Integrin α2β1 induces phosphorylation-dependent and phosphorylation-independent activation of phospholipase Cγ2 in platelets: role of Src kinase and Rac GTPase

Background: Platelet adhesion promoted by integrin alpha(2)beta(1) induces integrin alpha(IIb)beta(3) activation through the phospholipase C (PLC)-dependent stimulation of the small GTPase Rap1b. Objective: To analyze the mechanism of PLC activation downstream of alpha(2)beta(1) that is required for regulation of Rap1b and alpha(IIb)beta(3). Methods: Human and murine platelets were allowed to adhere to immobilized type I monomeric collagen through alpha(2)beta(1). Tyrosine phosphorylation of PLC gamma 2, PLC activation, accumulation of GTP-bound Rap1b and fibrinogen binding were measured and compared. Results: Integrin alpha(2)beta(1) recruitment induced an evident PLC activation that was concomitant with robust tyrosine phosphorylation of PLC gamma 2, and was suppressed in platelets from PLC gamma 2-knockout mice. Moreover, PLC gamma 2(-/-) platelets were unable to accumulate active Rap1b and to activate alpha(IIb)beta(3) upon adhesion through alpha(2)beta(1). Inhibition of Src kinases completely prevented tyrosine phosphorylation of PLC gamma 2 in adherent platelets, but did not affect its activation, and both Rap1b and alpha(IIb)beta(3) stimulation occurred normally. Importantly, alpha(IIb)beta(3)-induced phosphorylation and activation of PLC gamma 2, as well as accumulation of active Rap1b, were totally suppressed by Src inhibition. Integrin alpha(2)beta(1) recruitment triggered the Src kinase-independent activation of the small GTPase Rac1, and activation of Rac1 was not required for PLC gamma 2 phosphorylation. However, when phosphorylation of PLC gamma 2 was blocked by the Src kinase inhibitor PP2, prevention of Rac1 activation significantly reduced PLC gamma 2 activation, GTP-Rap1b accumulation, and alpha(IIb)beta(3) stimulation. Conclusions: Src kinases and the Rac GTPases mediate independent pathways for PLC gamma 2 activation downstream of alpha(2)beta(1).