Endothelial cell protein C receptor plays an important role in protein C activation in vivo

Blood ◽  
2001 ◽  
Vol 97 (6) ◽  
pp. 1685-1688 ◽  
Author(s):  
Fletcher B. Taylor ◽  
Glenn T. Peer ◽  
Marion S. Lockhart ◽  
Gary Ferrell ◽  
Charles T. Esmon
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cell ◽  
Protein C ◽  
Degradation Products ◽  
Activated Protein C ◽  
Vital Signs ◽  
Cell Protein ◽  
Increased Risk

Endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin-thrombomodulin complex about 5-fold in vitro. Augmentation is EPCR concentration dependent even when the EPCR concentration is in excess of the thrombomodulin. EPCR is expressed preferentially on large blood vessel endothelium, raising questions about the importance of protein C-EPCR interaction for augmenting systemic protein C activation. In these studies, this question was addressed directly by infusing thrombin into baboons in the presence or absence of a monoclonal antibody to EPCR that blocks protein C binding. Activated protein C levels were then measured directly by capturing the enzyme on a monoclonal antibody and assaying with chromogenic substrate. Blocking protein C-EPCR interaction resulted in about an 88% decrease in circulating activated protein C levels generated in response to thrombin infusion. Leukocyte changes, fibrinogen consumption, fibrin degradation products, and vital signs were similar between the animals infused with thrombin alone and those infused with thrombin and the anti-EPCR antibody. The results indicate that EPCR plays a major role in protein C activation and suggest that defects in the EPCR gene might contribute to increased risk of thrombosis.

Activated protein C variants with normal cytoprotective but reduced anticoagulant activity

Blood ◽  
2004 ◽  
Vol 104 (6) ◽  
pp. 1740-1744 ◽  
Author(s):  
Laurent O. Mosnier ◽  
Andrew J. Gale ◽  
Subramanian Yegneswaran ◽  
John H. Griffin
Keyword(s):  
Endothelial Cell ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Site Directed Mutagenesis ◽  
Bleeding Complications ◽  
Cell Protein ◽  
Cytoprotective Activity ◽  
Increased Risk ◽  
Antiapoptotic Activity

Abstract Recombinant activated protein C (APC), a well-defined anticoagulant enzyme, reduced mortality in severe sepsis patients in a phase 3 trial. However, 2 potent anticoagulants, antithrombin III and recombinant tissue factor pathway inhibitor, failed to do so, implying the physiologic relevance of APC's less well-defined anti-inflammatory and antiapoptotic activities. Recombinant APC therapy conveys an increased risk of serious bleeding complications due to APC anticoagulant activity. To generate recombinant APC variants with reduced risk of bleeding due to reduced anticoagulant activity, we dissected APC's anticoagulant activity from its cytoprotective activity by site-directed mutagenesis. Using staurosporine-induced endothelial cell apoptosis assays, we show here that Ala mutations (RR229/230AA and KKK191_ 193AAA) in 2 APC surface loops that severely reduce anticoagulant activity result in 2 APC variants that retain normal antiapoptotic activity that requires protease activated receptor-1 and endothelial cell protein C receptor. Thus, it is possible to reduce anticoagulant activity while preserving antiapoptotic activity of recombinant APC variants. We suggest that therapeutic use of such APC variants may reduce serious bleeding risks while providing the beneficial effects of APC acting directly on cells. (Blood. 2004;104: 1740-1744)

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.

In Vitro Protection of Human Endothelial Cells from Adenovirus Vector Toxicity by Activated Protein C.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3694-3694
Author(s):  
Jay Nelson Lozier ◽  
Felice D’Agnillo
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Endothelial Cell ◽  
Protein C ◽  
First Generation ◽  
Activated Protein C ◽  
Adenovirus Vector ◽  
Dose Dependent

Abstract High dose adenovirus vector administration in vivo has been associated with toxicity toward many cell types, including endothelial cells. Some of the prominent pathological features of an adenovirus vector death in a gene therapy trial included capillary leak syndrome and disseminated intravascular coagulation (DIC). We investigated the hypothesis that activated protein C (APC) might have a protective effect on primary human microvascular endothelial cells exposed to a first-generation adenovirus vector. We exposed primary human endothelial cells to a first-generation (E1, E3 deleted) adenovirus vector, AVC3FIX5 at concentrations ranging from 103 to 105 vector particles per cell and showed dose-dependent cell death as early as 6 hours (40% cell death at the highest dose). Phase contrast and immunofluorescence microscopy revealed that some cells died rapidly by primary necrosis while others died by apoptosis over a longer time course. By 40 hours, only 40% of the cells were viable. We then tested the effect of pretreatment of endothelial cells with APC concentrations ranging from 1 nM to 100 nM. Dose-dependent protection was seen in which cell death was reduced to 9 and 2 % at APC concentrations of 50 and 100 nM, respectively. We also tested the effect of timing of the APC treatment and showed that 1 hour pre-treatment or concurrent APC treatment were protective, but APC administered one hour after the adenovirus exposure was substantially less protective. This suggested that APC exerts its protective actions on endothelial cells either by interfering with early steps in the interaction of the vector with the cells, (e.g., vector entry) or by modulating death signaling pathways. It has been proposed that APC protects against cell damage in sepsis by interaction with the endothelial cell protein C receptor (EPCR) and protease activated receptor 1 (PAR1) on the endothelial cell surface to induce MCP-1 and other immunomodulatory genes by proteolytic signaling (Riewald et al., Science296:1880–1882, 2002). Other investigators have shown protective effects of APC for endothelial cells subjected to hypoxia through normalization of levels of p53, Bax, and Bcl-2 gene expression (Cheng et al., Nat Med9:338–342, 2003). The APC concentrations in our experiments that were maximally protective (50–100 nM) were of the same order of magnitude as was shown to be protective in vitro by these investigators. If APC can be shown to have a protective effect against adenovirus-induced endothelial cell toxicity and DIC in vivo, this may be a useful therapeutic strategy to explore as treatement of gene therapy vector toxicity. Cell Viability vs. APC Concentration Cell Viability vs. APC Concentration

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

scholarly journals Identification of the Protein C/Activated Protein C Binding Sites on the Endothelial Cell Protein C Receptor

2000 ◽  
Vol 276 (11) ◽  
pp. 8364-8370 ◽  
Author(s):  
Patricia C. Y. Liaw ◽  
Timothy Mather ◽  
Natalia Oganesyan ◽  
Gary L. Ferrell ◽  
Charles T. Esmon
Keyword(s):  
Endothelial Cell ◽  
Binding Sites ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein

Abstract 34: Endogenous Superoxide Dismutase Protects From Impaired Generation of Activated Protein C and Enhanced Susceptibility to Experimental Thrombosis in Mice

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Sanjana Dayal ◽  
Sean X Gu ◽  
Katinan M Wilson ◽  
Ryan Hutchins ◽  
Steven R Lentz
Keyword(s):  
Superoxide Dismutase ◽  
Protein C ◽  
Activated Protein C ◽  
Vena Cava ◽  
Oxidative Modification ◽  
Mrna Levels ◽  
Endothelial Protein C Receptor ◽  
Experimental Thrombosis

In vitro studies have suggested that reactive oxygen species such as superoxide can produce prothrombotic effects, including enhanced platelet activation, increased tissue factor (TF) expression, and an oxidative modification in thrombomodulin impairing its capacity to enhance the generation of activated protein C (APC) by thrombin. It is not known, however, if elevated levels of superoxide accelerate susceptibility to experimental thrombosis in vivo . We used mice genetically deficient in superoxide dismutase-1 (SOD1, an antioxidant enzyme that dismutates superoxide to hydrogen peroxide), to test the hypothesis that lack of SOD1 enhances susceptibility to thrombosis. Susceptibility to carotid artery thrombosis in a photochemical injury model demonstrated that Sod1-/- mice formed stable occlusions significantly faster than Sod1+/+ mice (P<0.05). In an inferior vena cava (IVC) stasis model Sod1- /- mice developed significantly larger thrombi 48 hours after IVC ligation (P<0.05 vs. Sod1+/+ mice). After activation with thrombin (0.5 U/ml) or convulxin (200 ng/ml), no differences in surface expression of P-selectin or binding of fibrinogen were observed between platelets from Sod1-/- and Sod1+/+ mice. The expression of TF mRNA in lung measured by real time qPCR showed similar levels in Sod1-/- and Sod1 +/+ mice. However, the activation of exogenous protein C by thrombin in lung homogenates was decreased in Sod1 -/- mice (P<0.05 vs. Sod1 +/+ mice). Further, in vivo generation of activated protein C in response to thrombin (40 U/Kg) infusion was significantly lower in Sod1-/- mice (P<0.05 vs. Sod1+/+ mice). No differences in mRNA levels for thrombomodulin or endothelial protein C receptor were detected in Sod1 -/- mice vs. Sod1 +/+ mice, suggesting that altered generation of activated protein C in Sod1-/- mice may be related to a direct oxidative effect on thrombomodulin. In accordance, thrombomodulin treated with xanthine/hypoxanthine showed 40% loss of ability to activate protein C that was overcome by addition of SOD and catalase (P<0.05). We conclude that endogenous SOD1 in mice protects from impaired generation of activated protein C and accelerated thrombosis.

Abstract 531: Tie-2/Cre--Mediated Conditional Deletion of Lipid Phosphate Phosphatase-3 Reveals Its Role in Endothelial Cell Apoptosis

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Ishita Chatterjee ◽  
Kishore K Wary
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Genome Wide Association Study ◽  
Vascular Endothelial Cell ◽  
Protein Levels ◽  
Conditional Deletion ◽  
Transmission Electron ◽  
Increased Risk

Rationale: A recent genome-wide association study (GWAS) has linked a frequently occurring variation in the LPP3 (also known as PPAP2b) loci to increased risk of coronary heart disease (CAD). However, the in vivo function of LPP3 in vascular endothelial cell is incompletely understood. Goal: To address the endothelial cell (EC) specific function of Lpp3 in mice. Results: Tie-2/Cre mediated Lpp3 deletion did not affect normal vasculogenesis in early embryonic development, in contrast, in late embryonic stages it led to impaired angiogenesis associated with hemorrhage, edema and late embryonic lethal phenotype. Immunohistochemical staining followed by microscopic analyses of mutant embryos revealed reduced fibronectin and VE-cadherin expression throughout different vascular bed, and increased apoptosis in CD31+ vascular structures. Transmission electron microscopy (TEM) showed the presence of apoptotic endothelial cells and disruption of adherens junctions in mutant embryos. LPP3-knockdown in vitro showed an increase in p53 and p21 protein levels, with concomitant decrease in cell proliferation. LPP3-knockdown also decreased transendothelial electrical resistance (TER), interestingly re-expression of ß-catenin cDNA into LPP3-depleted endothelial cells partially restored the effect of loss of LPP3. Conclusion: These results suggest the ability of LPP3 to regulate survival and apoptotic activities of endothelial cells during patho/physiological angiogenesis.

scholarly journals Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease-activated receptor-1 and endothelial cell protein C receptor

2003 ◽  
Vol 373 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Laurent O. MOSNIER ◽  
John H. GRIFFIN
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein ◽  
Protease Activated Receptor 1 ◽  
Induced Apoptosis ◽  
Apoptotic Effects ◽  
Dose Dependent

In a model of staurosporine-induced apoptosis using EAhy926 endothelial cells, inhibition of apoptosis by activated protein C was dose-dependent and required the enzyme's active site, implicating activated protein C-mediated proteolysis. Consistent with this implication, both protease-activated receptor-1 (PAR-1) and endothelial cell protein C receptor (EPCR) were required for the anti-apoptotic effects of activated protein C.

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