PAR4 activation involves extracellular loop 3 and transmembrane residue Thr153

Xu Han, Lukas Hofmann, Maria De la Fuente, Nathan Alexander, Krzysztof Palczewski, Marvin T. Nieman

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

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 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.

Original languageEnglish
Pages (from-to)2217-2228
Number of pages12
JournalBlood
Volume136
Issue number19
DOIs
StatePublished - 5 Nov 2020
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2020 by The American Society of Hematology.

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