PAR4 activation involves extracellular loop 3 and transmembrane residue Thr153

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.