a Department of Medical Sciences, Cardiology, University Hospital, Uppsala, Sweden
b Department of Cardiology, Sahlgrenska University Hospital, Gothenburg, Sweden
c Department of Cardiology, Karolinska Hospital, Stockholm, Sweden
d Department of Medicine and Cardiology, Aarhus University Hospital, Aarhus, Denmark
e Clinical Chemistry, University Hospital, Uppsala, Sweden
revised April 20, 2003; accepted April 24, 2003 * Correspondence: Jonas Oldgren, MD, Department of Medical Sciences, Cardiology, Uppsala University Hospital, SE-751 85 Uppsala, Sweden
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Abstract |
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Methods and results Three hundred and twenty patients were randomized to 72 h infusion with three different doses of inogatran, a direct thrombin inhibitor, or unfractionated heparin. There were no significant differences between the treatment groups in fibrinogen or C-reactive protein levels. Overall, the fibrinogen levels were significantly increased in the first 2496 h and still elevated at 30 days. The C-reactive protein levels showed a more pronounced increase during the first 2496 h, but then markedly decreased over 30 days. Troponin-positive compared to troponin-negative patients had higher fibrinogen and C-reactive protein levels up to 96 h, although there was an increase compared to pre-treatment levels in both groups. A high fibrinogen level (pre-treatment top tertile) was associated with an increased rate of death or myocardial (re-)infarction at 30 days, 13% vs 5.6%, P=0.03, and increased long-term mortality. A high C-reactive protein level was related to increased 30-day mortality, 4% vs 0%, P=0.01.
Conclusion Myocardial cell injury was related to a high degree of inflammation, only some of which is an acutephase response due to tissue damage. The rise in fibrinogen was sustained, which might reflect low grade inflammation with long-term risk of thrombosis. The transient elevation of C-reactive protein levels might indicate a propensity to a pronounced inflammatory response and is associated with increased mortality.
Key Words: Unstable coronary artery disease inflammation fibrinogen C-reactive protein troponin
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Introduction |
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Fibrinogen is an acute-phase protein directly involved both in platelet aggregation by cross-linking the glycoprotein IIb/IIIa-receptors on adjacent platelets,10 and in the coagulation cascade.11 C-reactive protein, another acute-phase protein, has an unclear biological function, but it has been suggested that C-reactive protein may also interact in the atherosclerotic process by activation of the complement system.12 The early levels of fibrinogen and C-reactive protein have in several studies been identified as indicators of increased short- and long-term risk for adverse clinical outcome in unstable coronary artery disease.7,1317 There is limited knowledge about the time-course and the influence of anticoagulant treatment of changesin fibrinogen and C-reactive protein in unstable coronary artery disease.7,18,19
The Thrombin Inhibition in Myocardial Ischemia (TRIM) study20 enrolled unstable coronary artery disease patients in Scandinavian centres during 1994 and 1995. The patients were randomized to three different doses of inogatran, a low molecular mass direct thrombin inhibitor,21,22 or standardunfractionated heparin, given as intravenous infusion for 72 h. The aim of the present substudy was to assess the degree of inflammatory activity, as reflected by the course of changes in fibrinogen and C-reactive protein levels in serial plasma samples, the influence of unfractionated heparin or different doses of inogatran on these levels, and their relation to manifestations of myocardial ischaemia.
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Methods |
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Patients were, within 24 h from the qualifying episode of chest pain, randomized to blinded treatment with unfractionated heparin or one of three different fixed doses of inogatran. Low, medium and high dose inogatran patients received intravenous bolus injections of 1.10; 2.75 and 5.50 mg respectively, followed by continuous infusion of 2.0; 5.0 and 10.0mg.h1respectively. Heparin was administered as a 5000 U intravenous bolus injection followed by infusion with 1200U.h1. All infusions were to be continued for 72 h. Aspirin was strongly recommended and given to 96% of the patients within the first day, but other platelet inhibitors and oral anticoagulants were notallowed.
Fibrinogen, C-reactive protein andtroponin T
Venous blood samples were obtained, preferably by direct venipuncture, into citrated tubes for analyses of fibrinogen and C-reactive protein and into heparin tubes for analysis of troponin T. Samples for analyses of fibrinogen and C-reactive protein were collected pre-treatment, during study drug infusion at 24 and 72 h and 24 h after cessation of infusion at 96 h, and finally at 30 days follow-up. For the analysis of troponin T, samples were drawn pre-treatment, at 6 and 12 h. The first 2 ml blood were disposed of and the samples were within30 min centrifuged at 2000 g for 20 min. Aliquots of 500 µl plasma in Eppendorf tubes were frozen and stored at 70°C until analysis.
Fibrinogen was analysed by rate nephelometry with a Beckman Array protein system (Beckman Instruments Inc). The assay was performed according to the recommendations of the manufacturer, except that goat antihuman fibrinogen (Atlantic Antibodies) was used. The assay was calibrated against a human plasma standard (Behring Diagnostics GmbH). C-reactive protein was analysed with the Immulite system, a chemiluminescent enzyme-labelled immunometric assay based on a ligand-labelled monoclonal antibody and separationby antiligand-coated solid phase (Immulite CRP, Diagnostic Products Corporation).23
Measurements of troponin T (ELISA Troponin(e) T) were carried out with an ES 300 analyser(Boehringer Mannheim GmbH). The discriminator value for myocardial cell injury recommended by the manufacturer was 0.1µg.l1,24 but evaluations were also performed using 0.06µg.l1as cut-off.
End-points
Clinical end-points were a composite of death or nonfatal myocardial (re-)infarction at 72 h (theend of infusion), 7 and 30 days. Myocardial(re-)infarction was diagnozed using standardclinical, ECG and cardiac marker criteria.20 Anindependent end-point committee evaluated all end-points.25
Long-term follow-up data were obtained from 286 of the 320 patients at a median of 29 months (range 1250 months). This information wasobtained from hospital records and local or national registries. If data in these sources were missing the information was collected by telephone interview.
Statistics
Differences in the levels of inflammation markers were judged with non-parametric tests, between-group comparisons with Mann-Whitney or Kruskal-Wallis tests as appropriate, and within-group differences between different time-points with Wilcoxon signed rank tests. The levels of inflammatory markers and their relation to clinicaloutcome were evaluated for the total substudy population. Fisher exact test (two-sided) or chi2tests as appropriate were used to judge significance of differences in proportions.
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Results |
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Baseline characteristics
Taking all treatment groups together, patients with high pre-treatment fibrinogen levels, i.e. in the top tertile, were older (median 68 vs 65 years, P=0.003) and had in higher proportion a history of stable angina >4 weeks (74% vs 62%, P=0.04), previous myocardial infarction (55% vs 42%, P=0.03) and congestive heart failure (26% vs 8%, P<0.001). Hypertension and diabetes mellitus were present in 39% and 17% of the patients, respectively, without differences in relation to pre-treatment fibrinogen levels. There were no differences in baseline characteristics in relation to pre-treatment C-reactive protein levels.
Time-course of changes in fibrinogen and C-reactive protein levels
Median time from onset of chest pain to randomization was 12 h, and 20% of the 320 patients were randomized within 6 h of symptom onset. Overall there was a significant increase in fibrinogen during the first 24 h after randomization, which seemed to reach its maximum after 7296 h (Table 1). After30 days the fibrinogen levels still remained significantly higher than pre-treatment. The increase in C-reactive protein levels was, as compared to changes in fibrinogen levels, more pronounced with a large upward dispersion during the first 2496 h (Table 1). In contrast to fibrinogen, the levels of C-reactive were markedly decreased from day 4 to day 30, when the level tended to be lower than pre-treatment.
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Clinical outcome in relation to fibrinogen and C-reactive protein
High fibrinogen levels, i.e. in the pre-treatment top tertile, were related to increased risk of ischaemic events at 30 days (Table 2). However, the rate of adverse ischaemic events tended to be lower during the ongoing anticoagulant treatment in patients with high pre-treatment fibrinogen, but within the first 4 days after cessation of treatment there was a clinical reactivation with a more than doubledischaemic event rate (Fig. 2). Thus, 12 of the 100 patients with pre-treatment levels of fibrinogenin the top tertile (and without ischaemic events during treatment) died or experienced a myocardial (re-)infarction from cessation of treatment to 30-days follow-up, as compared to seven (3.4%) of the 208 patients with lower pre-treatmentfibrinogen levels, P=0.003. Furthermore, a high pre-treatment level of fibrinogen was a predictor of long-term mortality (Fig. 3).
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Discussion |
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There are limited data concerning the inflammatory activity in patients with unstable angina in relation to signs of myocardial cell damage. Consistent with previous studies, the troponin-positive patients had significantly higher levels of fibrinogen and C-reactive protein pre-treatment and up to at least 96 h thereafter in the present study. This enhanced acute-phase reaction is probably induced by the myocardial cell damage.2628
In a previous study18 of unstable angina patients without troponin elevation, there was no increase in the levels of C-reactive protein up to 96 h after admission, despite ischaemic episodes during 24 h continuous ECG-monitoring in the majority of the patients. In contrast, elevated levels of C-reactive protein despite normal troponin T on admission were reported in another study of unstable angina patients.7 In eight of these 20 patients the C-reactive protein level was doubled at 2472 h after admission, and all of them had an inhospital major coronary event (death or myocardial infarction) or underwent urgent revascularization. The present study is thus the first to report a significant acute-phase response, with an early increase in fibrinogen and especially C-reactive protein, in a large number of unstable angina patients without any signs of myocardial cell damage. This acute-phase reaction indicates other sources of inflammatory activity than myocardial cell injury in the acute phase of unstable coronary artery disease.12 Recently, polymorphism in exon 2 of the C-reactive protein gene has been described, although noassociation with C-reactive protein regulation or concentration is known.29 One might speculate that differences in the acute-phase response in unstable coronary artery disease might reflect differences in the individual response to inflammatory stimuli.2,30 Interestingly, higher C-reactive protein levels have been reported in the offspring of patients with myocardial infarction.31 Thus, a low grade inflammatory activity, indicated by slight elevation of C-reactive protein and long-lasting fibrinogenelevation, might be associated with a propensity to a pronounced inflammatory response on plaqueruptures and/or thromboembolic myocardialdamage.
Several epidemiological studies have identified elevated levels of markers of inflammation, mainly C-reactive protein, as risk indicators for future cardiovascular events, both in apparently healthy men3235 and women.36 Furthermore, both fibrinogen and C-reactive protein have been found to indicate increased short- and long-term risk for adverse clinical outcome in unstable coronaryartery disease.7,1317
The differences in magnitude and time-courseof elevations of fibrinogen and C-reactive protein levels observed in the present study might indicate different underlying mechanisms of theirassociations to new ischaemic events in unstable coronary artery disease. A high fibrinogen level was related with a trend towards a lower rate of clinical events during ongoing anticoagulant treatment,but also marked clinical reactivation after cessation of treatment with a significantly higher rate of ischaemic events at 30 days. Fibrinogen is directly involved in the thrombotic process, both in platelet aggregation cross-linking the glycoprotein IIb/IIIa-receptors on adjacent platelets, and in the coagulation cascade where it is cleaved by thrombin to soluble fibrin which subsequently polymerises to form the fibrin network stabilising the platelet clot.11 The sustained high levels of fibrinogenmight thereby be associated with long-term risk of thrombosis and myocardial (re-)infarction.13
On the other hand, the C-reactive protein level was only related to increased mortality and not to myocardial (re-)infarction in the present as in other trials.13,16,37,38 Similar to these results, elevated levels of interleukin-6 have been related toincreased mortality, but not an increase in the combined end-point of death and myocardialinfarction, in patients with unstable coronaryartery disease.39 C-reactive protein is mainly regulated by interleukin-6, which is present in the atherosclerotic plaque and secreted by bothendothelial cells, smooth muscle cells, macrophages and T-cells.40 High levels of interleukin-6 may thereby reflect a greater atheroscleroticburden and/or increased inflammatory activity in the plaques. Thus, these plaques would be more vulnerable and prone to deeper fissuring, causing more severe thrombotic episodes.39 However, the largest increase in C-reactive protein was observed in patients with myocardial cell injury. Also the raised mortality was mainly seen in the top tertile of C-reactive protein. Therefore, much of the relation between the C-reactive protein level and mortality could be explained by an inflammatory reaction in the damaged myocardium.
In conclusion, myocardial cell injury, asdetected by elevated troponins, was associated with a higher degree of inflammation, only in part explained by an acute-phase response due to the tissue damage. The elevation of fibrinogen levels in the acute phase was sustained for at least 30 days, which might reflect a low grade inflammatorycondition in atherosclerotic lesions and a prothrombotic state, thus explaining the association between the fibrinogen level and the long-term risk for thrombosis and new ischaemic events. This risk seems diminished during treatment with thrombin inhibitors, but recurs early after cessation of treatment. The pronounced elevation of C-reactive protein levels in the acute phase was transient, which might indicate a propensity to a pronouncedinflammatory response at plaque ruptures and/or thromboembolic myocardial damage, both of which might be associated with increased mortality. The mechanisms of the inflammatory response in the acute phase of unstable coronary disease need to be further explored in order to understand their association with future cardiovascular incidents.
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Acknowledgments |
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References |
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