The alpha(2)beta(1) integrin is a necessary co-receptor for collagen-induced activation of Syk and the subsequent phosphorylation of phospholipase C gamma 2 in platelets

Although there are multiple potential collagen-binding proteins on platelets, the contribution of each to collagen-induced signaling events and platelet activation is unclear. We investigated which early platelet signaling events, if any, could be attributed specifically to the binding of calf ages to one of its receptors, the alpha(2) beta(1) integrin. Treatment of platelets with collagen induced a rapid activation of the non-receptor tyrosine kinase, Syk, as measured by an increase in phosphorylation and kinase activity, Collagen also induced the rapid phosphorylation of phospholipase C gamma 2 (PLC gamma 2). The phosphorylation of bath Syk and PLC gamma 2, as well as platelet aggregation, was blocked by an anti-alpha(2) beta(1) integrin monoclonal antibody (P1E6), demonstrating that collagen binding to alpha(2) beta(1) is necessary for signaling, Gross-linking of the alpha(2) beta(1) integrin with stimulatory monoclonal antibody against either the beta(1) or alpha(2) subunit stimulated the phosphorylation of bath Syk and PLC gamma 2. However, antibody stimulation was dependent on co-stimulation of the Fc gamma II receptor (CD32) since specific F(ab')(2) fragments did not induce Syk and PLC gamma 2 phosphorylation. Thus, these results suggest that occupancy of alpha(2) beta(1) by collagen is necessary, but that a co-receptor, in addition to alpha(2) beta(1), is required for these collagen-induced signaling events, Moreover, the P1E6 antibody did not inhibit all collagen-induced tyrosine phosphorylation events, demonstrating that collagen also induces phosphorylation events that are, independent of the alpha(2) beta(1) integrin. In addition to Syk and PLC gamma 2, we identified the Fc gamma II receptor (Fc gamma RII) as being rapidly phosphorylated in response to collagen stimulation even in the absence of antibodies. Finally, to determine if Syk activation precedes and directly contributes to the phosphorylation of PLC gamma 2, platelets were preincubated with the Syk-selective kinase inhibitor, piceatannol. A concentration of piceatannol that inhibited the phosphorylation and kinase activity of Syk, but had no effect on Src kinase activity, blocked the collagen-induced phosphorylation of PLC gamma 2 and also inhibited collagen-induced platelet aggregation. Our results begin to delineate a signaling pathway whereby occupancy of the alpha(2) beta(1) integrin is required, but not sufficient, for collagen-induced activation of Syk and the subsequent phosphorylation of PLC gamma 2. These events are. necessary for platelet activation and aggregation in response to collagen.