A novel protein C-factor VII chimera provides new insights into the structural requirements for cytoprotective PAR1 signaling

Background Activated protein C (APC) cell signaling is largely reliant upon its ability to mediate protease-activated receptor 1 (PAR1) proteolysis when bound to the endothelial cell protein C receptor (EPCR). Furthermore, EPCR-bound PC modulates PAR1 signaling by thrombin to induce APC-like endothelial cell (EC) cytoprotection. Objective The molecular determinants of EPCR-dependent cytoprotective PAR1 signaling remain poorly defined. To address this, a PC-factor VII (FVII) chimera (PCFVII−82) possessing FVII N-terminal domains and conserved EPCR binding was characterized. Methods APCFVII−82 anticoagulant activity was measured by calibrated automated thrombography and FVa degradation assays. APCFVII−82 signaling activity was characterised using reporter assays of PAR1 proteolysis and EC barrier integrity. APCFVII−82 anti-inflammatory activity was assessed by its inhibition of NF-κB activation and cytokine secretion from monocytes. Results PCFVII−82 was activated normally by thrombin on ECs, but was unable to inhibit plasma thrombin generation. Surprisingly, APCFVII−82 did not mediate EPCR-dependent PAR1 proteolysis, confer PAR1-dependent protection of thrombin-induced EC barrier disruption, or limit PAR1-dependent attenuation of IL-6 release from LPS-stimulated macrophages. Interestingly, EPCR occupation by active-site blocked APCFVII−82 was, like FVII, unable to mimic EC barrier stabilization induced by PC upon PAR1 proteolysis by thrombin. APCFVII−82 did, however, diminish LPS-induced NF-κB activation and TNF-α release from monocytes in an apolipopotein E receptor 2 (ApoER2)-dependent manner, with similar efficacy as wild type APC. Conclusions These data identify a novel role for APC light chain amino acid residues outwith the EPCR binding site in enabling cytoprotective PAR1 signaling. This article is protected by copyright. All rights reserved.