Abstract
Thrombin activates platelets through protease activated receptors (PARs). Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 also regulates PAR4 signaling. In response to thrombin (30–100 nM) or PAR4 activating peptide (AYPGKF), platelets from PAR3−/− mice had increased Gq signaling compared to wild type mice as demonstrated by a 1.6-fold increase in the maximum intracellular calcium (Ca2+) mobilization, an increase in phosphorylation level of protein kinase C (PKC) substrates, and a 2-fold increase of Ca2+ release from intracellular stores. Moreover, platelets from heterozygous mice (PAR3+/−) had an intermediate increase in maximum Ca2+ mobilization. Treatment of PAR3−/− mice platelets with P2Y12 antagonist (2MeSAMP) did not affect Ca2+ mobilization from PAR4 in response to thrombin or AYPGKF. The activation of RhoA-GTP downstream G12/13 signaling in response to thrombin was not significantly different between wild type and PAR3−/− mice. Since PAR3 influenced PAR4 signaling independent of agonist, we examined the direct interaction between PAR3 and PAR4 with bioluminescence resonance energy transfer (BRET). PAR3 and PAR4 form constitutive homodimers and heterodimers. In summary, our results demonstrate that in addition to enhancing PAR4 activation at low thrombin concentrations, PAR3 negatively regulates PAR4-mediated maximum Ca2+ mobilization and PKC activation in mouse platelets by physical interaction.
Highlights
Thrombin signaling in platelets is mediated by protease activated receptors (PARs)
Intracellular Ca2+ mobilization is increased in PAR32/2 mouse platelets We first determined if the absence of PAR3 affected Protease Activated Receptor 4 (PAR4) mediated intracellular Ca2+ mobilization in PAR32/2 platelets in response to thrombin
Heterozygous mice (PAR3+/2) had an intermediate value (1.1 nM with a 95% confidence interval of 0.5–3.7 nM). These results agree with published data showing that PAR3 is a cofactor for PAR4 activation at low thrombin concentrations [6]
Summary
Thrombin signaling in platelets is mediated by protease activated receptors (PARs). PARs are G-protein-coupled receptors (GPCR) that are activated via proteolytic cleavage of the extracellular N-terminus to initiate a variety of signaling cascades through activation of heterotrimeric G proteins [1,2]. Human platelets express PAR1 and PAR4 and cleavage of each receptor initiates signaling cascades [3]. It has been shown that PAR1 is directly coupled to Gi in human platelets and in COS7 cells transfected with PAR1 [9,10]. Other studies have shown that PAR1 and PAR4 do not couple directly to Gi, rather activation of the Gi pathway is mediated by secondary release of ADP, which acts on the Gi-coupled ADP receptor, P2Y12 [8,11,12]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
