Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor γ chain complex on human platelets

We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor gamma chain (FcR gamma) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcR gamma, Syk, and phospholipase C gamma 2 (PLC gamma 2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI-FcR gamma complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI-FcR gamma complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcR gamma, Syk, and PLC gamma 2 and recruited tyrosine-phosphorylated Syk to the GPVI-FcR gamma complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcR gamma and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI-FcR gamma complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family-specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcR gamma, Syk, and PLC gamma 2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI-FcR gamma complex.