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

Abstract

We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor γ chain (FcRγ) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRγ, Syk, and phospholipase Cγ2 (PLCγ2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI-FcRγ 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-FcRγ complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRγ Syk, and PLCγ2 and recruited tyrosine-phosphorylated Syk to the GPVI-FcRγ complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRγ and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI-FcRγ 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 FcRγ Syk, and PLCγ2, 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-FcRγ complex.