Residual platelet ADP reactivity after clopidogrel treatment is dependent on activation of both the unblocked P2Y1 and the P2Y12 receptor and is correlated with protein expression of P2Y12

Two ADP receptors have been identified on human platelets: P2Y1 and P2Y12. The P2Y12 receptor blocker clopidogrel is widely used to reduce the risks in acute coronary syndromes, but, currently, there is no P2Y1 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 P2Y12 and P2Y1 receptors was determined by co-incubation with the selective antagonists AR-C69931 and MRS2179 in vitro. P2Y1 and P2Y12 receptor expression on both RNA and protein level were determined, as well as the P2Y12 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 P2Y12 receptor and partly due to activation of the P2Y1 receptor, which is unaffected by clopidogrel. Moreover, a correlation between increased P2Y12 protein expression on platelets and decreased response to clopidogrel was noticed, r2=0.43 (P<0.05). No correlation was found between P2Y12 mRNA levels and clopidogrel resistance, indicating post-transcriptional mechanisms. To achieve additional ADP inhibition in platelets, antagonists directed at the P2Y1 receptor could be more promising than the development of more potent P2Y12 receptor antagonists

Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study

<p>Abstract</p> <p>Background</p> <p>Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA) and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment.</p> <p>Methods</p> <p>With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B₂(TXB₂)-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis.</p> <p>Results</p> <p>The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5'-diphosphate (ADP)-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r²= 0.49). In opposite, TXB₂-levels decreased with ASA compared to clopidogrel. Serum TXB₂and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN-induced activation measured by flow cytometry were lower for clopidogrel compared to ASA. Furthermore, adhesion to collagen was lower for ASA and clopidogrel combined compared with either drug alone.</p> <p>Conclusion</p> <p>The indirect pharmacodynamic measures of the effects of ASA and clopidogrel might be used together with ADP-induced activation and serum TXB₂for evaluation of anti-platelet treatment. This should be further evaluated in future clinical studies where screening opportunities with the adhesion assay will be optimised towards increased sensitivity to anti-platelet treatment.</p