Role of the P2Y12 gene polymorphism in platelet responsiveness to clopidogrel in healthy subjects

Clopidogrel is an inhibitor of platelet function. Its active metabolite irreversibly blocks the platelet ADP receptor P2Y12 [1] and prevents ischemic events in patients with atherosclerosis [2]. The antiplatelet effect of clopidogrel shows wide interindividual variability and in 5–30% of patients the recommended dose of 75 mg day fails to inhibit platelet aggregation ex vivo [3–5]. We examined the role of the gain-of-function polymorphism (H2 allele) of the P2Y12 gene [6], which encodes the clopidogrel target receptor, in the extent of clopidogrel pharmacodynamic response. To investigate the possible influence of the H2 allele free from confounding factors, such as medications or cardiovascular diseases, we evaluated clopidogrel responsiveness in young healthy subjects during a 1-week oral course of clopidogrel 75 mg day. Among 225 Caucasian men aged 18–35 years screened for the H1H2 genotype [7], we consecutively included 29 healthy volunteers (14 H1H1, 11 H1H2, and 4 H2H2). The study was approved by the Paris-Cochin ethics committee and all the subjects gave their written informed consent. The volunteers were asked to come to the Clinical Investigations Center each day at 09:00 hours where they were given a standard dose of clopidogrel 75 mg once daily for 7 days in the presence of medical staff. Blood samples for pharmacodynamic studies were drawn just before the first clopidogrel intake on days 1, 2, 3, 4, 5, and 7, and on days 9, 10, 11, and 14. Platelet response to 10 lmol L ADP was evaluated with platelet aggregation and vasodilator-stimulated phosphoprotein (VASP) phosphorylation, which is specific for P2Y12 inhibition. Aggregation studies were performed at baseline, on treatment, and after treatment completion at 37 C using a photometric method on a four-channel aggregometer (Regulest, Amneville, France). ADP-induced aggregation (10 lmol L final concentration, Sigma-Aldrich, St-Quentin Fallavier, France) was reported as maximal aggregation or as a percentage of baseline aggregation. VASP was measured in whole blood at baseline, on day 4 and on day 7 of clopidogrel intake using a flow cytometric assay (Platelet VASP , Diagnostica Stago, Biocytex, Marseille, France) adapted to a Cy-Flow apparatus (Partec GmbH, SteGeneviève-des-Bois, France). Results were expressed as a platelet reactivity index (PRI, %) [8]. All experiments were performed blindly to the P2Y12 genotype. The effect of the genotype on pharmacodynamic parameters was assessed at each time point by the Kruskal–Wallis test. When this test was significant (P < 0.05), paired comparisons of the three genotype groups were made usingWilcoxon’s test. Clopidogrel responsiveness was also evaluated in each genotype group across time using Friedman’s test. At baseline, ADP-induced platelet aggregation did not vary with the genotype: median values were 79.5% [interquartile range (IQR) 75.0; 81.0] in H1H1 subjects; 76.0% (IQR 72.0; 78.0) in H1H2 subjects; and 79.0% (IQR 76.5; 81.0) in H2H2 subjects (P 1⁄4 0.25). The aggregation fell significantly between days 1 and 7 of clopidogrel intake in the whole population [median at day 7 (IQR): 54% (45.5; 65.5), P < 0.0001], but the response did not differ between H1H1 subjects and H1H2 + H2H2 subjects (P 1⁄4 0.58). However, while median platelet aggregation significantly fell in H1H1 [54% (46; 68)] and H1H2 [49% (29; 59)] groups on day 7 of clopidogrel intake, no significant fall occurred in the H2H2 subjects [76% (65; 82), P < 0.05 vs. H1H1 and H1H2 groups] (Fig. 1). The inhibition of platelet aggregation relative to baseline was 7% (3; 9) in H2H2 subjects, 28% (15; 38) in H1H2 subjects, and 23% (11; 32) in H1H1 subjects (P 1⁄4 0.01 vs. H2H2 subjects). Interestingly, when individual responses to the 7-day oral course of clopidogrel were classified in quartiles of relative reduction in ADP-induced platelet aggregation from baseline (first quartile 0–13.8%; second quartile 13.9–30.8%; third quartile 30.9–42.2%; fourth quartile> 42.3%), the fourH2H2 homozygotes were situated in the first quartile, which may define poor responders although this was also the case in three of the H1H1 subjects. At day 7, the PRI (VASP phosphorylation assay) fell significantly after clopidogrel intake in the H1H1 group [PRI baseline: 76.9% (IQR 63.3; 89.4); PRI day 7: 46.3% (5.6; 67.5), P 1⁄4 0.0015] and in the H1H2 group [PRI baseline: 69.7% (58.1; 75.8); PRI day 7: 42.9% (16; 66.1), P 1⁄4 0.0051] with no significant difference between the H1H1 and H1H2 + H2H2 groups. However, the median fall in the PRI did not reach statistical significance in the H2H2 Correspondence: Pascale Gaussem, INSERM U 765, Service d’Hématologie Biologique A, Hôpital Européen Georges Pompidou, 20 rue Leblanc, 75908 Paris Cedex 15, France. Tel. : +33 1 56 09 39 36; fax: +33 1 56 09 39 13; e-mail: pascale. gaussem@egp.aphp. fr