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The structural basis for protease-activated receptor 4 (PAR4) activation was uncovered, revealing its ligand binding site and a role in venous thromboembolism (VTE) risk reduction.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • Protease-activated receptor 4 (PAR4) is crucial for sustained thrombin signaling in platelets and stable thrombus formation.
  • PAR4 activation occurs via N-terminal cleavage, exposing a tethered ligand, but its structural basis and ligand binding site (LBS) remain unknown.

Purpose of the Study:

  • To elucidate the molecular mechanism of tethered ligand-mediated PAR4 activation.
  • To identify the structural components of the PAR4 ligand binding site (LBS).
  • To investigate the role of PAR4 polymorphisms in venous thromboembolism (VTE).

Main Methods:

  • Hydrogen/deuterium exchange (H/D exchange) mass spectrometry.
  • Computational molecular modeling.
  • Platelet signaling assays.
  • Genetic analysis of a VTE cohort (INVENT consortium).

Main Results:

  • H/D exchange and modeling identified the LBS comprising transmembrane domains 3 (TM3) and 7 (TM7).
  • A key interaction between the tethered ligand (Gly48) and LBS (Thr153) was predicted and validated by mutation studies.
  • Extracellular loop 3 (ECL3) acts as a gatekeeper, with proline residues essential for PAR4 activation.
  • A common PAR4 polymorphism (P310L, rs2227376) was associated with a 15% reduced risk of VTE.

Conclusions:

  • The structural basis for PAR4 activation by its tethered ligand has been determined.
  • The ligand binding site involves TM3 and TM7, with ECL3 playing a critical gatekeeper role.
  • PAR4 genetic variation influences VTE risk, highlighting its therapeutic potential.