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 and PAR4) and two collagen receptors, the alpha(2)beta(1) integrin (alpha(2)beta(1)) and the glycoprotein VI (GPVI)/FcRgamma 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 (alpha2-CRP) containing the alpha(2)beta(1)-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to alpha2-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 alpha(2)beta(1)-dependent and GPVI/FcRgamma-independent as revealed in experiments with alpha(2)beta(1)- or FcRgamma-deficient mouse platelets. We further show that suboptimal activation of other platelet G(q)-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to alpha2-CRP. The enhanced alpha(2)beta(1)-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of alpha(IIb)beta(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 G(q)-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced alpha(2)beta(1) binding to collagens containing GFOGER sites.
"In platelets, most studies have been performed to understand the molecular mechanism behind integrin a IIb b 3 inside-out activation. Relatively limited information is available about the control of integrin a 2 b 1 (Chen and Kahn, 2003; Jung and Moroi, 2001; Marjoram et al., 2009; Pula and Poole, 2008; Wang et al., 2009) with conflicting observations about the possible contribution of PI3K (Jung and Moroi, 2001; Marjoram et al., 2009), and nothing is known about inside-out regulation of other integrins in platelets. "
[Show abstract][Hide abstract] ABSTRACT: Platelets bind annexin V when stimulated with combinations of platelet agonists such as collagen and thrombin. Previous studies have demonstrated significant heterogeneity of platelets binding annexin V. The relative role of the thrombin protease-activated receptors (PARs), PAR1 and PAR4, together with different methods of platelet preparation on annexin V binding to platelets is unclear. We therefore investigated the role of PAR1- and PAR4-activating peptides in combination with collagen-related peptide on annexin V binding. In diluted whole blood, PAR1- and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding. However, in washed platelets, PAR-activating peptides were less potent than thrombin at inducing annexin V binding. This difference was more pronounced when experiments were performed at 37 degrees C compared to room temperature. In studies of diluted whole blood, platelet rich plasma and washed platelets, platelets incubated at room temperature bound more annexin V than platelets incubated at 37 degrees C. We also saw a significant effect of time on annexin V binding, in that more annexin V bound to platelets with longer incubation times. In conclusion, PAR1 and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding in combination with collagen-related peptide in diluted whole blood and platelet rich plasma, but not in washed platelets. In addition, incubation temperature and time has a strong influence on annexin V binding to platelets. Thus variations in these conditions may explain the differences observed between previous studies.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate the in vitro antithrombotic effects of two PAR1 antagonists, ER121958 and SCH203099 on both SFLLR-induced platelet adhesion and aggregation and on the thrombin time in human and guinea-pig platelets. ER121958 inhibited SFLLR-induced guinea-pig and human platelet adhesion with the IC(50) values of 1.73nM and 2.91nM, respectively and SFLLR-induced guinea-pig and human platelet aggregation with the IC(50) values of 2.74nM and 11.9nM, respectively. Similarly, SCH203099 exhibited a non competitive profile of inhibition on both SFLLR-induced guinea-pig and human platelet adhesion with the IC(50) values of 93nM and 127nM, respectively or SFLLR-induced guinea-pig and human platelet aggregation with the IC(50) values of 1.74microM and 2.36microM, respectively. These two antagonists failed to prolong the thrombin time. Altogether, these results highlighted the potent anti-platelets properties of both ER121958 and SCH203099 in an in vitro model of aggregation as well as in a static model of adhesion without any effect on the last step of coagulation cascade. Moreover, this work emphasized that guinea-pig is a suitable animal model to study the role of PAR1 antagonists since the magnitude of the effects of ER121958 and SCH203099 on both SFLLR-induced platelet adhesion and aggregation were similar in both species.
European journal of pharmacology 10/2010; 644(1-3):188-94. DOI:10.1016/j.ejphar.2010.07.013 · 2.53 Impact Factor
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