Purinergic P2Y₁₂ receptor blockade inhibits shear-induced platelet phosphatidylinositol 3-kinase activation

Pathologically elevated shear stress triggers aspirin-insensitive platelet thrombosis. Signaling mechanisms involved in shear-induced platelet thrombosis are not well understood. To investigate these, we examined the hypothesis that functionally important platelet phosphatidylinositol 3-kinase (PI3-K) activity is stimulated by an in vitro shear stress of 120 dynes/cm² (shear rate of 6000 sec^-1). Phosphatidylinositol 3,4,5-trisphosphate (PIP₃) production was examined in washed human platelets subjected to pathological shear stress in a cone-plate viscometer. PIP₃ production peaks 30 s after shear begins and is initiated by von Willebrand factor (VWF) binding to the glycoprotein (Gp) lb-IX-V complex. Inhibiting PI3-K with wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) results in the inhibition of shear-induced platelet aggregation. In resting platelets, class IA PI3-K associates with the tyrosine kinase Syk. Within 30 s of beginning shear, PI3-K-associated Syk becomes tyrosine phosphorylated. Inhibiting Syk activation with piceatannol results in the inhibition of PIP₃ production and aggregation. Selective blockade of the P2Y₁₂ receptor results in the inhibition of Syk phosphorylation, PIP₃ production, and aggregation. These results indicate that shear-induced VWF binding to platelet GpIb-IX-V stimulates functionally important PI3-K activity. PI3-K activation is signaled by rapid feedback amplification that involves P2Y₁₂ receptor-mediated activation of Syk.