Physical and functional association of the Src family kinases Fyn and Lyn 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 gamma chain (FcRgamma) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRgamma, Syk, and phospholipase Cgamma2 (PLCgamma2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI-FcRgamma 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-FcRgamma complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRgamma, Syk, and PLCgamma2 and recruited tyrosine-phosphorylated Syk to the GPVI-FcRgamma complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRgamma and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI-FcRgamma 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 FcRgamma, Syk, and PLCgamma2, 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-FcRgamma complex.
Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
Y
Y
Ezumi
K
K
Shindoh
M
M
Tsuji
H
H
Takayama
D001792:Blood Platelets / Q000378:metabolism*; D006801:Humans; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D011940:Receptor Aggregation; D011961:Receptors, Fc / Q000378:metabolism*; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism*
D010980:Platelet Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D011961:Receptors, Fc; C098341:platelet membrane glycoprotein VI; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases