HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Rac, a small guanosine triphosphate–binding protein, and p21-activated kinase are activated during platelet spreading on collagen-coated surfaces: roles of integrin a2b1

In this study, the receptors and signals involved in collagen-induced platelet spreading were examined. It was found that platelet spreading on collagen (presenting a polygon shape with a number of filopodialike projections) was inhibited by the anti–integrin a2 antibody, suggesting the involvement of integrin a2b1 in this process. Studies with a glutathione-Stransferase fusion protein that binds specifically to activated Rac and in vitro p21-activated kinase (PAK) kinase assays revealed that Rac and PAK were activated during this collagen-activated process. Platelet spreading on collagen-coated surfaces was inhibited strongly by PP1 (a Src family kinase inhibitor) or weakly by wortmannin (a phosphatidylinositol 3-kinase [PI3-kinase] inhibitor) but not at all by Y-27632 (a Rho kinase inhibitor). The surfaces coated with anti–integrin a2b1 antibodies also induced platelet spreading (presenting an almost complete round shape) and activation of Rac and PAK, although more slowly than collagencoated surfaces. The antibody-induced responses were strongly inhibited by PP1 or wortmannin but not by Y-27632. The same concentration of Y-27632 inhibited collagen-induced shape change of platelets in suspension. These findings suggest that Rac and/or PAK activation, but not Rho, may play certain roles in platelet spreading via integrin a2b1 and that Src family kinases and PI3-kinase participate in these processes. Furthermore, the difference between spreading on collagen and the anti-integrin antibody suggests the involvement of other receptor(s) (in addition to the integrin a2b1) for collageninduced spreading, the most likely candidate being glycoprotein VI. (Blood. 2001; 98:3708-3716)