PAR4 activation involves extracellular loop 3 and transmembrane residue Thr153

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Abstract

Protease-activated receptor 4 (PAR4) mediates sustained thrombin signaling in platelets and is required for a stable thrombus. PAR4 is activated by proteolysis of the N terminus to expose a tethered ligand. The structural basis for PAR4 activation and the location of its ligand binding site (LBS) are unknown. Using hydrogen/deuterium exchange (H/D exchange), computational modeling, and signaling studies, we determined the molecular mechanism for tethered ligand–mediated PAR4 activation. H/D exchange identified that the LBS is composed of transmembrane 3 (TM3) domain and TM7. Unbiased computational modeling further predicted an interaction between Gly48 from the tethered ligand and Thr153 from the LBS. Mutating Thr153 significantly decreased PAR4 signaling. H/D exchange and modeling also showed that extracellular loop 3 (ECL3) serves as a gatekeeper for the interaction between the tethered ligand and LBS. A naturally occurring sequence variant (P310L, rs2227376) and 2 experimental mutations (S311A and P312L) determined that the rigidity conferred by prolines in ECL3 are essential for PAR4 activation. Finally, we examined the role of the polymorphism at position 310 in venous thromboembolism (VTE) using the International Network Against Venous Thrombosis (INVENT) consortium multi-ancestry genome-wide association study (GWAS) meta-analysis. Individuals with the PAR4 Leu310 allele had a 15% reduction in relative risk for VTE (odds ratio, 0.85; 95% confidence interval, 0.77-0.94) compared with the Pro310 allele. These data are consistent with our H/D exchange, molecular modeling, and signaling studies. In conclusion, we have uncovered the structural basis for PAR4 activation and identified a previously unrecognized role for PAR4 in VTE.

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Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

Amal Arachiche, María de la Fuente-Fernández, Marvin T. Nieman (2013)

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.