Endothelial protein C receptor and thrombomodulin facilitate protease-activated receptor 1 cleavage at Arginine-46 by thrombin and activated protein C.

Protease-activated receptor 1 (PAR1) has two cleavage sites for activation by coagulation proteases (Arg41 and Arg46). The cleavage of Arg46 by either activated protein C (APC) or thrombin leads to cytoprotective signaling; however, neither protease can cleave this site in the absence of their receptors, endothelial protein C receptor (EPCR) and thrombomodulin (TM), respectively. Arg41 is the preferred cleavage site for both proteases in the absence of receptors. The mechanism by which these receptors function as cofactors to catalyze the cleavage of PAR1-R46 by coagulation proteases is not known. Here, we hypothesized that both receptors alleviate inhibitory interactions of the P2-Leu45 residue on the extracellular domain of PAR1 with protease catalytic pockets. To test this hypothesis, we prepared a PAR1-R41A mutant in which P2-Leu45 of the receptor was substituted with a Pro. Both PAR1-R41A and PAR1-R41A-L45P constructs were transfected to PAR1-knockout EA.hy926 endothelial cells lacking or expressing EPCR or TM followed by monitoring the protease activation of receptors by signaling assays. Furthermore, WT or EPCR and TM expressing human embryonic kidney 293 cells were transfected with PAR1 cleavage reporter constructs carrying N-terminal NanoLuc luciferase and C-terminal enhanced YFP tags. Signaling and receptor cleavage assays indicated that both APC and thrombin cleave Arg46 in cells expressing PAR1-R41A-L45P, but not PAR1-41A, independent of their receptors. The catalytic activity of thrombin was >10-fold faster than APC in both assays. These results suggest that EPCR and TM function as cofactors to alleviate inhibitory interactions of P2-Leu45 of PAR1 with target proteases.