Activated platelets participate in arterial thrombosis by forming aggregates and potentiating the coagulation through exposure of procoagulant phosphatidylserine. The function of the two receptors for ADP, P2Y1 and P2Y12, is well-established in aggregation, but is incompletely understood in the platelet procoagulant response. We established that, in PRP from healthy subjects, ADP accelerated and potentiated tissue factor-induced throm-bin generation exclusively via stimulation of P2Y12 and not via P2Y1 receptors. The P2Y12 receptors also mediated the potentiating effect of PAR-1 stimulation on thrombin generation. Furthermore, ADP enhanced in a P2Y12-dependent manner the Ca2+ response induced by thrombin, which was either added externally or generated in-situ. This ADP effect was in part dependent of phosphoinositide 3-kinase and was paralleled by increased phosphatidylserine exposure. In PRP from (young) patients with either stroke or type-II diabetes, platelet-dependent thrombin generation was similarly enhanced by ADP or SFLLRN as in healthy subjects. In PRP from stroke patients of older age, the P2Y12-mediated contribution to thrombin generation was variably reduced by two weeks of clopidogrel medication. Remaining P2Y12 activity after medication correlated with remaining P2Y12-dependent P-selectin exposure, i.e. Ca2+-dependent secretion, likely due to incomplete antagonism of P2Y12 receptors. Together, these results indicate that physiological platelet agonists amplify phosphatidylserine exposure and subsequent thrombin generation by release of ADP and P2Y12-receptor stimulation. This P2Y12 response is accomplished by a novel Ca2+ signalling pathway. It is similarly active in platelets from control subjects and patients at thrombotic risk. Finally, the thrombo-gram method is useful for measuring incomplete P2Y12 inhibition with clopidogrel.
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