Abstract 367: PAR3 Negatively Regulates PAR4-Mediated Gq Signaling Without Affecting the G12/13 Pathway in Mouse Platelets
Protease activated receptors (PAR) are G-protein-coupled receptors (GPCR) that are activated by proteolytic cleavage of the N-terminus. Mouse platelets use PAR3 and PAR4 to respond to thrombin. PAR3 itself does not signal, but it is a cofactor that enhances cleavage and activation of PAR4 by thrombin ∼ 10-fold. PAR4 is the signaling thrombin receptor and is sufficient to induce platelet activation at >10 nM thrombin. The aim of this study is to determine how PAR3 regulates PAR4 signaling in mouse platelets. In this study we show that platelets from PAR3 knockout mice (PAR3-/-) had a 1.6-fold increase in the maximum calcium mobilization at thrombin concentrations above 10 nM, compared to wild type platelets. The PAR3+/- had an intermediate increase (∼ 1.2-fold) in the maximum calcium mobilization. PAR3-/- platelets also had an increase in PKC activation, as measured by serine phosphorylation of PKC substrates in response to thrombin (10-100nM). The PAR4 agonist peptide (AYPGKF) also induced an increase in maximum calcium mobilization and PKC activation in PAR3-/- platelets, indicating that PAR3 may be influencing PAR4 at the level of the receptor. PAR4 is coupled to Gq and G12/13. PAR3 influences PAR4-mediated calcium mobilization and PKC activation which are both downstream of Gq. We next, determined the influence of PAR3 on PAR4 mediated G12/13 signaling by measuring the activation of small GTPase RhoA protein in response to thrombin (1-100nM). The level of RhoA activation measured was not different in PAR3-/- compared to wild-type platelets in response to thrombin. In conclusion, PAR3 negatively regulates PAR4 signaling through the Gq pathway without affecting the G12/13 pathway. Since the increase of Gq signaling is independent of PAR4 agonist, PAR3 is influencing PAR4 signaling at the level of the receptors, perhaps by forming heterodimers. The crosstalk between platelet GPCRs may be a general mechanism for regulating platelet signaling and uncovering the details of these interactions may lead to novel insights for developing therapeutics.
- © 2012 by American Heart Association, Inc.