PHYSICAL AND FUNCTIONAL ASSOCIATION OF THE SRC FAMILY KINASES FYN ANDLYN WITH THE COLLAGEN RECEPTOR GLYCOPROTEIN VI-FC RECEPTOR-GAMMA CHAIN COMPLEX ON HUMAN PLATELETS

We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor ga mma chain (FcR gamma) in human platelets, is essential for collagen-st imulated tyrosine phosphorylation of FcR gamma, Syk, and phospholipase C gamma 2 (PLC gamma 2), leading to platelet activation. Here we inve stigated involvement of the Src family in the proximal signals through the GPVI-FcR gamma complex, using the snake venom convulxin from Crot alus durissus terrificus, which specifically recognizes GPVI and activ ates platelets through cross-linking GPVI. Convulxin-coupled beads pre cipitated the GPVI-FcR gamma complex from platelet lysates. Collagen a nd convulxin induced tyrosine phosphorylation of FcR gamma, Syk, and P LC gamma 2 and recruited tyrosine-phosphorylated Syk to the GPVI-FcR g amma complex. Using coprecipitation methods with convulxin-coupled bea ds 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 ass ociated with the GPVI-FcR gamma complex irrespective of stimulation. F urthermore, Fyn was rapidly activated by collagen or cross-linking GPV I. The Src family-specific inhibitor PP1 dose-dependently inhibited co llagen- or convulxin-induced tyrosine phosphorylation of proteins incl uding FcR gamma, Syk, and PLC gamma 2, accompanied by a loss of aggreg ation and ATP release reaction. These results indicate that the Src fa mily plays a critical role in platelet activation via the collagen rec eptor GPVI-FcR gamma complex.