P2Y12 polymorphisms and antiplatelet effects of aspirin in patients with coronary artery disease

Abstract

What is already known about this subject: * Genetic polymorphisms of the P2Y(12) ADP receptor on platelets have been shown to contribute to variability in platelet aggregation in healthy humans. * P2Y(12) ADP receptor polymorphisms are more frequently present in patients with vascular disease than in healthy people. * The majority of patients with vascular disease receive acetylsalicylic acid as an anti-aggregatory agent, which has also been shown to induce a variable response; however, the role of P2Y(12) ADP receptor polymorphisms in the platelet response to acetylsalicylic acid in patients with vascular disease has not yet been studied.

What this study adds: * The present data show that the platelet response to acetylsalicylic acid is independent of the presence or absence of P2Y(12) ADP receptor polymorphisms in patients with stable coronary artery disease who have had their first myocardial infarction. * This is important, as studies in healthy humans had suggested that carriers of P2Y(12) ADP receptor polymorphisms may be at increased risk of experiencing cardiovascular events. * However, the observed variability of the platelet response to the cyclooxygenase inhibitor acetylsalicylic acid (in our study) and to the P2Y(12) ADP receptor blocker clopidogrel (in a study by Angiolillo et al) in patients with coronary artery disease is clearly not determined by common P2Y(12) ADP receptor polymorphisms.

Aims: Recently, two genetic polymorphisms of the platelet ADP receptor P2Y(12) (haplotypes H2 and 34T) have been implicated in increased platelet aggregation and atherothrombotic risk. It was suggested that these polymorphisms contribute to a diminished response to antiplatelet drugs. Therefore, we investigated the effects of these polymorphisms on platelet aggregation in aspirin-treated patients with coronary artery disease (CAD).

Methods: Platelet aggregation was studied in platelet-rich plasma from 124 patients with CAD treated with 100 mg aspirin day(-1). P2Y(12) ADP receptor polymorphisms were determined by PCR-RFLP. The 52G > T polymorphism was used as tag-SNP for the H2 haplotype. Aggregation was induced by 1 mg l(-1) collagen. In a subgroup (n = 72), a concentration-response curve to collagen (0.5-10 mg l(-1)), aggregation at 2 micromol l(-1) ADP and 1 mmol l(-1) arachidonic acid were determined.

Results: Whereas arachidonic acid-induced aggregation was inhibited in all patients, collagen and ADP-induced aggregation were highly variable. However, aggregation did not differ significantly between carriers and noncarriers of the 52T-allele (1 mg l(-1) collagen: 32.7% (21.9-38.6%) vs. 32.5% (21.2-41.6%); P = 0.77; ADP: 33.1% (29.9-40.9%) vs. 39.1% (31.5-49.7%); P = 0.34), respectively. EC(50) values were 1.26 mg l(-1) (0.79-2.02) and 1.54 mg l(-1) (0.98-2.4) collagen in noncarriers and carriers of the H2 haplotype, respectively (P = 0.56). Moreover, the 34 degrees C > T polymorphism did not significantly affect any of the aggregatory responses.

Conclusions: Low-dose aspirin inhibits platelet aggregation to the same extent in patients carrying or not carrying the P2Y(12) H2 haplotype and/or the 34T allele. Our data do not support the hypothesis that these polymorphisms contribute to an attenuated antiplatelet effect of aspirin.

Figure 1

Study hypothesis. Platelet aggregation is mediated by multiple pathways. The two most important pathways are the cyclooxygenase (COX)-dependent formation of thromboxane A₂ (TxA₂), which binds to thromboxane receptors, and the granule secretion of ADP, which binds to ADP receptors (e.g. P2Y₁₂). Both pathways lead to GPIIb-IIIa receptor activation resulting in stable aggregate formation. During aspirin treatment, the TxA₂ mediated pathway is blocked, whereas the ADP mediated pathway is largely unaffected. In the presence of P2Y₁₂ receptor polymorphisms (H2 haplotype and/or 34°C > T), which were recently associated with increased platelet aggregation, aspirin treatment may be ineffective due to predominance of the ADP mediated pathway

Figure 2

Interindividual variability of maximal platelet aggregation in aspirin-treated patients with coronary artery disease (CAD). Maximal aggregation (%) in response to 1 mg l⁻¹ collagen (x-axis) was plotted against maximal aggregation (%) in response to 2 μmol l⁻¹ ADP (•), and 1 mmol l⁻¹ arachidonic acid (○), respectively, in 72 CAD patients treated with 100 mg aspirin day⁻¹. Collagen- and ADP-induced aggregation were significantly correlated (r = 0.63; P < 0.0001). Similarly, collagen- and arachidonic acid-induced aggregation were correlated (r = 0.73; P < 0.0001). However, aggregation induced by arachidonic acid was considered to be inhibited, because none of the patients had an aggregation response above 15% and subjects without aspirin therapy showed significantly higher maximal aggregation values (data not shown)

Figure 3

Maximal collagen-induced platelet aggregation in platelets with P2Y₁₂ genotypes in aspirin-treated patients with coronary artery disease (CAD). Maximal aggregation (%) in response to 1 mg l⁻¹ collagen in CAD patients treated with 100 mg aspirin day⁻¹. (A) patients carrying no 52T-allele (n = 86) or one or two 52T-alleles (n = 38). (B) patients carrying no 34T-allele (n = 60) or one or two 34T-alleles (n = 64)

Figure 4

Concentration-response curves for collagen-induced platelet aggregation subject to P2Y₁₂ genotypes in aspirin-treated patients with coronary artery disease (CAD). Maximal aggregation (%) in response to 0.5, 1, 2, 5 and 10 mg l⁻¹ collagen in CAD patients treated with 100 mg aspirin day⁻¹. (A) patients carrying no 52T-allele (n = 58) or carrying one or two 52T-alleles (n = 17; P = 0.56) (52 G/G, (▵); 52 G/T or 52 T/T, (○)). (B) patients carrying no 34T-allele (n = 33) or one or two 34T-alleles (n = 42; P = 0.25). Data are mean ± SEM (34 C/C, (▵); 34 C/T or 34 T/T, (○))

Figure 5

Maximal ADP-induced platelet aggregation subject to P2Y₁₂ genotypes in aspirin-treated patients with coronary artery disease (CAD). Maximal aggregation (%) in response to 2 μmol l⁻¹ ADP in CAD patients treated with 100 mg aspirin day⁻¹. (A) patients carrying no 52T-allele (n = 56) or one or two 52T-alleles (n = 16). (B) patients carrying no 34T-allele (n = 33) or one or two 34T-alleles (n = 39)

Figure 6

Maximal collagen-induced platelet aggregation in aspirin-treated patients with coronary artery disease (CAD) carrying both polymorphisms, 52T and 34T. Maximal aggregation (%) in response to 1 mg l⁻¹ collagen in CAD patients treated with 100 mg aspirin day⁻¹ carrying neither the 52G > T polymorphism nor the 34°C > T polymorphism (n = 35), and aspirin-treated patients carrying both polymorphisms, 52G > T and 34°C > T (n = 13)