Inhibition of the platelet ADP receptor, P2Y12, has been shown to reduce thrombin-stimulated exposure of platelet phosphatidylserine and tissue factor (TF)-dependent thrombin generation in human platelet rich plasma (

). Platelets stimulated by the combination of convulxin (Cvx) and thrombin develop two populations of platelets, one with low coagulation factor binding and a second with high levels of coagulation factor binding (
). Platelets in the population with increased coagulation factor binding have been termed COAT platelets. Previously we have demonstrated that the percentage of COAT platelets correlates with increased thrombin generation on Cvx plus thrombin-stimulated platelets compared to platelets stimulated with thrombin alone (
). In this study we investigated whether inhibition of the platelet P2Y12 receptor reduced the development of the COAT platelets in addition to reducing thrombin generation.

METHOD: A well established cell-based model of hemostasis was used. Components included: LPS-stimulated monocytes expressing TF, coagulation factors (II, V, VII, VIII, IX, X, XI) at plasma concentrations, coagulation inhibitors (AT, TFPI) at plasma concentrations, platelets from normal donors, and calcium chloride. Thrombin generation in the system was measured by cleavage of a chromogenic substrate and compared to a standard curve. Model systems without the addition of Cvx (20 ng/ml) were compared to model system with the addition of Cvx with and without a 2-methylthiol AMP (2MeSAMP, an inhibitor of the P2Y12 receptor). To determine the percentage of COAT platelets formed, samples were removed at 9.5 minutes after the model system was initiated, fixed in 1% paraformaldehyde, and analyzed on FACScan flow cytometer for binding of factor V using a polyclonal primary and secondary.

RESULTS: In the absence of 2MeSAMP approximately 30% of Cvx plus thrombin-stimulated platelets were COAT platelets. In the presence of 2MeSAMP, none of the platelets were COAT platelets. 2MeSAMP led to a significant reduction in the peak and rate of thrombin generation in the presence of Cvx but no change in the total amount of thrombin generated as measure by the area under the curve.

CONCLUSIONS: Inhibition of the platelet P2Y12 receptor prevents COAT platelet formation. The absence of COAT platelet formation correlates with a reduction in both the rate of thrombin generation and the peak of thrombin generated. COAT platelet formation and up-regulated thrombin generation induced by the combination thrombin plus moderate concentrations of Cvx are P2Y12-dependent.

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