Phosphatidylinositol 3,4,5-Trisphosphate-dependent Stimulation of Phospholipase C-γ2 Is an Early Key Event in FcγRIIA-mediated Activation of Human Platelets

Platelets express a single class of Fcγ receptor (FcγRIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. FcγRIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-γ2 (PLC-γ2), and calcium signaling. Here, we show that in response to FcγRIIA cross-linking, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) is rapidly produced, whereas phosphatidylinositol (3,4)-bisphosphate accumulates more slowly, demonstrating a marked activation of phosphoinositide 3-kinase (PI 3-kinase). Inhibition of PI 3-kinase by wortmannin or LY294002 abolished platelet secretion and aggregation, as well as phospholipase C (PLC) activation, indicating a role of this lipid kinase in the early phase of platelet activation. Inhibition of PLCγ2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin αIIb/β3 engagement. In contrast, the stable association of PLCγ2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLCγ2 was able to preferentially interact in vitro with PtdIns(3,4,5)P3. Finally, exogenous PtdIns(3,4,5)P3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P3 play a key role in the activation and adequate location of PLCγ2 induced by FcγRIIA cross-linking.