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A novel protein C–factor VII chimera provides new insights into the structural requirements for cytoprotective protease‐activated receptor 1 signaling
Author(s) -
Gleeson E. M.,
McDonnell C. J.,
Soule E. E.,
Willis Fox O.,
Rushe H.,
Rehill A.,
Smith O. P.,
O'Donnell J. S.,
Preston R. J. S.
Publication year - 2017
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.13807
Subject(s) - endothelial protein c receptor , protein c , thrombin , microbiology and biotechnology , proteolysis , signal transduction , chemistry , receptor , cell signaling , chimera (genetics) , biology , biochemistry , immunology , platelet , gene , enzyme
Essentials The basis of cytoprotective protease‐activated receptor 1 (PAR1) signaling is not fully understood. Activated protein C chimera (APC FVII‐82 ) was used to identify requirements for PAR1 signaling. APC FVII‐82 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 PAR 1 signaling by thrombin to induce APC ‐like EC cytoprotection. Objective The molecular determinants of EPCR ‐dependent cytoprotective PAR 1 signaling remain poorly defined. To address this, a PC –factor VII chimera ( PC FVII ‐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 PAR 1 proteolysis and EC barrier integrity. APC FVII ‐82 anti‐inflammatory activity was assessed according to its inhibition of nuclear factor‐κB ( NF ‐κB) activation and cytokine secretion from monocytes. Results PC FVII ‐82 was activated normally by thrombin on EC s, but was unable to inhibit plasma thrombin generation. Surprisingly, APC FVII ‐82 did not mediate EPCR ‐dependent PAR 1 proteolysis, confer PAR 1‐dependent protection of thrombin‐induced EC barrier disruption, or limit PAR 1‐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 PAR 1 proteolysis by thrombin. APC FVII ‐82 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 PAR 1 signaling.