scholarly journals Factor VIIa binding to endothelial cell protein C receptor protects vascular barrier integrity in vivo

2014 ◽  
Vol 12 (5) ◽  
pp. 690-700 ◽  
Author(s):  
J. Sundaram ◽  
S. Keshava ◽  
R. Gopalakrishnan ◽  
C. T. Esmon ◽  
U. R. Pendurthi ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Protein C ◽  
Factor Viia ◽  
Cell Protein ◽  
Barrier Integrity ◽  
Vascular Barrier Integrity

scholarly journals Inactivation of Factor VIIa by Antithrombin In Vitro, Ex Vivo and In Vivo: Role of Tissue Factor and Endothelial Cell Protein C Receptor

PLoS ONE ◽  
2014 ◽  
Vol 9 (8) ◽  
pp. e103505 ◽  
Author(s):  
Rit Vatsyayan ◽  
Hema Kothari ◽  
Nigel Mackman ◽  
Usha R. Pendurthi ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Endothelial Cell ◽  
Tissue Factor ◽  
Protein C ◽  
Factor Viia ◽  
Ex Vivo ◽  
Cell Protein

scholarly journals Factor VIIa binding to endothelial cell protein C receptor: Differences between mouse and human systems

2012 ◽  
Vol 107 (05) ◽  
pp. 951-961 ◽  
Author(s):  
Prosenjit Sen ◽  
Curtis A. Clark ◽  
Ramakrishnan Gopalakrishnan ◽  
Ulla Hedner ◽  
Charles T. Esmon ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Plasma Levels ◽  
Protein C ◽  
Factor Viia ◽  
Cell Protein ◽  
Dependent Manner ◽  
Binding Studies ◽  
High Concentrations

SummaryRecent in vitro studies have shown that the zymogen and activated form of factor (F)VII bind to endothelial cell protein C receptor (EPCR). At present, there is no evidence that FVIIa binds to EPCR on vascular endothelium in vivo in the presence of circulating protein C, a primary ligand for EPCR. The present study was carried out to investigate the interaction of murine and human ligands with murine EPCR both in vivo and in vitro. Measurement of endogenous plasma levels of FVII in wild-type, EPCR-deficient and EPCR-over expressing mice showed slightly lower levels of FVII in EPCR-over expressing mice. However, infusion of high concentrations of competing ligands, either human APCi or FVIIai, to EPCR-over expressing mice failed to increase plasma levels of mouse FVII whereas they increased the plasma levels of protein C by two- to three-fold. Examining the association of exogenously administered mouse FVIIa or human FVIIa by immunohistochemistry revealed that human, but not murine FVIIa, binds to the murine endothelium in an EPCR-dependent manner. In vitro binding studies performed using surface plasmon resonance and endothelial cells revealed that murine FVIIa binds murine EPCR negligibly. Human FVIIa binding to EPCR, particularly to mouse EPCR, is markedly enhanced by availability of Mg2+ ions. In summary, our data show that murine FVIIa binds poorly to murine EPCR, whereas human FVIIa binds efficiently to both murine and human EPCR. Our data suggest that one should consider the use of human FVIIa in mouse models to investigate the significance of FVIIa and EPCR interaction.

scholarly journals FVIIa (Factor VIIa) Induces Biased Cytoprotective Signaling in Mice Through the Cleavage of PAR (Protease-Activated Receptor)-1 at Canonical Arg41 (Arginine41) Site

2020 ◽  
Vol 40 (5) ◽  
pp. 1275-1288 ◽  
Author(s):  
Vijay Kondreddy ◽  
Usha R. Pendurthi ◽  
Xiao Xu ◽  
John H. Griffin ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein C ◽  
Factor Viia ◽  
Vascular Endothelial Cell ◽  
Cell Protein ◽  
Mouse Strains ◽  
Biased Agonism ◽  
Protease Activated Receptor 1

Objective: Recent studies showed that FVIIa (factor VIIa), upon binding to EPCR (endothelial cell protein C receptor), elicits endothelial barrier stabilization and anti-inflammatory effects via activation of PAR (protease-activated receptor)-1–mediated signaling. It is unknown whether FVIIa induces PAR1-dependent cytoprotective signaling through cleavage of PAR1 at the canonical site or a noncanonical site, similar to that of APC (activated protein C). Approach and Results: Mouse strains carrying homozygous R41Q (canonical site) or R46Q (noncanonical site) point mutations in PAR1 (QQ41-PAR1 and QQ46-PAR1 mice) were used to investigate in vivo mechanism of PAR1-dependent pharmacological beneficial effects of FVIIa. Administration of FVIIa reduced lipopolysaccharide-induced inflammation, barrier permeability, and VEGF (vascular endothelial cell growth factor)-induced barrier disruption in wild-type (WT) and QQ46-PAR1 mice but not in QQ41-PAR1 mice. In vitro signaling studies performed with brain endothelial cells isolated from WT, QQ41-PAR1, and QQ46-PAR1 mice showed that FVIIa activation of Akt (protein kinase B) in endothelial cells required R41 cleavage site in PAR1. Our studies showed that FVIIa cleaved endogenous PAR1 in endothelial cells, and FVIIa-cleaved PAR1 was readily internalized, unlike APC-cleaved PAR1 that remained on the cell surface. Additional studies showed that pretreatment of endothelial cells with FVIIa reduced subsequent thrombin-induced signaling. This process was dependent on β-arrestin1. Conclusions: Our results indicate that in vivo pharmacological benefits of FVIIa in mice arise from PAR1-dependent biased signaling following the cleavage of PAR1 at the canonical R41 site. The mechanism of FVIIa-induced cytoprotective signaling is distinctly different from that of APC. Our data provide another layer of complexity of biased agonism of PAR1 and signaling diversity.

Endotoxin and thrombin elevate rodent endothelial cell protein C receptor mRNA levels and increase receptor shedding in vivo

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1687-1693 ◽  
Author(s):  
Jian-Ming Gu ◽  
Yasuhiro Katsuura ◽  
Gary L. Ferrell ◽  
Paula Grammas ◽  
Charles T. Esmon
Keyword(s):  
Cell Culture ◽  
Septic Shock ◽  
Endothelial Cell ◽  
Protein C ◽  
Fold Increase ◽  
Cell Protein ◽  
Thrombin Inhibitor ◽  
Mrna Levels ◽  
Soluble Epcr

The endothelial cell protein C receptor (EPCR) facilitates protein C activation by the thrombin-thrombomodulin complex. Protein C activation has been shown to be critical to the host defense against septic shock. In cell culture, tumor necrosis factor- (TNF-) down-regulates EPCR expression, raising the possibility that EPCR might be down-regulated in septic shock. We examined EPCR mRNA and soluble EPCR levels in mice and rats challenged with lethal dose 95 levels of endotoxin. Toxic doses of TNF- failed to alter EPCR mRNA levels in mice. Rather than EPCR mRNA levels falling in response to endotoxin, as predicted from cell-culture experiments, they rose approximately 3-fold 6 hours after exposure to endotoxin before returning toward baseline levels at 24 hours after exposure. Soluble EPCR levels rose approximately 4-fold. Infusion of hirudin, a specific thrombin inhibitor, before endotoxin exposure almost completely blocked the increase in EPCR mRNA and soluble EPCR. Consistent with the idea that the responses were mediated by thrombin, thrombin infusion (5 U/kg of body weight for 3 hours) resulted in an approximately 2-fold increase in EPCR mRNA and soluble EPCR. Incubation of rat endothelial cells with thrombin or murine protease-activated receptor 1 agonist peptide resulted in a 2-fold increase in EPCR mRNA. These results indicate that thrombin plays a major role in up-regulating EPCR mRNA and shedding in vivo.

scholarly journals A critical role of endothelial cell protein C receptor in the intestinal homeostasis in experimental colitis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Vijay Kondreddy ◽  
Shiva Keshava ◽  
Charles T. Esmon ◽  
Usha R. Pendurthi ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Endothelial Cell ◽  
Epithelial Cells ◽  
Protein C ◽  
Factor Viia ◽  
Activity Index ◽  
Critical Role ◽  
Experimental Colitis ◽  
Cell Protein ◽  
Bodyweight Loss ◽  
Colon Mucosa

AbstractCrohn’s disease and ulcerative colitis are the two forms of disorders of the human inflammatory bowel disease with unknown etiologies. Endothelial cell protein C receptor (EPCR) is a multifunctional and multiligand receptor, which is expressed on the endothelium and other cell types, including epithelial cells. Here, we report that EPCR is expressed in the colon epithelial cells, CD11c+, and CD21+/CD35+ myeloid cells surrounding the crypts in the colon mucosa. EPCR expression was markedly decreased in the colon mucosa during colitis. The loss of EPCR appeared to associate with increased disease index of the experimental colitis in mice. EPCR−/− mice were more susceptible to dextran sulfate sodium (DSS)-induced colitis, manifested by increased weight loss, macrophage infiltration, and inflammatory cytokines in the colon tissue. DSS treatment of EPCR−/− mice resulted in increased bleeding, bodyweight loss, anemia, fibrin deposition, and loss of colon epithelial and goblet cells. Administration of coagulant factor VIIa significantly attenuated the DSS-induced colon length shortening, rectal bleeding, bodyweight loss, and disease activity index in the wild-type mice but not EPCR−/− mice. In summary, our data provide direct evidence that EPCR plays a crucial role in regulating the inflammation in the colon during colitis.

Factor VIIa Bound to Endothelial Cell Protein C Receptor Activates Protease Activated Receptor 1-Mediated Cell Signaling and Barrier-Protective Response In Endothelial Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 346-346
Author(s):  
Prosenjit Sen ◽  
Ramakrishnan Gopalakrishnan ◽  
Hema Kothari ◽  
Curtis Clark ◽  
Usha Pendurthi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Cell Signaling ◽  
Protective Effect ◽  
Protein C ◽  
Factor Viia ◽  
Stress Fibers ◽  
Cell Protein ◽  
Mapk Activation

Abstract Abstract 346 Endothelial cell protein C receptor (EPCR) is the cellular receptor for protein C and activated protein C (APC). In addition to controlling coagulation by modulating the protein C-mediated anticoagulant pathway, EPCR has been shown to play a critical role in supporting APC-induced cell signaling, which could be responsible for some of the non-hemostatic functions of EPCR and APC. Recent studies from our laboratory and others have shown that factor VIIa (FVIIa), a coagulation factor whose primary function is to initiate tissue factor (TF)-dependent coagulation, also binds to EPCR on endothelium. At present, the physiological significance of this interaction is unclear. APC binding to EPCR has been shown to provide cytoprotective effects via protease activated receptor (PAR) 1-mediated cell signaling. In earlier studies using exogenously expressed PAR1 and PAR2 reporter constructs in a heterologus cell model system, we were unable to find measurable n-terminal cleavage (activation) of PARs by FVIIa bound to EPCR. It is possible that transfected PAR constructs may segregate differently on the cell surface membrane than that of endogenous PARs, and thus may have decreased susceptibility for cleavage by FVIIa-EPCR. In the present study, we have investigated whether FVIIa, upon binding to EPCR on endothelial cells, activates endogenous PAR1 and induces PAR1-mediated cell signaling. To determine whether FVIIa cleaves endogenously expressed PAR1 on endothelial cells, unperturbed cultures of human umbilical vein endothelial cells (HUVEC) were exposed to varying concentrations of FVIIa (0-40 nM) and the cleavage of PAR1 at the cell surface was measured quantitatively in a cell-surface ELISA using a cleavage-specific PAR1 monoclonal antibody. The data show that FVIIa, in a dose- and time-dependent manner, cleaves PAR1 on endothelial cells. FVIIa cleavage of PAR1 on endothelial cells is dependent on FVIIa binding to EPCR, as prevention of FVIIa binding to EPCR by pretreating HUVEC with EPCR polyclonal antibody completely abolished FVIIa cleavage of PAR1. Similarly, silencing EPCR with EPCR-specific siRNA fully attenuated FVIIa cleavage of PAR1. FVIIa cleavage of PAR1 on endothelial cells is independent of TF as pretreatment of HUVEC with anti-TF antibodies or transduction of HUVEC with adenovirus encoding TF had no significant effect on FVIIa cleavage of PAR1. The efficiency of PAR1 cleavage by FVIIa appears to be comparable to that of APC, as both at 10 nM cleave PAR1 to a similar extent. FVIIa (10 nM) cleaves only a fraction of PAR1 (∼25 to 30%) on endothelial cell surface; increasing either FVIIa concentration or duration of treatment has not resulted in additional cleavage of remaining PAR1. Low expression of PAR2 in endothelial cells and lack of cleavage specific antibodies to PAR2 prevented us from determining whether FVII bound to EPCR also cleaves PAR2. FVIIa (10 nM) induced p44/42 MAPK activation in HUVEC and this activation was dependent on EPCR and PAR1 but not PAR2, as silencing EPCR or PAR1 but not PAR2 attenuated FVIIa-induced p44/42 MAPK phosphorylation. In additional studies, FVIIa (10 nM) was found to elicit protection against thrombin-induced barrier disruption in endothelial cells as analyzed in a dual-chamber system using Evans blue-labeled BSA or measurements of transendothelial electrical resistance. FVIIa-induced barrier-protective effect is EPCR-dependent. F-actin staining of HUVEC exposed to thrombin showed formation of transcellular actin stress fibers, cellular contractions and paracellular gap formation. Pretreatment of HUVEC with FVIIa maintained actin at the cell periphery, and reduced formation of central stress fibers and paracellular gaps. FVIIa-induced p44/42 MAPK activation and barrier protective effect are mediated via Rac1, as specific inhibitors against Rac1 or transduction of Rac1 dominant negative mutant abolished these FVIIa-induced effects. Consistent with in vitro findings, in vivo studies in mice showed that administration of FVIIa prior to LPS attenuated the LPS-induced vascular leakage in lung and kidney. Overall, our present data provide strong and convincing evidence that FVIIa bound to EPCR on endothelial cells activates PAR1-mediated cell signaling and provides a barrier protective effect. These findings are novel and assume a great clinical significance as FVIIa is used prophylactically for prevention of bleeding in hemophiliacs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Factor VIIa bound to endothelial cell protein C receptor activates protease activated receptor-1 and mediates cell signaling and barrier protection

Blood ◽  
2011 ◽  
Vol 117 (11) ◽  
pp. 3199-3208 ◽  
Author(s):  
Prosenjit Sen ◽  
Ramakrishnan Gopalakrishnan ◽  
Hema Kothari ◽  
Shiva Keshava ◽  
Curtis A. Clark ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Signaling ◽  
Protective Effect ◽  
Protein C ◽  
Factor Viia ◽  
Mitogen Activated Protein Kinase ◽  
Cell Protein ◽  
Mapk Activation ◽  
Protease Activated Receptor 1

Abstract Recent studies have shown that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C, but the physiologic significance of this interaction is unclear. In the present study, we show that FVIIa, upon binding to EPCR on endothelial cells, activates endogenous protease activated receptor-1 (PAR1) and induces PAR1-mediated p44/42 mitogen-activated protein kinase (MAPK) activation. Pretreatment of endothelial cells with FVIIa protected against thrombin-induced barrier disruption. This FVIIa-induced, barrier-protective effect was EPCR dependent and did not involve PAR2. Pretreatment of confluent endothelial monolayers with FVIIa before thrombin reduced the development of thrombin-induced transcellular actin stress fibers, cellular contractions, and paracellular gap formation. FVIIa-induced p44/42 MAPK activation and the barrier-protective effect are mediated via Rac1 activation. Consistent with in vitro findings, in vivo studies using mice showed that administration of FVIIa before lipopolysaccharide (LPS) treatment attenuated LPS-induced vascular leakage in the lung and kidney. Overall, our present data provide evidence that FVIIa bound to EPCR on endothelial cells activates PAR1-mediated cell signaling and provides a barrier-protective effect. These findings are novel and of great clinical significance, because FVIIa is used clinically for the prevention of bleeding in hemophilia and other bleeding disorders.

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