Platelet αIIbβ3 (GPIIb-IIIa), a noncovalently associated heterodimer, is a prototypic integrin that functions as a physiologic receptor for fibrinogen and von Willebrand factor. αIIbβ3 plays a crucial role in platelet aggregation, a key event of hemostatic plug formation and pathologic thrombus formation. During thrombogenesis, the affinity of αIIbβ3 for macromolecular ligands is dynamically regulated. After exposure to subendothelial matrix, several mediators such as ADP and thromboxane A2, or shear stress, platelets becomes activated and inside-out signaling that induces a high-affinity state of αIIbβ3 for soluble ligands (αIIbβ3 activation) are generated. Previous studies revealed that activation of αIIbβ3 is a reversible process. When platelets are stimulated with weak agonists such as adenosine diphosphate (ADP) and epinephrine in the absence of fibrinogen, αIIbβ3 gradually looses its binding capacity. In contrast, thrombin can induce long-lasting αIIbβ3 activation in the absence of fibrinogen. Although much attention has been directed to the nature of inside-out signaling, the mechanisms by which αIIbβ3 keep in the high-affinity state still remains elusive.

In this study, we have demonstrated a critical role of endogenous ADP via its P2Y12 receptor in the maintenance of αIIbβ3 activation. Washed platelets adjusted to 50 x 106/ml were stimulated with 0.2 U/ml thrombin or 5 mM U46619 under static conditions. After the 15-min stimulation, 1 mM AR-C69931MX (a P2Y12 antagonist), 1 mM A3P5P (a P2Y1 antagonist) or buffer alone was added to the suspensions for additional 5 min. The platelet suspensions were then incubated with FITC-PAC1 and PE-anti-CD62P for 30 min and analyzed in a flow cytometer. AR-C69931MX and A3P5P attenuated the number of activated αIIbβ3 about 95% and 45%, respectively on the already activated platelets with thrombin- or U46619 without inhibiting CD62P expression. In an another set of experiments, platelets stimulated with thrombin or U46619 for 15min were then diluted to 1:100 with buffer containing the same agonist concentration (0.5 x 106/ml). In these conditions the number of activated αIIbβ3 was also markedly decreased (~85% reduction). Furthermore, the reduction in activated αIIbβ3 by the dilution was reversed by the addition of exogenous ADP in a dose-dependent fashion. HPLC analysis revealed that the amounts of ADP released from thrombin- and U46619-stimulated platelets were 2.6 and 0.75 mmol/1011platelets, respectively, and these values were comparable with ADP doses required for sustained αIIbβ3 activation in the diluted platelet suspension. Thus, released endogenous ADP plays a critical role in the maintenance of αIIbβ3 activation, and certain platelet concentrations are needed for this action. We also examined Rap 1b activation during the maintenance of αIIbβ3 activation. Thrombin induced sustained Rap 1b activation in the absence of ligand. However, AR-C69931MX disrupted the sustained Rap 1b activation. Thus, there was a close relationship between the maintenance αIIbβ3 activation and Rap 1b activation.

Our data provide that the continuous interaction between released ADP and P2Y12 receptor is critical for the maintenance of αIIbβ3 activation.

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