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 computationalmodeling 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 consistentwith 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.
|Number of pages||12|
|State||Published - 5 Nov 2020|
Bibliographical noteFunding Information:
This study was supported by a grant from the National Institutes of Health (NIH) National Heart Lung and Blood Institute (NHLBI) (HL098217) (M.T.N.) and the American Heart Association (15GRNT25090222) (M.T.N.). X.H. received research funding from the American Heart Association 2018 Predoctoral Fellowship (18PRE33960396) and is cofunded by the Schwab Charitable Fund. This research was supported in part by grant R24EY027283 to K.P. from the NIH National Eye Institute and the Immune Core Facility in the CWRU/UH Center for AIDS Research (NIH grant: P30 AI036219). The authors also acknowledge support from an RPB unrestricted grant to the Department of Ophthalmology, University of California, Irvine. The INVENT Consortium is supported in part by NIH, NHLBI grant HL134894.
© 2020 by The American Society of Hematology.