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 Fc gamma receptor (Fc gamma RIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. Fc gamma RIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-gamma 2 (PLC-gamma 2), and calcium signaling. Here, we show that in response to Fc gamma RIIA cross-linking, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P-3) 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 PLC gamma 2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin alpha(IIb)/beta(3) engagement. In contrast, the stable association of PLC gamma 2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLC gamma 2 was able to preferentially interact in vitro with PtdIns(3,4,5)P-3. Finally, exogenous PtdIns(3,4,5)P-3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P-3 play a key role in the activation and adequate location of PLC gamma 2 induced by Fc gamma RIIA cross-linking.