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

J Thromb Haemost. 2017 Nov;15(11):2198-2207. doi: 10.1111/jth.13807. Epub 2017 Sep 21.

Abstract

Essentials The basis of cytoprotective protease-activated receptor 1 (PAR1) signaling is not fully understood. Activated protein C chimera (APCFVII-82 ) was used to identify requirements for PAR1 signaling. APCFVII-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 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 (PCFVII-82 ) possessing FVII N-terminal domains and conserved EPCR binding was characterized. Methods Activated PC-FVII chimera (APCFVII-82 ) anticoagulant activity was measured with calibrated automated thrombography and activated FV degradation assays. APCFVII-82 signaling activity was characterized by the use of reporter assays of PAR1 proteolysis and EC barrier integrity. APCFVII-82 anti-inflammatory activity was assessed according to its inhibition of nuclear factor-κB (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 interleukin-6 release from lipopolysaccharide (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 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.

Keywords: anticoagulant; endothelium; protein engineering; protein C; thrombin.

MeSH terms

  • Animals
  • Binding Sites
  • Blood Coagulation
  • Capillary Permeability
  • Endothelial Cells / metabolism*
  • Endothelial Protein C Receptor / metabolism
  • Factor VII / chemistry
  • Factor VII / genetics
  • Factor VII / metabolism*
  • HEK293 Cells
  • Humans
  • Inflammation / metabolism
  • Inflammation / prevention & control*
  • Interleukin-6 / metabolism
  • LDL-Receptor Related Proteins / metabolism
  • Macrophages / metabolism*
  • Mice
  • Monocytes / metabolism*
  • NF-kappa B / metabolism
  • Protein Binding
  • Protein C / chemistry
  • Protein C / genetics
  • Protein C / metabolism*
  • Protein Interaction Domains and Motifs
  • RAW 264.7 Cells
  • Receptor, PAR-1 / chemistry
  • Receptor, PAR-1 / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Signal Transduction
  • Structure-Activity Relationship
  • Thrombin / metabolism
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • Endothelial Protein C Receptor
  • Interleukin-6
  • LDL-Receptor Related Proteins
  • NF-kappa B
  • PROCR protein, human
  • Protein C
  • Receptor, PAR-1
  • Recombinant Fusion Proteins
  • Tumor Necrosis Factor-alpha
  • interleukin-6, mouse
  • low density lipoprotein receptor-related protein 8
  • Factor VII
  • Thrombin