A novel protein C-factor VII chimera provides new insights into the structural requirements for cytoprotective protease-activated receptor 1 signaling.

UNLABELLED

Essentials The basis of cytoprotective protease-activated receptor 1 (PAR1) signaling is not fully understood. Activated protein C chimera (APC ) was used to identify requirements for PAR1 signaling. APC did not initiate PAR1 signaling, but conferred monocyte anti-inflammatory activity. APC-specific light chain residues are required for cytoprotective PAR1 signaling.

SUMMARY

Background Activated protein C (APC) cell signaling is largely reliant upon its ability to mediate protease-activated receptor (PAR) 1 proteolysis when bound to the endothelial cell (EC) protein C (PC) receptor (EPCR). Furthermore, EPCR-bound PC modulates PAR1 signaling by thrombin to induce APC-like EC cytoprotection. Objective The molecular determinants of EPCR-dependent cytoprotective PAR1 signaling remain poorly defined. To address this, a PC-factor VII chimera (PC ) possessing FVII N-terminal domains and conserved EPCR binding was characterized. Methods Activated PC-FVII chimera (APC ) anticoagulant activity was measured with calibrated automated thrombography and activated FV degradation assays. APC signaling activity was characterized by the use of reporter assays of PAR1 proteolysis and EC barrier integrity. APC anti-inflammatory activity was assessed according to its inhibition of nuclear factor-κB (NF-κB) activation and cytokine secretion from monocytes. Results PC was activated normally by thrombin on ECs, but was unable to inhibit plasma thrombin generation. Surprisingly, APC did not mediate EPCR-dependent PAR1 proteolysis, confer PAR1-dependent protection of thrombin-induced EC barrier disruption, or limit PAR1-dependent attenuation of interleukin-6 release from lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, EPCR occupation by active site-blocked APC was, like FVII, unable to mimic EC barrier stabilization induced by PC upon PAR1 proteolysis by thrombin. APC did, however, diminish LPS-induced NF-κB activation and tumor necrosis factor-α release from monocytes in an apolipoprotein E receptor 2-dependent manner, with similar efficacy as wild-type APC. Conclusions These findings identify a novel role for APC light chain amino acid residues outside the EPCR-binding site in enabling cytoprotective PAR1 signaling.