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

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. 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.

Gleeson EM ，McDonnell CJ ，Soule EE ，Willis Fox O ，Rushe H ，Rehill A ，Smith OP ，O'Donnell JS ，Preston RJS ... -  《-》

Occupancy of human EPCR by protein C induces β-arrestin-2 biased PAR1 signaling by both APC and thrombin.

Activation of protease-activated receptor 1 (PAR1) by activated protein C (APC) and thrombin elicits paradoxical cytoprotective and cytotoxic signaling responses in vascular endothelial cells through cleavage of the receptor at Arg-46 and Arg-41 protease recognition sites, respectively. It has been reported that unlike a disruptive G-protein-mediated PAR1 signaling by thrombin, APC induces a protective β-arrestin-2 biased PAR1 signaling by unknown mechanisms. We hypothesize that the occupancy of endothelial protein C receptor (EPCR) by the Gla-domain of protein C/APC is responsible for the β-arrestin-2 biased PAR1 signaling independent of the protease cleavage site. To test this hypothesis, we monitored the signaling specificity of thrombin in endothelial cells in response to lipopolysaccharide (LPS) with or without pretreatment of cells with protein C-S195A. The PAR1-dependent recruitment of β-arrestin-2 in response to LPS by both APC and thrombin was analyzed by functional, gene silencing, and signaling assays. Results indicate that similar to APC, thrombin exerts cytoprotective effects via β-arrestin-2 biased PAR1 signaling. Similar to APC, thrombin triggered β-arrestin-2-dependent recruitment of disheveled 2 (Dvl-2) in PC-S195A pretreated cells. Further studies in HeLa cells transfected with PAR1 constructs revealed that EPCR occupancy initiates β-arrestin-2 biased PAR1 signaling independent of the protease cleavage sites. We demonstrate that EPCR occupancy recruits G-protein coupled receptor kinase 5, thereby inducing β-arrestin-2 biased PAR1 signaling by both APC and thrombin. In support of a physiological relevance for these results, intraperitoneal administration of PC-S195A conferred a cytoprotective effect for thrombin in an in vivo inflammatory model.

Roy RV ，Ardeshirylajimi A ，Dinarvand P ，Yang L ，Rezaie AR ... -  《-》

Factor VII, EPCR, aPC Modulators: novel treatment for neuroinflammation.

Inflammation and coagulation are linked and pathogenic in neuroinflammatory diseases. Protease-activated receptor 1 (PAR1) can be activated both by thrombin, inducing increased inflammation, and activated protein C (aPC), inducing decreased inflammation. Modulation of the aPC-PAR1 pathway may prevent the neuroinflammation associated with PAR1 over-activation. We synthesized a group of novel molecules based on the binding site of FVII/aPC to the endothelial protein C receptor (EPCR). These molecules modulate the FVII/aPC-EPCR pathway and are therefore named FEAMs-Factor VII, EPCR, aPC Modulators. We studied the molecular and behavioral effects of a selected FEAM in neuroinflammation models in-vitro and in-vivo. In a lipopolysaccharide (LPS) induced in-vitro model, neuroinflammation leads to increased thrombin activity compared to control (2.7 ± 0.11 and 2.23 ± 0.13 mU/ml, respectively, p = 0.01) and decreased aPC activity (0.57 ± 0.01 and 1.00 ± 0.02, respectively, p < 0.0001). In addition, increased phosphorylated extracellular regulated kinase (pERK) (0.99 ± 0.13, 1.39 ± 0.14, control and LPS, p < 0.04) and protein kinase B (pAKT) (1.00 ± 0.09, 2.83 ± 0.81, control and LPS, p < 0.0002) levels indicate PAR1 overactivation, which leads to increased tumor necrosis factor-alpha (TNF-α) level (1.00 ± 0.04, 1.35 ± 0.12, control and LPS, p = 0.02). In a minimal traumatic brain injury (mTBI) induced neuroinflammation in-vivo model in mice, increased thrombin activity, PAR1 activation, and TNF-α levels were measured. Additionally, significant memory impairment, as indicated by a lower recognition index in the Novel Object Recognition (NOR) test and Y-maze test (NOR: 0.19 ± 0.06, -0.07 ± 0.09, p = 0.03. Y-Maze: 0.50 ± 0.03, 0.23 ± 0.09, p = 0.02 control and mTBI, respectively), as well as hypersensitivity by hot-plate latency (16.6 ± 0.89, 12.8 ± 0.56 s, control and mTBI, p = 0.01), were seen. FEAM prevented most of the molecular and behavioral negative effects of neuroinflammation in-vitro and in-vivo, most likely through EPCR-PAR1 interactions. FEAM is a promising tool to study neuroinflammation and a potential treatment for a variety of neuroinflammatory diseases.

Golderman V ，Ben-Shimon M ，Maggio N ，Dori A ，Gofrit SG ，Berkowitz S ，Qassim L ，Artan-Furman A ，Zeimer T ，Chapman J ，Shavit-Stein E ... -  《Journal of Neuroinflammation》

Protease activated receptor 1 (PAR-1) activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand.

Bae JS ，Rezaie AR 《THROMBOSIS AND HAEMOSTASIS》

Protease-activated receptor-1 cleaved at R46 mediates cytoprotective effects.

Activated protein C (aPC) mediates powerful cytoprotective effects through the protease-activated receptor-1 (PAR1) that translate into reduced harm in mouse injury models. However, it remains elusive how aPC-activated PAR1 can mediate cytoprotective effects whereas thrombin activation does the opposite. We hypothesized that aPC and thrombin might induce distinct active conformations in PAR1 causing opposing effects. We analyzed antibody binding to, and cleavage and signalling of PAR1 in either endogenously expressing endothelial or overexpressing 293T cells. In thrombin-cleaved PAR1 neither the tethered ligand nor the hirudin-like domain were available for anti-PAR1 ATAP2 and WEDE15 binding unless the tethered ligand was quenched. In contrast, aPC irreversibly prevented ATAP2 binding while not affecting WEDE15 binding. Reporter constructs with selective glutamine substitutions confirmed R41 as the only thrombin cleavage site in PAR1, whereas aPC preferentially cleaved at R46. Similarly, we report distinct cleavage sites on PAR3, K38 for thrombin and R41 for aPC. A soluble peptide corresponding to R46-cleaved PAR1 enhanced the endothelial barrier function and reduced staurosporine toxicity in endothelial as well as in 293T cells if PAR1 was expressed. Overexpression of PAR1 variants demonstrated that cleavage at R46 but not R41 is required for cytoprotective aPC signaling. We provide a novel concept on how aPC and thrombin mediate distinct effects. We propose that the enzyme-specific cleavage sites induce specific conformations which mediate divergent downstream effects. This unexpected model of PAR1 signaling might lead to novel therapeutic options for the treatment of inflammatory diseases.

Schuepbach RA ，Madon J ，Ender M ，Galli P ，Riewald M ... -  《-》