Platelet P2Y₁₂ receptors enhance signalling towards procoagulant activity and thrombin generation

Summary

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.

Keywords

ADP - coagulation - platelets - purinergic receptors

• References

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