percutaneous transluminal coronary angioplasty. Clin Cardiol. 23: 776–80. 166. Jernberg T, Stridsberg M, Venge P, et al. (2002). N-terminal pro brain natriuretic peptide on 121 admission for early risk stratification of patients with chest pain and no ST-segment elevation. J Am Coll Cardiol. 40:437–45. 167. Omland T, de Lemos JA, Morrow DA, et al. (2002). Prognostic value of N-terminal pro-atrial and pro-brain natriuretic peptide in patients with acute coronary syndromes. Am J Cardiol. 89:463–5. 168. Berger R, Huelsman M, Strecker K, et al. (2002). B-type natriuretic peptide predicts sudden death in patients with chronic heart failure. Circulation. 105:2392–7. 169. Richards AM, Doughty R, Nicholls MG, et al. (2001). Plasma N-terminal pro-brain natriuretic peptide and adrenomedullin: prognostic utility and prediction of benefit from carvedilol in chron- ic ischemic left ventricular dysfunction. Australia-New Zealand Heart Failure Group. J Am Coll Cardiol. 37:1781–7. 170. Morrow DA, Antman EM, Tanasijevic M, et al. (2000). Cardiac troponin I for stratification of early outcomes and the efficacy of enoxaparin in unstable angina: a TIMI-11B substudy. J Am Coll Cardiol. 36:1812–7. 171. Ridker PM, Rifai N, Pfeffer MA, et al. (1999). Long-term effects of pravastatin on plasma con- centration of C-reactive protein. The Cholesterol and Recurrent Events (CARE) Investigators. Circulation. 100:230–5. About the author Stefan James received the Israel Hwasser Award from the Upsala Medical Association for the best thesis in clinical medicine in the academic year 2002/2003. He is at present Consultant at the Dept. of Cardiology, Uppsala University Hospital For correspondence: Stefan James, M.D., Ph.D. Dept. of Cardiology, Thoraxcenter, University Hospital, SE-751 85 Uppsala, Sweden Telephone: +46 18 6112818 Fax: +46 18 506638 E-mail: stefan.james@akademiska.se 122