Paxillin Becomes Tyrosine-Phosphorylated and Binds Csk in a GpIIb-IIIa-Dependent Manner Following GPVI-Induced Activation of Murine Platelets.

Abstract

Though Src family kinases (SFKs) play a critical role in collagen-induced platelet activation, little is known about how SFK activity is regulated following exposure of platelets to collagen. In resting cells, SFKs are maintained in an inactive conformation, in part, via intramolecular interactions between their SH2 domain and a C-terminal tyrosine residue whose phosphorylation state is controlled by the C-terminal Src kinase, Csk. Access of Csk to SFKs, in turn, is regulated by recruitment of Csk, via its SH2 domain, to one or more tyrosine-phosphorylated Csk-binding proteins, which include Csk binding protein (Cbp/PAG) itself, paxillin, and its closely-related paxillin family member, Hic-5. Recent studies have shown that human platelets possess only two Csk-binding proteins: Cbp/PAG and Hic-5, and that Hic-5 can become tyrosine-phosphorylated when platelets are stimulated with a variety of platelet agonists, including, thrombin, U46619, and collagen. The purpose of the present investigation was to characterize the complement of Csk-binding proteins in murine platelets and to begin to determine their role in regulating collagen-induced platelet activation. Murine platelets, like human platelets, were found to express Hic-5, and in addition contained two other Csk-binding proteins: paxillin and leupaxin. Of these, both paxillin and Hic-5 became tyrosine phosphorylated, and paxillin was shown to be able to recruit Csk in a time-dependent manner following exposure of murine platelets to collagen and the GPVI/FcRg chain collagen receptor-specific agonist, CRP. These data suggest that the function of Hic-5 in human platelets may be performed by both Hic-5 and paxillin in mice. Finally, both paxillin tyrosine phosphorylation and Csk recruitment were blocked by agents that interfered with either platelet granule secretion or with integrin engagement, consistent with the notion that members of the paxillin family function as integrin-dependent negative feedback regulators of platelet adhesion and spreading.