Suboptimal Activation of Protease-activated Receptors

Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 andPAR4) and two collagen receptors, the 2 1 integrin ( 2 1) and the glycoprotein VI (GPVI)/FcR chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide ( 2CRP) containing the 2 1-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to 2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was 2 1-dependent and GPVI/FcR -independent as revealed in experiments with 2 1or FcR -deficient mouse platelets. We further show that suboptimal activation of other platelet Gq-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to 2-CRP. The enhanced 2 1-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of IIb 3 integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet Gq-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced 2 1 binding to collagens containing GFOGER sites.