Residual platelet ADP reactivity after clopidogrel treatment is dependent on activation of both the unblocked P2Y(1) and the P2Y (12) receptor and is correlated with protein expression of P2Y (12)

Abstract

Two ADP receptors have been identified on human platelets: P2Y(1) and P2Y(12). The P2Y(12) receptor blocker clopidogrel is widely used to reduce the risks in acute coronary syndromes, but, currently, there is no P2Y(1) blocker in clinical use. Evidence for variable responses to clopidogrel has been described in several reports. The mechanistic explanation for this phenomenon is not fully understood. The aim of this study was to examine mechanisms responsible for variability of 2MeS-ADP, a stable ADP analogue, induced platelet reactivity in clopidogrel-treated patients. Platelet reactivity was assessed by flow cytometry measurements of P-selectin (CD62P) and activated GpIIb/IIIa complex (PAC-1). Residual 2MeS-ADP activation via the P2Y(12) and P2Y(1) receptors was determined by co-incubation with the selective antagonists AR-C69931 and MRS2179 in vitro. P2Y(1) and P2Y(12) receptor expression on both RNA and protein level were determined, as well as the P2Y(12) H1 or H2 haplotypes. Our data suggest that the residual platelet activation of 2MeS-ADP after clopidogrel treatment is partly due to an inadequate antagonistic effect of clopidogrel on the P2Y(12) receptor and partly due to activation of the P2Y(1) receptor, which is unaffected by clopidogrel. Moreover, a correlation between increased P2Y(12) protein expression on platelets and decreased response to clopidogrel was noticed, r(2)=0.43 (P<0.05). No correlation was found between P2Y(12) mRNA levels and clopidogrel resistance, indicating post-transcriptional mechanisms. To achieve additional ADP inhibition in platelets, antagonists directed at the P2Y(1) receptor could be more promising than the development of more potent P2Y(12) receptor antagonists.

Figure 1

Graph showing the increased inhibition of residual ADP-induced P-selectin positive platelets (a) and expression of activated GPIIb/IIIa (b) after the further blocking of the P2Y₁₂ receptor with AR-C 69931 (10 μM) and the P2Y₁ receptor with MRS 2179 (10 μM) and the blocking of both receptors with a combination of both antagonists. These effects are seen in platelets already blocked by clopidogrel and aspirin. Blocking the P2Y₁₂ receptor further with AR-C 69931 (10 μM) significantly inhibited the percentage of P-selectin positive platelets by 35.8±3.5% (mean ± SEM) (n =19) and the expression of activated GPIIb/IIIa by 43.5±3.4%. A significantly greater inhibition was seen when the P2Y₁ antagonist MRS 2179 (10 μM) was used, where the inhibition of P-selectin increased to 67.3±4.4% and the inhibition of activated GPIIb/IIIa increased to 54.2±2.6%. Combining the antagonists yielded an even greater significant inhibition: 78.4±3.8% for P-selectin and 72.2±2.5% for activated GPIIb/IIIa. There were significance differences between the groups, P <0.01, in all cases (n =19). Repeated-measures ANOVA with Bonferroni’s post-test were used to compare data

Figure 2

The relative expression and GAPDH-normalized expression of P2Y₁₂ (a) and P2Y₁ (b) protein correlated with the percentage of P-selectin positive platelets after stimulation with 1 μM of 2MeS-ADP. In (a) there is a significant correlation of P-selectin positive platelets with P2Y₁₂ protein expression, with r²=0.43 (P <0.05) (n =14). In (b) there is no significant correlation of P-selectin positive platelets and P2Y₁ protein expression (r²=0.002, P=0.88, n=14) (pos plts positive platelets)