Thrombomodulin Switches Signaling and Protease-Activated Receptor 1 Cleavage Specificity of Thrombin

Background: Cleavage of the extracellular domain of PAR1 (protease-activated receptor 1) by thrombin at Arg41 and by APC (activated protein C) at Arg46 initiates paradoxical cytopathic and cytoprotective signaling in endothelial cells. In the latter case, the ligand-dependent coreceptor signaling by EPCR (endothelial protein C receptor) is required for the protective PAR1 signaling by APC. Here, we investigated the role of thrombomodulin in determining the specificity of PAR1 signaling by thrombin. Methods: We prepared a PAR1 knockout (PAR1(-/-)) EA.hy926 endothelial cell line by CRISPR/Cas9 and transduced PAR1(-/-) cells with lentivirus vectors expressing PAR1 mutants in which either Arg41 or Arg46 was replaced with an Ala. Furthermore, human embryonic kidney 293 cells were transfected with wild-type or mutant PAR1 cleavage reporter constructs carrying N-terminal Nluc (NanoLuc luciferase) and C-terminal enhanced yellow fluorescent protein tags. Results: Characterization of transfected cells in signaling and receptor cleavage assays revealed that, upon interaction with thrombomodulin, thrombin cleaves Arg46 to elicit cytoprotective effects by a beta-arrestin-2 biased signaling mechanism. Analysis of functional data and cleavage rates indicated that thrombin-thrombomodulin cleaves Arg46>10-fold faster than APC. Upon interaction with thrombin, the cytoplasmic domain of thrombomodulin recruited both beta-arrestin-1 and -2 to the plasma membrane. Thus, the thrombin cleavage of Arg41 was also cytoprotective in thrombomodulin-expressing cells by beta-arrestin-1-biased signaling. APC in the absence of EPCR cleaved Arg41 to initiate disruptive signaling responses like thrombin. Conclusions: These results suggest that coreceptor signaling by thrombomodulin and EPCR determines the PAR1 cleavage and signaling specificity of thrombin and APC, respectively.