Activated protein C protects vascular endothelial cells from apoptosis in malaria and in sepsis

Abstract We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by protein C switches the PAR-1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells. To determine whether the occupancy of EPCR by protein C renders thrombin a protective enzyme, thus up-regulating the expression of signaling molecules in the antiinflammatory pathways, we investigated the effects of thrombin and thrombin receptor agonist peptides (TRAP) on TNF-a-stimulated HUVECs in the absence and presence of the catalytically inactive protein C-S195A by monitoring the expression of cell surface adhesion molecules (VCAM- 1, ICAM-1 and E-selectin), adhesion of neutrophils to cytokine-stimulated endothelial cells, regulation of the Rho family of small GTPases and the activation of nuclear factorkB (NF-kB) pathway. Analysis of the results indicates that both thrombin and TRAP initiate proinflammatory responses in endothelial cells, thus neither thrombin nor TRAP influenced the proinflammatory effects of TNF-a in the absence of the protein C mutant. Interestingly, however, the occupancy of EPCR by the protein C mutant switched the PAR-1-dependent signaling specificity of thrombin and TRAP, thus leading to inhibition of the expression of all three adhesion molecules as well as the binding of neutrophils to TNFa-stimulated endothelial cells. Furthermore, similar to activated protein C, both thrombin and TRAP activated Rac1 and inhibited the activation of RhoA and NF-kB pathways in response to TNF-a in cells pretreated with protein C-S195A. Based on these results we conclude that when EPCR is bound by its natural ligand protein C, the cleavage of PAR-1 by thrombin initiates antiinflammatory responses in vascular endothelial cells.

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Protective cross talk between activated protein C and TNF signaling in vascular endothelial cells: implication of EPCR, noncanonical NF-κB, and ERK1/2 MAP kinases

AJP Cell Physiology ◽

10.1152/ajpcell.00003.2010 ◽

2011 ◽

Vol 300 (4) ◽

pp. C833-C842 ◽

Cited By ~ 22

Author(s):

Christophe Guitton ◽

Alice Cottereau ◽

Nathalie Gérard ◽

Thibaut Quillard ◽

Annabelle Chauveau ◽

...

Keyword(s):

Endothelial Cells ◽

Cross Talk ◽

Protein C ◽

Map Kinases ◽

Vascular Endothelial Cells ◽

Activated Protein C ◽

Vascular Endothelial ◽

Endothelial Protein C Receptor ◽

Protein Levels ◽

Antiinflammatory Effects

Activated protein C (APC) is a natural anticoagulant protease that displays cytoprotective and antiinflammatory activities and has been demonstrated to reduce mortality of patients with severe sepsis. However, APC signaling is not fully understood. This study further investigated the antiinflammatory effects of APC in vascular endothelial cells (EC) and examined the cross talk between APC and TNF signaling. Analysis of the regulatory mechanisms mediated by APC on vascular human EC shows that APC impairs TNF signaling by triggering a preemptive activation of intracellular pathways. We found that APC signaling causes a moderate but significant induction of cell adhesion molecules (CAMs) including VCAM-1 at mRNA and protein levels. Activation of the noncanonical NF-κB and ERK1/2 are both pivotal to APC signaling leading to VCAM-1 expression. APC upregulates TNF receptor-associated factor 2 (TRAF2) and phosphorylates NF-κB p65 at Ser276 and Ser536 independently of IκB degradation. The ultimate protective antiinflammatory effect of APC in response to TNF is associated with a sustained activation of ERK1/2 and Akt while phosphorylation of NF-κB p65 is precluded. Inhibitors of ERK (PD98059 and U0126) abolish the antiinflammatory signal mediated by APC. Blocking antibodies and silencing assays also suggest that, in EC, protease-activated receptor 1 and endothelial protein C receptor (EPCR) both conduct ERK activation and VCAM-1 induction in response to APC. To conclude, APC protects EC by attenuating CAM expression during inflammation. APC engages a regulatory cross talk involving EPCR, ERK, and NF-κB that impairs TNF signaling.

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Thrombin inhibits nuclear factor κB and RhoA pathways in cytokine-stimulated vascular endothelial cells when EPCR is occupied by protein C

Thrombosis and Haemostasis ◽

10.1160/th08-09-0568 ◽

2009 ◽

Vol 101 (03) ◽

pp. 513-520 ◽

Cited By ~ 37

Author(s):

Jong-Sup Bae ◽

Alireza R. Rezaie

Keyword(s):

Endothelial Cells ◽

Adhesion Molecules ◽

Protein C ◽

Vascular Endothelial Cells ◽

Nuclear Factor Κb ◽

Small Gtpases ◽

Vascular Endothelial ◽

Nuclear Factor ◽

Endothelial Protein C Receptor ◽

Tnf Α

SummaryThe occupancy of endothelial protein C receptor (EPCR) by protein C switches the protease activated receptor 1 (PAR-1)-dependent signalling specificity of thrombin from a permeability enhancing to a barrier protective response in vascular endothelial cells. In this study, the modulatory effects of thrombin and thrombin receptor agonist peptides (TRAP) on tumour necrosis factor (TNF)-α-stimulated HUVECs in the absence and presence of the catalytically inactive protein C-S195A were evaluated by monitoring the expression of cell surface adhesion molecules (VCAM-1, ICAM-1 and E-selectin), adhesion of freshly isolated neutrophils to cytokine-stimulated endothelial cells, regulation of the Rho family of small GTPases and the activation of nuclear factor-κB (NF-κB) pathway. The analysis of results indicate that both thrombin and TRAP initiate proinflammatory responses in endothelial cells, thus neither PAR-1 agonist in-fluenced the proinflammatory effects of TNF-α in the absence of the protein C mutant. Interestingly, however, the occupancy of EPCR by the protein C mutant switched the PAR-1-dependent signaling specificity of thrombin, thus leading to thrombin inhibition of the expression of all three adhesion molecules as well as the binding of neutrophils to TNF-α-activated endothelial cells. Furthermore, similar to activated protein C, both thrombin and TRAP activated Rac1 and inhibited the activation of RhoA and NF-κB pathways in response to TNF-α in cells pre-treated with protein C-S195A. Based on these results we conclude that when EPCR is ligated by protein C, the cleavage of PAR-1 by thrombin initiates antiinflammatory responses, thus leading to activation of Rac1 and inhibition of RhoA and NF-κB signalling cascades in vascular endothelial cells.

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The ligand occupancy of endothelial protein C receptor switches the protease-activated receptor 1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells

Blood ◽

10.1182/blood-2007-06-096651 ◽

2007 ◽

Vol 110 (12) ◽

pp. 3909-3916 ◽

Cited By ~ 152

Author(s):

Jong-Sup Bae ◽

Likui Yang ◽

Chandrashekhara Manithody ◽

Alireza R. Rezaie

Keyword(s):

Endothelial Cells ◽

Protein C ◽

Vascular Endothelial Cells ◽

Activated Protein C ◽

Catalytic Efficiency ◽

Endothelial Protein C Receptor ◽

New Paradigm ◽

Caveolin 1 ◽

Protease Activated Receptor 1 ◽

Protective Signaling

AbstractRecent studies have indicated that activated protein C (APC) may exert its cytoprotective and anti-inflammatory activities through the endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on vascular endothelial cells. Noting that (1) the activation of protein C on endothelial cells requires thrombin, (2) relative to APC, thrombin cleaves PAR-1 with approximately 3 to 4 orders of magnitude higher catalytic efficiency, and (3) PAR-1 is a target for the proinflammatory activity of thrombin, it is not understood how APC can elicit a protective signaling response through the cleavage of PAR-1 when thrombin is present. In this study, we demonstrate that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that its occupancy by the γ-carboxyglutamic acid (Gla) domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through coupling of PAR-1 to the pertussis toxin–sensitive Gi-protein. Thus, when EPCR is bound by protein C, the PAR-1 cleavage-dependent protective signaling responses in endothelial cells can be mediated by either thrombin or APC. These results provide a new paradigm for understanding how PAR-1 and EPCR participate in protective signaling events in endothelial cells.

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The Ligand Occupancy of Endothelial Protein C Receptor Switches the PAR-1 Dependent Signaling Specificity of Thrombin from a Disruptive to a Protective Response in Endothelial Cells.

Blood ◽

10.1182/blood.v110.11.1746.1746 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1746-1746

Author(s):

Alireza R. Rezaie ◽

Jong-Sup Bae ◽

Likui Yang ◽

Chandrashekhara Manithody

Keyword(s):

Endothelial Cells ◽

Protein C ◽

Vascular Endothelial Cells ◽

Activated Protein C ◽

Catalytic Efficiency ◽

Endothelial Protein C Receptor ◽

New Paradigm ◽

Caveolin 1 ◽

Gi Protein ◽

Protective Signaling

Abstract It has been hypothesized that activated protein C (APC) exerts its cytoprotective and antiinflammatory activities through the endothelial protein C receptor (EPCR)-dependent cleavage of protease activated receptor 1 (PAR-1) on vascular endothelial cells. Noting that the activation of protein C on endothelial cells requires thrombin, relative to APC, thrombin cleaves PAR-1 with ∼3–4-orders of magnitude higher catalytic efficiency, and PAR-1 is a target for the proinflammatory activity of thrombin, it is not understood how APC can elicit a protective signaling response through the cleavage of PAR-1 when thrombin is present. In this study, we demonstrate that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that its occupancy by the Gla-domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through coupling of PAR-1 to the pertussis toxin sensitive Gi-protein. Thus, when EPCR is bound by protein C/APC, the PAR-1 cleavage-dependent protective signaling responses in endothelial cells can be mediated by either thrombin or APC. These results provide a new paradigm for understanding how PAR-1 and EPCR participate in protective signaling events in endothelial cells.

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Thrombomodulin-mediated catabolism of protein C by pleural mesothelial and vascular endothelial cells

Thrombosis and Haemostasis ◽

10.1160/th07-03-0223 ◽

2007 ◽

Vol 98 (09) ◽

pp. 627-634 ◽

Cited By ~ 1

Author(s):

Alireza Rezaie ◽

Steven Idell ◽

Alexei Iakhiaev

Keyword(s):

Endothelial Cells ◽

Protein C ◽

Vascular Endothelial Cells ◽

Degradation Products ◽

Cell Types ◽

Model Systems ◽

Vascular Endothelial ◽

Wild Type ◽

Endothelial Protein C Receptor ◽

Gla Domain

SummaryPleural mesothelial and vascular endothelial cells express protein C (PC) pathway components including thrombomodulin (TM) and endothelial protein C receptor (EPCR) and activate PC by the thrombin-TM dependent mechanism.We used these cells as model systems to identify molecules involved in endocytosis and degradation of PC. We find that mesothelial and endothelial cells can bind, internalize and degrade PC.Addition of thrombin markedly induced degradation of PC by these cells in a TM-dependent fashion, implicating the involvement of the thrombin-TM complex in internalization and degradation of PC. This observation defines a novel function for the thrombin-TM complex as a degradation receptor for PC and suggests that PC is degraded concurrent with its activation.A PC Gla-domain mutant, which is unable to bind to the EPCR, was degraded by the cells to a lesser extent than wild-type PC, implicating the PC degradation concurrent with its activation. Consistent with the role of thrombin-TM complex as a degradation receptor, the catalytically inactive thrombin-S195A also induced PC degradation though to a lesser extent than wild-type thrombin.This suggests that generation of activated PC (APC) can contribute to accumulation of degradation products, but is not essential for the thrombin-induced degradation of PC. The thrombin-TMmediated degradation of PC by both cell types suggest a previously unrecognized mechanism, which can contribute to PC consumption.This mechanism may be pathophysiologically relevant and can contribute to an acquired PC deficiency in conditions characterized by sustained thrombin generation.

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Polymorphisms in the endothelial protein C receptor gene and thrombophilia

Thrombosis and Haemostasis ◽

10.1160/th07-01-0071 ◽

2007 ◽

Vol 98 (09) ◽

pp. 564-569 ◽

Cited By ~ 28

Author(s):

Pilar Medina ◽

Silvia Navarro ◽

Amparo Estellés ◽

Francisco España

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Protein C ◽

Blood Clotting ◽

Receptor Gene ◽

Vascular Endothelial Cell ◽

Activated Protein C ◽

Vascular Endothelial ◽

Endothelial Protein C Receptor ◽

Clotting Cascade

SummaryThe protein C anticoagulant pathway plays a crucial role as a regulator of the blood clotting cascade. Protein C is activated on the vascular endothelial cell membrane by the thrombin-thrombomodulin complex. Once formed, activated protein C (APC) down-regulates thrombin formation by inactivating factors (F)Va and FVIIIa. Endothelial protein C receptor (EPCR) is able to bind protein C and increase the rate of protein C activation. Normal APC generation depends on the precise assemblage, on the surface of endothelial cells, of thrombin, thrombomodulin, protein C and EPCR.Therefore, any change in the efficiency of this assemblage may cause reduced/increased APC generation and modify the risk of thrombosis. This review highlights the different mutations/polymorphisms reported in the EPCR gene and their association with the risk of thrombosis.

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M.629 Homocysteine inhibits lysyl oxidase (LOX) and downregulates LOX expression in vascular endothelial cells

Atherosclerosis ◽

10.1016/s0021-9150(04)90627-2 ◽

2004 ◽

Vol 5 (1) ◽

pp. 146

Author(s):

B RAPOSO

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Lysyl Oxidase ◽

Vascular Endothelial

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Anti-metalloproteinase-9 Activities of Selected Indonesian Zingiberaceae Rhizome Extracts in Lipopolysaccharide-induced Human Vascular Endothelial Cells In Vitro

Planta Medica ◽

10.1055/s-0031-1282610 ◽

2011 ◽

Vol 77 (12) ◽

Author(s):

Y Yanti ◽

N Steven ◽

A Wiharja ◽

S Fajarianto

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Vascular Endothelial ◽

Metalloproteinase 9

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The Endothelial Cell and the Factor VIII Bypassing Activity of Prothrombin Complex Concentrate

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647034 ◽

1988 ◽

Vol 60 (02) ◽

pp. 226-229 ◽

Cited By ~ 2

Author(s):

Jerome M Teitel ◽

Hong-Yu Ni ◽

John J Freedman ◽

M Bernadette Garvey

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Factor Viii ◽

Prothrombin Complex Concentrate ◽

Vascular Endothelial Cells ◽

Vascular Endothelial ◽

Monolayer Cultures ◽

Coagulant Activity ◽

Time Courses ◽

Privileged Site

SummarySome classical hemophiliacs have a paradoxical hemostatic response to prothrombin complex concentrate (PCC). We hypothesized that vascular endothelial cells (EC) may contribute to this “factor VIII bypassing activity”. When PCC were incubated with suspensions or monolayer cultures of EC, they acquired the ability to partially bypass the defect of factor VIII deficient plasma. This factor VIII bypassing activity distributed with EC and not with the supernatant PCC, and was not a general property of intravascular cells. The effect of PCC was even more dramatic on fixed EC monolayers, which became procoagulant after incubation with PCC. The time courses of association and dissociation of the PCC-derived factor VIII bypassing activity of fixed and viable EC monolayers were both rapid. We conclude that EC may provide a privileged site for sequestration of constituents of PCC which express coagulant activity and which bypass the abnormality of factor VIII deficient plasma.

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