A novel proteinC-factorVII chimera provides new insights into the structural requirements for cytoprotective protease-activated receptor1 signaling

作者：Gleeson E M; Mcdonnell C J; Soule E E; Fox O Willis; Rushe H; Rehill A; Smith O P; O'Donnell J S; Preston R J S^*

来源：Journal of Thrombosis and Haemostasis, 2017, 15(11): 2198-2207.

DOI：10.1111/jth.13807

• anticoagulant
• endothelium
• proteinC
• protein engineering
• thrombin

Background Activated proteinC (APC) cell signaling is largely reliant upon its ability to mediate protease-activated receptor (PAR)1 proteolysis when bound to the endothelial cell (EC) proteinC (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-factorVII chimera (PCFVII-82) possessing FVII N-terminal domains and conserved EPCR binding was characterized.
Methods Activated PC-FVII chimera (APC(FVII-82)) anticoagulant activity was measured with calibrated automated thrombography and activated FV degradation assays. APC(FVII-82) signaling activity was characterized by the use of reporter assays of PAR1 proteolysis and EC barrier integrity. APC(FVII-82) anti-inflammatory activity was assessed according to its inhibition of nuclear factor-B (NF-B) 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, APC(FVII-82) 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(FVII-82) was, like FVII, unable to mimic EC barrier stabilization induced by PC upon PAR1 proteolysis by thrombin. APC(FVII-82) did, however, diminish LPS-induced NF-B activation and tumor necrosis factor- release from monocytes in an apolipoproteinE receptor2-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.

• 出版日期2017-11