Platelets express a single class of Fc␥ receptor (Fc␥RIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. Fc␥RIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-␥2 (PLC-␥2), and calcium signaling. Here, we show that in response to Fc␥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␥2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin ␣ IIb / 3 engagement. In contrast, the stable association of PLC␥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␥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␥2 induced by Fc␥RIIA cross-linking.
