Published online before print April 20, 2006, doi: 10.1161/01.ATV.0000222908.78873.36
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© 2006 American Heart Association, Inc.
Thrombosis |
Soluble CD40L Levels Are Regulated by the –3459 A>G Polymorphism and Predict Myocardial Infarction and the Efficacy of Antithrombotic Treatment in Non-ST Elevation Acute Coronary Syndrome
From the Department of Medical Sciences, Clinical Chemistry (A.M., A.S.), Cardiology, Uppsala Clinical Research Centre (B.L., L.W.), Uppsala University, Sweden.
Correspondence to Agneta Siegbahn, Department of Medical Sciences, Clinical Chemistry, Uppsala University, S-751 85 Uppsala, Sweden. E-mail agneta.siegbahn{at}akademiska.se
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Abstract |
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Objectives— Current evidence suggests the CD40–CD40L pathway as a key process in the development, progression, and outcome of acute coronary syndrome (ACS). The aim was to investigate the prognostic importance of soluble (s) CD40L levels, single nucleotide polymorphisms (SNP) in the CD40LG gene, and the relation between sCD40L and SNPs in patients with acute coronary syndromes (ACS).
Methods and Results— Samples were obtained on admission from 2359 patients with non-ST elevation ACS randomized to an early invasive versus a conservative and to placebo controlled long-term dalteparin treatment in the FRISC-II study. The –3459 A>G SNP was identified as a novel regulator of sCD40L levels (P=0.001). In the placebo-treated group, sCD40L levels above median were associated with a 2.5-fold increased risk of myocardial infarction (MI) (P
0.001) but not with raised mortality. In the dalteparin treated group, sCD40L showed no association with MI (P=0.75). Consequently, dalteparin treatment was effective in reducing the risk of MI only in patients with sCD40L levels above median. A combined assessment of troponin-T and sCD40L complemented the prognostic information on risk of MI.
Conclusions— We identified a SNP in the CD40LG gene as a novel regulator of sCD40L plasma concentrations. Soluble CD40L levels above median reflect a prothrombotic state, which can be managed with the use of intense anti-thrombotic treatments.
Elevation of soluble CD40L was associated with myocardial infarction in the FRISC-II trial, which enrolled 2457 patients with acute coronary syndromes. A prolonged dalteparin treatment was more beneficial in patients with elevated sCD40L. The –3459 A>G SNP in the CD40LG gene was identified as a novel regulator of sCD40L levels.
Key Words: ACS • CD40L • myocardial infarction • outcome • SNP • thrombosis
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Introduction |
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Acute coronary syndrome (ACS) is a common complication of atherosclerosis in which inflammation is persistent because of activated cells within coronary lesions and to the systemic response thereof.1,2
During the past decade, the treatment strategies in ACS patients have substantially changed by the use of a combination of intense anti-thrombotic treatments in combination with early invasive coronary procedures. However, not all patients have the same benefit of all treatments. Thus, echocardiography signs of ischemia and biomarkers such as troponin-T (TnT) and IL-6 have been shown useful for the identification of patients with the largest benefits of early invasive treatment or anticoagulant treatments.3–6 To further improve the risk stratification and tailoring of treatment in ACS patients, new markers with predictive power for both short-term and long-term outcomes and that are able to discriminate between the risk of new thrombotic events such as myocardial infarction (MI) and the more unspecific risks of mortality are needed.7
CD40 is expressed on a variety of immune cells within atherosclerotic lesions including endothelial cells, smooth muscle cells, and monocytes/macrophages, whereas CD40L is largely expressed on CD4+ T-cells and activated platelets.8–11 Blocking experiments in hyperlipidemic mice have shown that the size of coronary plaques as well as inflammatory cell content can be decreased by using an inhibitory antibody against CD40L.12 In addition to the immunoregulatory properties by CD40–CD40L interaction, evidence suggests that tissue factor, the initiator of blood coagulation, is upregulated through CD40 ligation by activated lymphocytes and platelets.13 Membrane-bound CD40L may be proteolytically cleaved to form soluble CD40L (sCD40L). This form is present in the circulation and likely has similar activities in vivo as the membrane bound form.14 The source of sCD40L in blood is still not fully elucidated, although the general view is that activated platelets are the most important source.8
Elevated sCD40L levels at hospital admission have been associated with clinical outcomes in patients with ACS within a 6- or 10-month follow-up period.15,16 However, the previous studies used retrospective data or composite primary end points. Furthermore, CD40LG polymorphisms in relation to sCD40L levels and outcomes in ACS have not previously been studied. The aims of the present study were therefore to investigate whether elevated levels of sCD40L are associated with increased risk of death or MI, respectively, and if the risk increase conferred by elevated sCD40L could be reduced by an invasive treatment or a prolonged treatment with low-molecular-weight heparin. Moreover, the aim was to identify single nucleotide polymorphisms (SNP) in the CD40LG gene with a correlation to outcomes and/or to sCD40L levels.
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Methods |
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The patients in the present study participated in the Scandinavian multicenter trial FRISC-II, which compared the benefits of an early invasive procedure over an early noninvasive strategy and a 90-day prolonged treatment with a low-molecular-weight heparin (dalteparin) versus placebo.17,18 Patients were eligible if they had symptoms of ischemia that could be verified by electrocardiography or increased biochemical markers and if the last period of chest pain had occurred within 48 hours from assignment to open-label dalteparin (study randomization). All patients received basic treatment with aspirin and at least 5 days with subcutaneous open-label dalteparin. If not contraindicated, patients were randomized within 72 hours from admission to invasive/noninvasive treatment and secondly to long-term dalteparin/placebo. The present study included the patient group without contraindications to early invasive procedures, in which a complete 24-month follow-up was available19 (Figure 1). Of those, 2359 patients had plasma samples available for the analysis of sCD40L. The end point of MI was defined by the occurrence of 2 of 3 criteria, which were typical chest pain, diagnostic echocardiography recording (mainly new Q-wave), and raised CK, CK-B, CK-MB, or CK-MBmass according to the criteria of the local hospital. All causes of mortality were recorded. Events during the first 6 months were recorded and evaluated by an independent End Point Committee, whereas the information after this time were collected by investigator reports, outpatient visits, or telephone contact with surviving patients.19 The FRISC-II study protocol, including genetic studies, was approved by the regional ethical review board at Uppsala University.
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Blood Sampling
Whole blood and plasma samples were obtained at study inclusion and stored in –70°C until analysis. At least 60% of the samples were taken within 48 hours from onset of symptoms and 85% within 72 hours. Plasma levels of sCD40L were measured in citrated blood using the enzyme-linked immunosorbent assay technique (Bender MedSystems, Burlingame, Calif). Intra- and interassay coefficients of variation were 6.9% to 15.0% and 8.0% to 16%, respectively. Procedures for determination of plasma CRP, IL-6, pro-B-type natriuretic peptide, and TnT have previously been published.3,19,20 Creatinine clearance was calculated from serum creatinine using the Cockcroft-Gault algorithm.21 Prothrombin fragment 1+2 and D-dimer were previously measured and investigated in a subgroup of 591 FRISC-II participants.22
SNP Selection and Genotyping
The 6 SNPs investigated in this project were selected from the dbSNP NCBI database (supplemental Table II, available online at http://atvb.ahajournals.org). The statistical power to address study aims were considered in the SNP selection process and thus SNPs with a minor allele frequency <5% were ruled out. For genotyping, we used the 12-plex GenomeLab SNPStream system (Beckman Coulter) or the homogeneous template directed dye terminator assay with fluorescence polarization detection for individual SNPs. The primers for polymerase chain reaction and minisequencing were designed using the Autoprimer software (http://www.autoprimer.com; Beckman Coulter Inc). Primer sequences are available from the authors on request. The quality of the genotype data were assessed by testing for Hardy-Weinberg equilibrium using the 
2 distribution for each assay. All SNPs conferred to Hardy-Weinberg equilibrium. The overall genotype call rate was 97%, and the accuracy was 99.3% according to duplicate analysis of, on average, 13% of the total genotypes. DNA samples were available from 2205 patients. The SNP genotyping was performed by the SNP technology platform in Uppsala.
Data Analyses
The distribution of sCD40L was skewed and thus median values and 25th to 75th percentiles are presented. Group differences in levels of sCD40L were tested using the Mann-Whitney U test. Before the dichotomization of sCD40L levels we performed a trend analysis of sCD40L quartiles in relation to outcome. The MI incidence in quartiles 1 and 2 were at the same level, and lower, than in quartiles 3 and 4, also at a similar level. Therefore, sCD40L levels above and below median was used for subsequent analyses. Moreover, the predictive value of sCD40L above median was equal in patients randomized to noninvasive and invasive treatment but significantly different in patients randomized to placebo compared with those in the dalteparin-treated group. Tests of independence for categorical factors were performed using the 2 distribution with 2-sided exact probability values. The logistic regression models used adjusted for age, sex, invasive treatment, smoking, diabetes, hypertension, previous MI, ST-depression at entry, use of aspirin, angiotensin-converting enzyme inhibitor, ß-blocker and statins, cholesterol >5.5 mmol/L, TnT 
0.03 µg/L, C-reactive protein 10 mg/L, creatinin clearance, and pro-B-type natriuretic peptide. All odds ratios that appear in the text were corrected against these factors. The probability of myocardial infarction was estimated using the Kaplan-Meier method and factors were compared using log-rank testing; 
=0.05 was used as significance level. The statistics were calculated with the assistance of Biostatistician Lars Berglund at the Uppsala Clinical Research Center. We calculated statistics in SPSS for Windows XP 12.1.
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Results |
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Soluble CD40L Plasma Concentrations
In total, 7% (n=173) of the patients had undetectable (<95 pg/mL) sCD40L. The median level of sCD40L was 290 (25th to 75th: 190 to 490) pg/mL. Soluble CD40L above median level was associated with female gender and diabetes, but not with elevation of CRP, IL-6, or TnT in plasma. In contrast, patients with sCD40L above median were admitted with higher plasma levels of prothrombin fragment 1+2 and d-dimer. The baseline characteristics of the FRISC-II patients according to sCD40L levels below/above median are shown in Table 1.
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Soluble CD40L and Survival
The sCD40L median levels among survivors and patients deceased within the 24-month follow-up period were 290 (190 to 490) pg/mL and 280 (200 to 520) pg/mL, respectively (P=0.59). Accordingly, the median sCD40L level was similar in survivors and deceased patients also within the different treatment groups of dalteparin, placebo, invasive, and noninvasive. Thus, sCD40L levels above median did not represent an increased risk of death.
Soluble CD40L and Association With MI
The median sCD40L at admission was 330 (200 to 560) pg/mL in patients who experienced MI during follow-up and 280 (190 to 480) pg/mL in those who did not (P=0.005). This difference was evident also among the 723 patients with TnT levels <0.03 µg/L, 410 (240 to 700) pg/mL and 290 (190 to 470) pg/mL, respectively (P=0.01).
Analyses with respect to treatment groups revealed that sCD40L levels were strongly associated with subsequent MI in patients treated with placebo, with an 8% incidence of MI among patients with sCD40L below and a 15% incidence in those above median (P<0.001). In contrast, patients on dalteparin had a nonsignificant 1.5% difference in MI incidence between sCD40L below and above median (Figure 2). The association between MI and sCD40L levels was tested in a logistic regression model, adjusted for several other cofactors, with the result that sCD40L was still associated with MI in the placebo group (odds ratio=2.50, P<0.001), whereas no significant association with MI among patients on dalteparin was observed (supplemental Table I, available online at http://atvb.ahajournals.org).
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The relationship between sCD40L above median and MI was similar in patients who were assigned statin treatment at discharge (n=1040, 43%), and in those who were not.
Efficacy of Long-Term Dalteparin and Early Invasive Treatment According to sCD40L Levels
In the main report of the FRISC-II medical study, a short-term benefit of prolonged dalteparin therapy was demonstrated, which persisted only during the ongoing 90 days of treatment.17 At the 24-month follow-up, the incidence of MI was similar in patients treated with placebo and dalteparin.19 The results of the present study showed that in patients with sCD40L above median, the dalteparin treatment seemed to be associated with a reduced risk of MI, also at 24 months (adjusted odds ratio 0.7, P=0.06) (Table 2). Moreover, no relapse was apparent in this patient group, which is in contrast to patients with sCD40L below median treated with dalteparin for 90 days (Figure 2). In women with sCD40L above median, the prolonged dalteparin treatment seemed even more efficient in reducing the MI risk (adjusted odds ratio, 0.5; 95% confidence interval, 0.3 to 0.9) (P=0.03).
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Because patients with sCD40L above median level represented a high-risk patient group, the absolute difference in MI incidence between noninvasive and early invasive treatment was 5.0% in this group and only 2.2% in those with sCD40L below median (Table 2). However, the relative risk reduction by early invasive treatment was similar in patients with sCD40L below and above median, adjusted odds ratio 0.8, 95% confidence interval (0.5 to 1.0) and 0.7 (0.5 to 0.9), respectively.
Biomarker Combinations in Prognosis and Treatment Selections
Both sCD40L and TnT correlated with incidence of MI in the FRISC-II cohort but were not intercorrelated. We found that a combined assessment of sCD40L with TnT complemented the prognostic information on absolute risk of MI and death/MI (Table 3). Elevation of both biomarkers, however, did not seem to indicate a better relative efficacy of early invasive or prolonged dalteparin treatment (Table 2).
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Coagulation and inflammation are interconnected processes and therefore we investigated whether elevation of CRP in combination with sCD40L also provided prognostic information superior to that of either marker by itself. However, this combination did not add prognostic information on MI in comparison to sCD40L on its own (data not shown).
The results of these combined assessments suggest that TnT was informative concerning the risk of death and MI and the efficacy of invasive treatment, whereas sCD40L was associated with the risk of MI and the efficacy of prolonged dalteparin treatment.
CD40LG Gene Variations
The CD40LG gene is positioned on chromosome X q26.3-q27.1. Thus, only female heterozygotes are present, whereas males are hemizygous for either allele. Analysis of linkage disequilibrium showed a considerable covariation between alleles, as assessed by D' (not shown) and R2 (supplemental Table II). Of 6 SNPs studied, the –3459 A>G SNP was the one with the closest correlation to sCD40L levels (P=0.001) (Figure 3). This SNP was also the most frequent (23.6% of the patients carried the G allele in hemizygous, heterozygous or homozygous form, n=521). Partial correlations with sCD40L levels were observed for remaining SNPs, which were likely explained by the degree of covariation with –3459 A>G (supplemental Table II). Haplotypes from all genotyped SNPs were reconstructed but did not prove better markers for sCD40L levels than the –3459 A>G SNP (data not shown). Although significantly associated with high sCD40L levels, the –3459 G allele did not confer an increased risk of MI or mortality (data not shown).
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Discussion |
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The present study greatly outnumbers previous studies investigating sCD40L in patients with ACS and also allows for distinction between mortality and MI end points. Our investigation further established the importance of sCD40L in the pathophysiology of ACS and its usefulness as a biomarker in this disease. We identified the –3459 A<G SNP in the CD40LG gene, which proved to be a novel determinant of sCD40L plasma levels, suggesting increased CD40L expression on cell surfaces and, thus, an enhanced CD40 interaction.
Soluble CD40L correlated with the composite of MI and death in a substudy of the OPUS-TIMI16 trial.15 Our results supported a strong correlation between sCD40L and MI but not for cardiovascular death. The reason might be that age and comorbidities eg, diabetes, previous MI, and cardiac and renal dysfunction might be stronger predictors of a poor survival than a new thrombotic event in patients with ACS.
Soluble CD40L levels also identified a subgroup of patients who benefited from a prolonged treatment with dalteparin. Thus, it seems likely that raised sCD40L is reflective of a prothrombotic state, induced by extensive platelet activation which include degranulation and surface expression of CD40L, possibly augmented by direct upregulation of tissue factor through the CD40–CD40L pathway.13,23–25 This is in accordance with the increased thrombin generation assessed by prothrombin fragment 1+2 and d-dimer in our patients admitted with sCD40L levels above median. Previously, elevation of sCD40L has been linked to prothrombotic states in studies of diabetes, hypercholesterolemia, and peripheral arterial disease.26–29
Elevated sCD40L levels have also been shown to be of importance for the efficacy of platelet inhibition. In a subgroup of the CAPTURE trial, sCD40L >60th percentile identified patients with a benefit of the gpIIb/IIIa inhibitor abciximab.16 Thus, raised levels of sCD40L seem to indicate a prothrombotic state with a potential and specific benefit for more intense antithrombotic treatment. Today, patients with NSTE-ACS receive clopidogrel in addition to ASA on admission. Whether sCD40L levels are of prognostic relevance also in these patients remains to be elucidated.
In agreement with a smaller study, the combination of TnT and sCD40L was clearly additive in the assessment of the absolute risk of MI and death in the current trial.15 However, the combined assessment of these 2 biomarkers did not seem to improve estimation of the relative treatment efficacy of either early invasive or prolonged anticoagulant treatment than each biomarker by itself. These results emphasize that elevated sCD40L in patients with ACS reflects a prothrombotic state that is not dependent on the degree of cardiac ischemia and that can be addressed by assigning anti-thrombotic treatments.
In this study, carriers of the –3459 G allele had &10% higher sCD40L levels on admission. The –3459 G allele did not represent an increased risk of death or MI. Because numerous other factors influence the outcome of patients with ACS, this finding speaks for a nonsignificant role of this polymorphism in the acute thrombotic situation. However, it has been shown that the CD40–CD40L system is involved also in the conversion of stable to unstable plaques, likely induced by tissue factor activation on interaction between T-lymphocytes and macrophages and by the production of matrix metalloproteinases or proinflammatory cytokines2,30 Thus, the long-term consequences of –3459 G allele carriage may still be of significance in ACS and related diseases.
The role of CD40–CD40L in plaque destabilizing mechanisms and in the promotion of inflammation and procoagulant activity in monocytes is well-established, whereas the role of soluble CD40L in the atherothrombosis of patients with ACS is less well-known.30,31 Recently, the Dallas heart study, which is a population-based cross-sectional study, reported the absence of a correlation between sCD40L levels and asymptomatic coronary atherosclerosis as assessed by artery calcium score and aortic plaque burden by magnetic resonance imaging.32 In agreement with the Dallas heart investigation, our results imply that the usefulness of sCD40L as prognostic marker seems to be restricted to patients with a present prothrombotic state.
Conclusions
In conclusion, sCD40L seems to be a potential biomarker of a raised risk of thrombotic events that indicates an increased risk of MI in patients with acute coronary syndrome. The level is modified by genetic variations although the identified regulating locus does not relate to clinical outcome. In the clinical setting of ACS, the level of sCD40L and troponin-T provide valuable tools to identify patients in whom anti-thrombotic and early invasive treatments are particularly useful in reducing the risk of myocardial infarction. Our results designate sCD40L as a biomarker reflecting a prothrombotic state.
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Acknowledgments |
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We gratefully acknowledge the work of Laboratory Assistant Birgitta Fahlström for plasma sample analyses. Furthermore, we show our gratitude to the FRISC-II investigators.
Sources of Funding
This study was supported by grants from the Swedish Research Council, the Swedish Heart–Lung foundation, and the King Gustaf V and Queen Victoria jubilee foundation. The SNP analysis of the present study was supported by the K&A Wallenberg foundation via Wallenberg Consortium North (WCN).
Disclosures
None.
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Footnotes |
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Original received January 25, 2006; final version accepted April 3, 2006.
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References |
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