Critical role of ADP interaction with P2Y12 receptor in the maintenance of αIIbβ3 activation:: association with Rap1B activation

Objective: Platelet integrin alpha(IIb)beta(3) plays a crucial role in platelet aggregation, and the affinity of alpha(IIb)beta(3) for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which alpha(IIb)beta(3) maintains its high-affinity state. Methods and results: Washed platelets adjusted to 50 x 10(3) mu L-1 were stimulated with 0.2 U mL(-1) thrombin or 5 mu m U46619 under static conditions. After the completion of alpha(IIb)beta(3) activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated alpha(IIb)beta(3) was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 mu m AR-C69931MX (a P2Y(12) antagonist) or 1 mm A3P5P (a P2Y(1) antagonist) disrupted the sustained alpha(IIb)beta(3) activation by similar to 92% and similar to 38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 mu L-1 also disrupted the sustained alpha(IIb)beta(3) activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5'-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL(-1) thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. Conclusions: Our data indicate that the continuous interaction between released ADP and P2Y(12) is critical for the maintenance of alpha(IIb)beta(3) activation.