Journal
JOURNAL OF THROMBOSIS AND HAEMOSTASIS
Volume 7, Issue 7, Pages 1200-1206Publisher
WILEY
DOI: 10.1111/j.1538-7836.2009.03444.x
Keywords
integrins; phospholipase C; platelets; Rac; Rap1b; Src kinases
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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).
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