The thrombin mutant Trp215Ala/Glu217Ala induces cytoprotective signaling in endothelial cells by cleavage of Arg46 of protease-activated receptor 1.

BACKGROUND: The thrombin mutant W215A/E217A (WE-thrombin) is currently undergoing clinical trials to assess its therapeutic potential as an antithrombotic and cytoprotective drug. Mechanistically, it is thought that WE-thrombin exerts its protective effects indirectly through thrombomodulin (TM)-dependent activation of protein C (APC). APC exerts protective effects by both enzymatically inactivating procoagulant cofactors (F)Va and FVIIIa in the anticoagulant pathway and cleaving Arg46 of protease-activated receptor 1 (PAR1; PAR1-Arg46) in the anti-inflammatory pathway. We recently discovered that upon binding to TM, thrombin cleaves PAR1-Arg46 with ∼10-fold greater efficiency than APC, thereby eliciting cytoprotective signaling effects in endothelial cells. OBJECTIVES: In this study, we investigated the hypothesis that WE-thrombin may have a direct, PAR1-dependent cytoprotective signaling function. METHODS: We evaluated the direct signaling and PAR1-Arg46 cleavage functions of WE-thrombin by transfecting PAR1 knockout endothelial cells or human embryonic kidney 293 cells with a PAR1 construct in which Arg46 is intact, but Arg41 has been replaced with Ala (PAR1-R41A). RESULTS: We discovered that WE-thrombin elicits direct cytoprotective signaling in transfected endothelial cells by cleaving PAR1-Arg46. This conclusion was supported by a PAR1-Arg46 cleavage-reporter assay using human embryonic kidney 293 cells transfected with both TM- and NanoLuc luciferase-labeled PAR1-R41A constructs. CONCLUSION: These results suggest that direct WE-thrombin activation of PAR1 through cleavage of Arg46 may be primarily responsible for the cytoprotective signaling function of WE-thrombin, independent of its role as a protein C activator.