Corinne Frere

High post-treatment platelet reactivity identified low-responders to dual antiplatelet therapy at increased risk of recurrent cardiovascular events after stenting for acute coronary syndrome.

Summary.  Background and objectives: Low response to antiplatelet therapy may be a risk factor for the development of ischemic complications in patients with non‐ST segment elevation acute coronary syndrome (NSTE ACS) undergoing coronary stenting. Methods: We prospectively studied the platelet response to both clopidogrel and aspirin in 106 NSTE ACS consecutive patients undergoing percutaneous coronary intervention (PCI) with stenting. A single post‐treatment blood sample was obtained just before PCI and analyzed by platelet aggregometry using both ADP and arachidonic acid (AA) as agonists to explore the responses to clopidogrel and aspirin, respectively. Patients were divided into quartiles according to the ADP or AA induced maximal intensity of platelet aggregation. Patients of the highest quartile (quartile 4) were defined as the ‘low‐responders’. Results: Twelve recurrent cardiovascular (CV) events occurred during the 1‐month follow‐up. Clinical outcome was significantly associated with platelet response to clopidogrel [Quartile 4 vs. 1, 2, 3: OR (95% CI) 22.4 (4.6–109)]. Low platelet response to aspirin was significantly correlated with clopidogrel low response (P = 0.003) but contributed less to CV events [OR (95%CI): 5.76 (1.54–35.61)]. Conclusions: A post‐treatment ADP‐induced platelet aggregation performed just before PCI identifies low responders to dual antiplatelet therapy with an increased risk of recurrent CV events.

Effect of Cytochrome P450 Polymorphisms on Platelet Reactivity After Treatment With Clopidogrel in Acute Coronary Syndrome

Genetic polymorphisms of cytochrome P450 (CYP) isoforms may promote variability in platelet response to clopidogrel. This study was conducted to analyze, in 603 patients with non-ST elevation acute coronary syndromes, the effect of CYP3A4, CYP3A5, and CYP2C19 gene polymorphisms on clopidogrel response and post-treatment platelet reactivity assessed by adenosine diphosphate (ADP)-induced platelet aggregation, vasodilator-stimulated phosphoprotein phosphorylation index, and ADP-induced P-selectin expression. The CYP2C19*2 polymorphism was significantly associated with ADP-induced platelet aggregation, vasodilator-stimulated phosphoprotein phosphorylation index, and ADP-induced P-selectin expression in recessive (p <0.01, p <0.007, and p <0.06, respectively) and codominant (p <0.08, p <0.0001, and p <0.009, respectively) models, but the CYP3A4*1B and CYP3A5*3 polymorphisms were not. The CYP2C19*2 allele carriers exhibited the highest platelet index levels in multivariate analysis (p = 0.03). After covariate adjustment, the CYP2C19*2 allele was more frequent in clopidogrel nonresponders, defined by persistent high post-treatment platelet reactivity (ADP-induced platelet aggregation >70%; p = 0.03). In conclusion, the present data suggest that the CYPC19*2 allele influences post-treatment platelet reactivity and clopidogrel response in patients with non-ST elevation acute coronary syndromes.

The CYP2C19*17 allele is associated with better platelet response to clopidogrel in patients admitted for non-ST acute coronary syndrome.

Clopidogrel is a thienopyridine derivative that inhibits ADPinduced platelet aggregation. It is an inactive pro-drug that requires several biotransformation steps before it acquires its antiplatelet effect [1,2]. After intestinal absorption, clopidogrel requires oxidation by hepatic cytochrome P450 (CYP) to generate an active metabolite. This active metabolite inhibits platelet activation through irreversible binding to the platelet ADP receptor, P2Y12. Numerous studies have reported inter-individual variability in platelet response to clopidogrel, which is of clinical relevance with regard to patient treatment [3]. However, the mechanisms underlying this variability remain unclear. Putative explanations for variable clopidogrel response include genetic variations that influence the liver metabolism of clopidogrel. Several functional polymorphisms in genes encoding CYP have already been evaluated. Previous studies reported that the CYP3A4*1B and CYP3A5*3 polymorphisms could not explain the variability in the platelet inhibitory effects of clopidogrel [4,5]. In contrast, recent data have shown that the CYP2C19*2 loss-of-function allele is associated with a marked decrease in platelet response to clopidogrel in young healthy male volunteers [6] and in patients with coronary artery disease [7,8]. Moreover, this polymorphism has recently been associated with a poor clinical prognosis [9–11] and with an increased risk of thrombosis after coronary stent placement [12]. Recently, novel allelic variants of the CYP2C19 have been described including the CYP2C19*17 which is associated with ultrarapid enzyme activity and increased medication metabolism including omeprazole [13]. The aim of the present study was to evaluate the impact of these novel allelic variants of the CYP2C19 on platelet response to clopidogrel in patients suffering from non-ST elevation acute coronary syndrome (NSTE ACS). We therefore retrospectively evaluated 598 patients with NSTE ACS after the administration of a 600-mg clopidogrel loading dose, and the impact of CYP2C19*4 CYP2C19*5, CYP2C19*6 and CYP2C19*17 on platelet response to clopidogrel, as assessed using the maximal intensity of 10 lMADPinduced platelet aggregation (ADP-Ag) and by the platelet reactivity index vasodilator stimulated phosphoprotein assay (PRI VASP). All subjects analyzed in this retrospective study participated in a larger study conducted by the same organization to examine the effects of genetic polymorphisms involved in clopidogrel metabolism in NSTE ACS patients [7]. The baseline characteristics of the patients were: age 64.7 ± 12 years, 453 (76%) males, 169 (28%) had diabetes, 332 (56%) suffered from hypertension, 321 (54%) were dyslipidemic, 263 (44%) were current smokers, 336 (45%) were receiving statins, 271 (45%) were treated with betablockers and the body mass index (BMI) was 26.9 ± 4.3 kg.m. All patients were genotyped for CYP2C19*4, CYP2C19*5, CYP2C19*6 and CYP2C19*17. No significant deviations from Hardy–Weinberg equilibrium were observed for any of the genetic variants. Table 1 summarizes the observed frequencies of the CYP variants evaluated in the studied population. CYP2C19*4, CYP2C19*5 and CYP2C19*6 were very rare variants, whereas CYP2C19*17 was relatively common. The baseline characteristics of the patients did not differ between the different groups of genotypes. None of the polymorphisms (CYP2C19*4, CYP2C19*5 or CYP2C19*6) influenced platelet response to clopidogrel (C. Frere, T. Cuisset, B. Gaborit, M.-C. Alessi, J.-S. Hulot, unpublished data). In contrast, the CYP2C19*17 genotype was significantly associated with PRI VASP values, both in recessive (P = 0.02) and codominant (P = 0.0073) models (Table 2). After adjustment for factors that influence platelet reactivity (i.e. age, gender, BMI, diabetes and smoking status), this association remained Correspondence: Corinne Frere, Inserm, U626, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France. Tel.: +33 491324504; fax: +33 491254336. E-mail: corinne.frere@mail.ap-hm.fr

Relationship between aspirin and clopidogrel responses in acute coronary syndrome and clinical predictors of non response.

OBJECTIVES We have prospectively investigated the association between aspirin and clopidogrel responses and the clinical predictors of non response. METHODS 635 Non ST Elevation Acute Coronary Syndrome (NSTE ACS) patients were included and received loading doses of 250 mg aspirin and 600 mg clopidogrel. We analyzed post-treatment maximal intensity of arachidonic acid and ADP-induced platelet aggregation (AA-Ag and ADP-Ag) and Platelet Reactivity Index of VAsodilator-Stimulated Phosphoprotein (PRI VASP). Aspirin and clopidogrel non responses were defined respectively by AA-Ag>30% and ADP-Ag>70%. RESULTS Aspirin non responders patients had significantly higher ADP-Ag and PRI VASP than aspirin responders: 63.7+/-15.9% vs 55+/-19% (p=0.0001) and 73.6+/-13.3% vs 53+/-23% (p=0.0001) respectively and the rate of clopidogrel non responders was higher among aspirin non responders than aspirin responders: 36.7% vs 22.7% (p=0.003). In addition, clopidogrel non responders had significantly higher AA-Ag and rate of aspirin non responders than clopidogrel responders: 21.6+/-24% vs 10.3+/-19% (p=0.0001) and 22.8% vs 12.9% (p=0.003) respectively. Age and Body Mass Index (BMI) were significantly associated with non response to Clopidogrel (p=0.035 and 0.02 respectively) and diabetes mellitus by trend (p=0.07). CONCLUSION We observed a relationship between aspirin and clopidogrel non responses and an association between age, BMI and diabetes mellitus and clopidogrel response.

Biological and genetic factors influencing plasma factor VIII levels in a healthy family population: results from the Stanislas cohort

The mechanisms underlying the variability of factor VIII (FVIII) levels are still poorly understood. The only receptor of FVIII identified so far is the lipoprotein receptor‐related protein (LRP), which is thought to be involved in FVIII degradation. We aimed to characterize biological and genetic factors related to FVIII variability, focusing on coding polymorphisms of the LRP gene and polymorphisms potentially detected by molecular screening of the LRP‐binding domains of the FVIII gene. Plasma FVIII coagulant activity (FVIII:C) and von Willebrand factor (VWF:Ag) antigen levels were measured in a sample of 100 healthy nuclear families (200 parents and 224 offspring). The ABO blood group and the three coding polymorphisms of the LRP gene (A217V, D2080N and C766T) were genotyped. Lipids and anthropometric factors poorly contributed to the variability of FVIII:C (<5%). A strong effect of ABO blood groups on FVIII:C levels was observed that remained significant after adjustment for VWF:Ag levels (P = 0·02). These two factors explained more than 50% of FVIII:C variability. After adjustment for VWF:Ag and ABO blood groups, a residual resemblance for FVIII:C persisted between biological relatives (ρ = 0·13 ± 0·06 between parents and offspring, ρ = 0·24 ± 0·09 between siblings) compatible with an additional genetic influence. The N allele of the LRP/D2080N polymorphism was associated with decreased levels of FVIII:C (90·4 ± 8·7 vs. 102·2 ± 3·5 IU/dl, P = 0·03) and VWF:Ag levels (109·1 ± 11·2 vs. 125·4 ± 4·4 IU/dl, P = 0·02). No polymorphism was detected in the LRP‐binding domains of the FVIII gene. This study reinforces the hypothesis of a genetic influence of FVIII levels beyond the influence of VWF:Ag and ABO blood groups. The D2080N polymorphism of the LRP gene weakly contributed to the variability of FVIII:C levels in this healthy population.

Predictive Values of Post-Treatment Adenosine Diphosphate–Induced Aggregation and Vasodilator-Stimulated Phosphoprotein Index for Stent Thrombosis After Acute Coronary Syndrome in Clopidogrel-Treated Patients

A low response to clopidogrel has been associated with an increased risk of stent thrombosis. However, the definition of a nonresponse to clopidogrel remains controversial, and different tests have been used to assess the clopidogrel response. The present study was designed to assess the predictive value of adenosine diphosphate (ADP)-induced platelet aggregation (ADP-Ag) and the Platelet Reactivity Index of vasodilator-stimulated phosphoprotein for the occurrence of stent thrombosis in patients admitted for non-ST-elevation acute coronary syndrome undergoing percutaneous coronary intervention. A total of 598 consecutive patients with non-ST-elevation acute coronary syndrome undergoing coronary stenting were prospectively included. They received 600 mg of clopidogrel >or=12 hours before percutaneous coronary intervention. Acute or subacute definite or probable stent thrombosis occurred in 11 patients (1.8%). These patients had significantly greater ADP-Ag compared to patients free of stent thrombosis (68 +/- 14% vs 56 +/- 19%, p = 0.002) but only a trend toward a greater Platelet Reactivity Index of vasodilator-stimulated phosphoprotein (62 +/- 14% vs 53 +/- 23%, p = 0.19). The construction of receiver operating characteristic curves to examine the most predictive value of ADP-Ag for stent thrombosis gave a threshold of ADP-Ag of >67% to identify low responders. These patients were at a greater risk of stent thrombosis than the clopidogrel responders (4.3% vs 0.8%, odds ratio 5.8, 95% confidence interval 1.9 to 24.6, p = 0.003). In conclusion, in patients with non-ST-elevation acute coronary syndrome undergoing percutaneous coronary intervention, ADP-Ag is a good parameter to identify clopidogrel nonresponders who are at increased risk of stent thrombosis, with a cutoff value of ADP-Ag of >67%.

TAFI gene haplotypes, TAFI plasma levels and future risk of coronary heart disease: the PRIME Study

Summary.  Objectives: To evaluate the association of thrombin‐activatable fibrinolysis inhibitor (TAFI) gene polymorphisms with the risk of coronary heart disease (CHD) and with TAFI levels measured by a newly developed enzyme‐linked immunosorbent assay (ELISA) (TAFI‐1B1), shown to be a reliable method for detecting quantitative variations in circulating TAFI. Patients/Methods: Six polymorphisms (C‐2599G, G‐438A, Ala147Thr, Thr325Ile, C + 1542G and T + 1583A) were genotyped and baseline plasma concentrations of TAFI were measured in a nested case–control design as part of the Prospective Epidemiological Study of Myocardial Infarction (PRIME) Study. Participants from France and Northern Ireland who had developed a CHD event during a 5‐year follow‐up (n = 321) were compared with 645 population‐ and age‐matched control subjects. Results: In France, the Thr147 allele was more frequent in cases than in controls (0.41 vs. 0.32; P = 0.02), whereas the reverse was observed in Northern Ireland (0.33 vs. 0.38; P = 0.19) (P = 0.01 for interaction). No other polymorphism was associated with CHD risk. Consistent with the results derived from the single‐locus analysis, haplotype analysis revealed that the haplotype carrying the Thr147 allele was associated with increased risk of CHD in France while the reverse tended to hold in the Northern Ireland population. Single‐locus and haplotype analyses revealed that two polymorphisms, C‐2599G and Ala147Thr (or T + 1583A that is in nearly complete association with it), had additive effects on TAFI levels and explained >18% of TAFI variability. This effect was homogeneous in France and Northern Ireland, and in cases and controls who exhibited similar TAFI levels. Conclusions: Thrombin‐activatable fibrinolysis inhibitor gene polymorphisms are strongly associated to plasma TAFI levels, but the relation to CHD risk is less clear.

Weak and non-independent association between plasma TAFI antigen levels and the insulin resistance syndrome

Summary.  Increased plasma thrombin‐activatable fibrinolysis inhibitor (TAFI) levels were recently shown to be a part of the insulin resistance syndrome. We investigated the relationship between plasma TAFI antigen levels and insulin resistance markers and compared these results with those obtained for PAI‐1 and fibrinogen which are known to be closely related to insulin resistance syndrome and fat mass, respectively. Eighty‐nine obese females had 1.3‐, 1.2‐, and 3‐fold higher circulating TAFI, fibrinogen and PAI‐1, respectively, compared with 64 lean females. Univariate analysis showed that the significance level for association between TAFI or fibrinogen concentrations and insulin resistance markers was lower than the significance level for association between PAI‐1 and insulin resistance markers. Nevertheless, TAFI, fibrinogen, and PAI‐1 plasma levels were significantly associated to each other. In linear stepwise ascendant analysis, insulin resistance markers accounted for 50% of the interindividual variability of plasma PAI‐1 and only for 10% of plasma TAFI and 13% of fibrinogen variability. The contribution of insulin resistance markers to plasma TAFI antigen levels variability disappeared when PAI‐1 or fibrinogen was entered in the statistical model. TAFI mRNA was detected in the liver but not in adipose tissue and endothelial cells. No TAFI mRNA was detected in normal or atherosclerotic vessels either. These results suggest that elevated TAFI antigen levels found in obese subjects are not independently associated with the metabolic markers of the insulin resistance syndrome. Increased plasma TAFI antigen levels in obesity might reflect a specific pathway of regulation at the liver level.

Clopidogrel response: Head-to-head comparison of different platelet assays to identify clopidogrel non responder patients after coronary stenting

OBJECTIVES We investigated the agreement between different platelet tests to identify clopidogrel non response. BACKGROUND Biological definition of clopidogrel non response remains controversial. Different platelet tests have been linked with recurrent ischemic events and proposed for daily practice. METHODS We prospectively investigated the agreement of platelet tests to isolate clopidogrel non response in patients receiving high 150 mg clopidogrel maintenance dose after coronary stenting. Clopidogrel response was assessed with ADP-induced aggregation (ADP-Ag) (non response if >70%), Platelet reactivity index VASP (PRI VASP) (non response if >50%) and Verify Now Point-of-care assay (VN) (non response if PRU > 240 AU). RESULTS Seventy consecutive patients were included. The rates of non-responders were respectively: 13% (n = 9) with the ADP-Ag, 39% (n = 27) with the PRI VASP and 33% (n = 23) with the VN. We observed significant correlation between different platelet tests assessing clopidogrel response: r = 0.55 (p < 0.0001) for ADP-Ag and PRI VASP, r = 0.64 (p < 0.0001) for ADP-Ag and VN and r = 0.59 (p < 0.0001) for PRI VASP and VN. However, using the most common thresholds, the agreement between the difference tests was poor: 0.35 for ADP-Ag and PRI VASP, 0.36 for ADP-Ag and VN and 0.46 for PRI VASP and VN. CONCLUSION This study showed that assessment of platelet function inhibition by clopidogrel is highly test-specific. Indeed, our results demonstrated a poor agreement between different platelet assays and suggested that identification of clopidogrel non responders is test-dependent.

Lack of effect of chronic kidney disease on clopidogrel response with high loading and maintenance doses of clopidogrel after Acute Coronary Syndrome

Dual antiplatelet therapy with aspirin and clopidogrel is currently the gold standard therapy for patients undergoing percutaneous coronary interventions and/or after acute coronary syndrome [1–3]. Several biological studies have described a broad inter individual variability of biological response to clopidogrel [4]. The clinical relevance of this biological entity has been demonstrated in different clinical setting such as acute coronary syndrome or elective PCI [5–7]. Mechanisms underlying this variability of response remain unclear and multifactorial [4]. Chronic Kidney Disease (CKD) has been linked with a lower efficacy of clopidogrel in large randomized controlled trials [8,9] and recent biological studies proposed CKD as a factor of low response to clopidogrel [10,11]. Accordingly, patients with CKD have poor outcomes after percutaneous coronary intervention (PCI), including stent thrombosis [12–15]. In a prospective cohort of patients admitted for NSTE ACS, we decided to assess the effect of CKD on high doses clopidogrel effect in the acute phase after 600 mg and one month after high 150 mg maintenance dose. Consecutive patients admitted for NSTE ACS to our hospital were eligible for this prospective study. Patients received a 600 mg loading dose of clopidogrel and 250 mg of aspirin at least 12 hours before coronary angiography and assessment of clopidogrel response. After PCI, all patients received aspirin 75 mg and clopidogrel 150 mg at discharge. After onemonth, platelet function tests were repeated to assess chronic response to clopidogrel. We decided to repeat platelet function while patients were in their maintenance phase of treatment for at least 30 days to overcome the variability in pharmacodynamic profiles known to occur during the initial days and weeks after initiation of therapy [4]. Patients gave written informed consent for participation. Blood samples for testing platelet reactivity were drawn in the catheterization laboratory from a six-French arterial sheath before the coronary angiography and repeated after one month from a venous catheter.