A critical role of lipid rafts in the organization of a key FcgammaRIIa-mediated signaling pathway in human platelets.

The involvement of platelet FcgammaRIIa in heparin-associated thrombocytopenia (HIT) is now well established. However, the precise sequence of molecular events initiated by FcgammaRIIa cross-linking in platelets remains partly characterized. We investigated here the role of lipid rafts in the spatio-temporal organization of the FcgammaRIIa-dependent signaling events. Upon cross-linking, FcgammaRIIa relocated in rafts where the kinase Lyn and the adapter LAT were among the major phosphotyrosyl proteins. Upon stimulation by HIT sera, the second messenger phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) accumulated in rafts in a P(2)Y(12) adenosine diphosphate (ADP) receptor-dependent manner. PtdIns(3,4,5)P(3) was then essential to specifically recruit phospholipase Cgamma2 (PLCgamma2) to these membrane microdomains. Controlled disruption of rafts by methyl beta-cyclodextrin reversibly abolished PtdIns(3,4,5)P(3) production, PLC activation and platelet responses induced by FcgammaRIIa cross-linking without affecting the tyrosine phosphorylation events. This work demonstrates that platelet rafts are essential for the integration of a key signaling complex leading to the rapid production of PtdIns(3,4,5)P(3) and in turn PLCgamma2 activation during HIT.