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

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.

Signaling by Activated Protein C in Endothelial Cells Is PAR1 Dependent but Distinct from Thrombin Signaling.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 221-221 ◽  
Author(s):  
Matthias Riewald ◽  
Clemens Feistritzer ◽  
Wolfram Ruf
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein C ◽  
Biological Effects ◽  
Activated Protein C ◽  
Cell Protein ◽  
Protein Levels ◽  
Thrombospondin 1 ◽  
Anti Inflammatory

Abstract Thrombin initiates fibrin formation and platelet activation, and activates protein C, generating activated protein C (APC) that inhibits blood coagulation by a negative feedback loop. Thrombin has also proinflammatory effects through activation of cellular protease activated receptor-1 (PAR1). Endothelial cell protein C receptor (EPCR) can bind both protein C and APC and activation of EPCR-bound protein C is enhanced. Results from animal models and clinical trials indicate that APC has potent protective effects in systemic inflammation that are independent from its well established anticoagulant function and recombinant APC was recently approved to treat patients with severe sepsis. The molecular basis for APC’s anti-inflammatory effects is incompletely understood. We have identified PAR1 and EPCR as part of a novel APC signaling pathway in quiescent endothelial cells, raising the question how the same receptor PAR1 can mediate both pro- and anti-inflammatory effects. In an overexpression system in PAR-deficient fibroblasts, wildtype PAR2 but not a PAR2 variant with an Arg36 to Ala substitution at the P1 position was activated by APC, indicating that APC can activate PAR2 in addition to PAR1 through a canonical cleavage mechanism. Therefore, we tested whether endothelial cell PAR2 can be activated by APC under conditions where endogenous PAR2 expression is upregulated. Even when PAR2 expression was highly upregulated in inflammatory cytokine-stimulated human umbilical endothelial cells (HUVECs), signaling by APC was strictly dependent on PAR1 cleavage and signaling. Consistent with these results in HUVECs, intravenous injection of APC in wildtype, PAR1−/−, and PAR2−/− mice demonstrated that PAR1 is the major murine receptor that mediates induction of the transcript for monocyte chemoattractant protein-1 in the lung in response to APC. This indicates that indeed the same receptor PAR1 mediates signaling by APC and thrombin both in vitro and in vivo. To test the possibility that APC diminishes proinflammatory thrombin-PAR1 signaling by downregulating cellular levels of functional PAR1, we tested whether preincubation with APC can desensitize Erk1/2 phosphorylation by thrombin. Phospho-Erk1/2 was induced by APC dependent upon PAR1 cleavage, but APC-pretreated cells still responded to PAR1-dependent thrombin signaling, suggesting that only a fraction of the cellular PAR1 is subject to cleavage by APC. These results indicate that APC does not block thrombin signaling by desensitation at the receptor level. Large-scale gene expression profiling demonstrated that APC and thrombin had specific effects on gene expression in tumor necrosis factor α (TNFα )-perturbed endothelial cells that were not detected in quiescent cells. Transcripts for several proapoptotic genes including p53 and thrombospondin-1 were downregulated by APC but not by thrombin or PAR1 agonist peptides in TNFα-stimulated HUVECs. Western blotting confirmed that in TNFα-perturbed HUVECs pretreatment with APC significantly reduced the increase in cellular p53 protein levels in response to the cytotoxic doxorubicin. This APC effect was dependent on EPCR binding and PAR1 cleavage. Thrombospondin-1 protein levels were similarly downregulated by APC but upregulated by thrombin. Both down- and upregulation by APC and thrombin, respectively, were PAR1 dependent. These findings demonstrate that the same receptor on the same cell type can mediate opposite biological effects and they suggest that EPCR cosignaling may modify PAR1-dependent APC signaling in endothelial cells.

Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4393-4401 ◽  
Author(s):  
James B. Rance ◽  
George A. Follows ◽  
Peter N. Cockerill ◽  
Constanze Bonifer ◽  
David A. Lane ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Binding Sites ◽  
Hepg2 Cells ◽  
Protein C ◽  
Cell Protein ◽  
Hypersensitive Site ◽  
Flanking Region

Abstract The human endothelial cell protein C receptor (hEPCR) is normally expressed by the endothelium of large blood vessels, but the molecular basis for its in vivo specificity is uncertain. In this study, DNaseI hypersensitive site mapping demonstrated the presence of a hypersensitive site in the 5′ flanking region of the hEPCR gene in endothelial cells and certain transformed cells (HeLa and U937) known to express hEPCR in vitro. Conversely, this site was only weakly hypersensitive in HepG2 cells, cells which do not express hEPCR mRNA. Functional analysis of this 5′ flanking region by in vivo dimethylsulfate footprinting in cultured endothelial cells identified multiple regions, containing high and low homology consensus Sp1 binding sequences, that were protected from methylation in endothelial cells. These sequences were not protected in HepG2 cells. Reporter gene analysis of this region in endothelial cells demonstrated the presence of promoter activity conferred by the proximal 572 bp but failed to identify a functional TATA-box. This promoter was inactive in HepG2 cells. Electrophoresis mobility shift assays using endothelial cell nuclear extracts identified Sp1 family proteins binding to sites that were protected during footprinting. Sp1 sites were identified in regions at –368, –232, –226, –201, –146, and –102 bp relative to the translation start site. With the exception of the site at –102 bp, each identified Sp1 binding site made a positive contribution to reporter gene expression, although no individual site was critically important. We conclude that transcription factor binding to Sp1 binding sites in the 5′ flanking region is critical for normal hEPCR gene expression in endothelial cells.

scholarly journals Mechanisms by Which Soluble Endothelial Cell Protein C Receptor Modulates Protein C and Activated Protein C Function

2000 ◽  
Vol 275 (8) ◽  
pp. 5447-5452 ◽  
Author(s):  
Patricia C. Y. Liaw ◽  
Pierre F. Neuenschwander ◽  
Mikhail D. Smirnov ◽  
Charles T. Esmon
Keyword(s):  
Endothelial Cell ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein

scholarly journals Zinc Modulates the Interaction of Protein C and Activated Protein C with Endothelial Cell Protein C Receptor

2010 ◽  
Vol 285 (26) ◽  
pp. 20410-20420 ◽  
Author(s):  
Prosenjit Sen ◽  
Sanghamitra Sahoo ◽  
Usha R. Pendurthi ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Endothelial Cell ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein

scholarly journals Homocysteine, an atherogenic stimulus, reduces protein C activation by arterial and venous endothelial cells

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 895-901 ◽  
Author(s):  
GM Rodgers ◽  
MT Conn
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein C ◽  
Vascular Endothelial Cell ◽  
Activated Protein C ◽  
Cell Protein ◽  
Blood Levels ◽  
Factor V ◽  
Elevated Blood ◽  
Cultured Endothelial Cells

Abstract Elevated blood levels of homocysteine are associated with atherosclerosis and thrombotic disease. We previously reported that treatment of cultured endothelial cells with homocysteine increased endogenous factor V activity by activation of the cofactor. Because endothelial cell-associated factor Va would be regulated by the protein C mechanism, the ability of homocysteine-treated arterial and venous endothelial cells to activate protein C was investigated. Both arterial and venous endothelial cells activated protein C; 0.6 mmol/L homocysteine reduced endothelial cell protein C activation by 12%. Maximal inhibition (90%) of protein C activation occurred with 7.5 to 10 mmol/L homocysteine after 6 to 9 hours of incubation. Metabolism of homocysteine was not accelerated by cultured endothelial cells. Investigation of the mechanism(s) by which homocysteine reduced protein C activation indicated that the metabolite did not induce an inhibitor to activated protein C, but in low concentrations acted as a competitive inhibitor to thrombin. These data suggest that perturbation of the vascular endothelial cell protein C mechanism by homocysteine may contribute to the thrombotic tendency seen in patients with elevated blood levels of this metabolite.

Structural and Functional Implications of the Intron/Exon Organization of the Human Endothelial Cell Protein C/Activated Protein C Receptor (EPCR) Gene: Comparison With the Structure of CD1/Major Histocompatibility Complex 1 and 2 Domains

Blood ◽  
1999 ◽  
Vol 94 (2) ◽  
pp. 632-641 ◽  
Author(s):  
Rachel E. Simmonds ◽  
David A. Lane
Keyword(s):  
Major Histocompatibility Complex ◽  
Endothelial Cell ◽  
Secondary Structure ◽  
Untranslated Region ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein ◽  
Major Histocompatibility ◽  
Initiation Point ◽  
Histocompatibility Complex

Abstract The endothelial cell protein C/activated protein C receptor (EPCR) is located primarily on the surface of the large vessels of the vasculature. In vitro studies suggest that it is involved in the protein C anticoagulant pathway. We report the organization and nucleotide sequence of the human EPCR gene. It spans approximately 6 kbp of genomic DNA, with a transcription initiation point 79 bp upstream of the translation initiation (Met) codon in close proximity to a TATA box and other promoter element consensus sequences. The human EPCR gene has been localized to 20q11.2 and consists of four exons interrupted by three introns, all of which obey the GT-AG rule. Exon I encodes the 5′ untranslated region and the signal peptide, and exon IV encodes the transmembrane domain, the cytoplasmic tail, and the 3′ untranslated region. Exons II and III encode most of the extracellular region of the EPCR. These exons have been found to correspond to those encoding the 1 and 2 domains of the CD1/major histocompatibility complex (MHC) class I superfamily. Flanking and intervening introns are of the same phase (phase I) and the position of the intervening intron is identically located. Secondary structure prediction for the amino acid sequence of exons II and III corresponds well with the actual secondary structure elements determined for the 1 and 2 domains of HLA-A2 and murine CD1.1 from crystal structures. These findings suggest that the EPCR folds with a β-sheet platform supporting two -helical regions collectively forming a potential binding pocket for protein C/activated protein C.

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)

Sign in / Sign up

Export Citation Format

Share Document