Protease-activated receptor-1 cleaved at R46 mediates cytoprotective effects

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Pharmacology

Background:

• Activated protein C (aPC) exhibits cytoprotective effects via protease-activated receptor-1 (PAR1), contrasting with thrombin's detrimental effects.
• The mechanism by which aPC-activated PAR1 confers protection, while thrombin activation leads to harm, remains unclear.

Purpose of the Study:

• To investigate the hypothesis that aPC and thrombin induce distinct active conformations in PAR1, resulting in opposing cellular responses.
• To elucidate the differential signaling pathways initiated by aPC and thrombin through PAR1.

Main Methods:

• Analysis of antibody binding, cleavage patterns, and downstream signaling of PAR1 in endothelial cells and 293T cells.
• Utilized reporter constructs with selective glutamine substitutions to identify specific cleavage sites on PAR1 and PAR3.

Main Results:

• Thrombin cleavage at R41 and aPC cleavage at R46 were identified as distinct sites on PAR1, influencing receptor conformation and accessibility.
• aPC irreversibly altered ATAP2 binding, while thrombin did not, indicating distinct conformational changes.
• Cleavage at R46 of PAR1 was essential for the cytoprotective signaling induced by aPC, enhancing endothelial barrier function and reducing toxicity.

Conclusions:

• aPC and thrombin induce distinct PAR1 conformations through enzyme-specific cleavage sites (R46 for aPC, R41 for thrombin).
• These divergent conformations mediate opposing downstream effects, providing a novel mechanistic understanding of PAR1 signaling.
• This discovery opens avenues for developing targeted therapies for inflammatory diseases by modulating PAR1 signaling pathways.