Critical Role of Endogenous ADP Via P2Y12 Receptor in Maintenance of α_Iibβ₃ Activation.

Platelet α_IIbβ₃ (GPIIb-IIIa), a noncovalently associated heterodimer, is a prototypic integrin that functions as a physiologic receptor for fibrinogen and von Willebrand factor. α_IIbβ₃ plays a crucial role in platelet aggregation, a key event of hemostatic plug formation and pathologic thrombus formation. During thrombogenesis, the affinity of α_IIbβ₃ 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 α_IIbβ₃ for soluble ligands (α_IIbβ₃ activation) are generated. Previous studies revealed that activation of α_IIbβ₃ is a reversible process. When platelets are stimulated with weak agonists such as adenosine diphosphate (ADP) and epinephrine in the absence of fibrinogen, α_IIbβ₃ gradually looses its binding capacity. In contrast, thrombin can induce long-lasting α_IIbβ₃ activation in the absence of fibrinogen. Although much attention has been directed to the nature of inside-out signaling, the mechanisms by which α_IIbβ₃ 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 α_IIbβ₃ 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 α_IIbβ₃ 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 α_IIbβ₃ was also markedly decreased (~85% reduction). Furthermore, the reduction in activated α_IIbβ₃ 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 α_IIbβ₃ activation in the diluted platelet suspension. Thus, released endogenous ADP plays a critical role in the maintenance of α_IIbβ₃ activation, and certain platelet concentrations are needed for this action. We also examined Rap 1b activation during the maintenance of α_IIbβ₃ 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 α_IIbβ₃ activation and Rap 1b activation.

Our data provide that the continuous interaction between released ADP and P2Y₁₂ receptor is critical for the maintenance of α_IIbβ₃ activation.