Evidence for the involvement of p59fyn and p53/56lyn in collagen receptor signalling in human platelets

Abstract

The binding of collagen to platelet glycoprotein VI (GPVI) leads to the subsequent activation of phospholipase Cγ2 through a pathway that is dependent on the Fc receptor γ (FcR γ) chain and the tyrosine kinase p72syk. We have investigated the role of platelet Src-family kinases in this signalling pathway. The selective Src-family kinase inhibitor PP1 prevented collagen-stimulated increases in whole-cell tyrosine phosphorylation and tyrosine phosphorylation of the FcR γ chain and p72syk. A similar set of observations was made for a collagen-related peptide (CRP), which binds to GPVI but not to the integrin α2β1 (GPIa/IIa). These effects were seen at a concentration of PP1 that inhibited platelet aggregation, dense granule release and Ca2+ mobilization induced by CRP, but not aggregation and Ca2+ mobilization mediated by the G-protein-coupled receptor agonist thrombin. After stimulation by CRP or collagen, the Src-family kinases p59fyn and p53/56lyn became associated with several tyrosine-phosphorylated proteins including the FcR γ chain. This was not true of the other platelet Src-family kinases. The association between the FcR γ chain and p59fyn was also seen under basal conditions, and was stable only in the weak detergent Brij96 but not in Nonidet P40, suggesting a non-SH2-dependent interaction. These results provide strong evidence for the involvement of p59fyn and p53/56lyn in signalling via GPVI, with p59fyn possibly acting upstream of FcR γ chain phosphorylation.