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 ◽  
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.

scholarly journals 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 ◽  
2007 ◽  
Vol 110 (12) ◽  
pp. 3909-3916 ◽  
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.

Factor X/Xa Elicits Protective Signaling Responses In Endothelial Cells Directly Via PAR-2 and Indirectly Via EPCR-Dependent Recruitment of PAR-1

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2115-2115
Author(s):  
Alireza R. Rezaie ◽  
Jong-Sup Bae ◽  
Likui Yang
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Conflicts Of Interest ◽  
Factor Viia ◽  
Factor X ◽  
Endothelial Protein C Receptor ◽  
Caveolin 1 ◽  
Gla Domain ◽  
Protective Signaling ◽  
Dependent Interaction

Abstract Abstract 2115 We recently demonstrated that the Gla-domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier protective response if endothelial cells were pre-incubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells, also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a crosstalk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla-domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cell painting with an engineered EPCR to augment the protein C system

2015 ◽  
Vol 114 (12) ◽  
pp. 1144-1155 ◽  
Author(s):  
Eveline A. M. Bouwens ◽  
Fabian Stavenuiter ◽  
Laurent O. Mosnier
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Activated Protein C ◽  
Wild Type ◽  
Endothelial Protein C Receptor ◽  
Gpi Anchor ◽  
Akt Phosphorylation ◽  
Caveolin 1 ◽  
Dose Dependent

SummaryThe protein C (PC) system conveys beneficial anticoagulant and cytoprotective effects in numerous in vivo disease models. The endothelial protein C receptor (EPCR) plays a central role in these pathways as cofactor for PC activation and by enhancing activated protein C (APC)-mediated protease-activated receptor (PAR) activation. During inflammatory disease, expression of EPCR on cell membranes is often diminished thereby limiting PC activation and APC’s effects on cells. Here a caveolae-targeting glycosylphosphatidylinositol (GPI)-anchored EPCR (EPCR-GPI) was engineered to restore EPCR’s bioavailability via “cell painting.” The painting efficiency of EPCR-GPI on EPCR-depleted endothelial cells was time- and dose-dependent. The EPCR-GPI bioavailability after painting was long lasting since EPCR surface levels reached 400 % of wild-type cells after 2 hours and remained > 200 % for 24 hours. EPCR-GPI painting conveyed APC binding to EPCR-depleted endothelial cells where EPCR was lost due to shedding or shRNA. EPCR painting normalised PC activation on EPCR-depleted cells indicating that EPCR-GPI is functional active on painted cells. Caveolin-1 lipid rafts were enriched in EPCR after painting due to the GPI-anchor targeting caveolae. Accordingly, EPCR painting supported PAR1 and PAR3 cleavage by APC and augmented PAR1-dependent Akt phosphorylation by APC. Thus, EPCR-GPI painting achieved physiological relevant surface levels on endothelial cells, restored APC binding to EPCR-depleted cells, supported PC activation, and enhanced APC-mediated PAR cleavage and cytoprotective signalling. Therefore, EPCRGPI provides a novel tool to restore the bioavailability and functionality of EPCR on EPCR- depleted and -deficient cells.

Glutamic Acid 192 of Activated Protein C Restricts the Specificity of PAR-1 Cleavage in Endothelial Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1752-1752
Author(s):  
Jong-Sup Bae ◽  
Alireza R. Rezaie
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Receptor Activation ◽  
Cell Permeability ◽  
Paradoxical Effect ◽  
Endothelial Protein C Receptor ◽  
Proinflammatory Response

Abstract It is known that residue 192 (chymotrypsin numbering) plays a key role in determining the P3 binding specificity of coagulation proteases. This residue is a Glu in both thrombin and activated protein C. Previous studies have indicated that the substrates containing an acidic residue at the P3 position are not recognized efficiently by either thrombin or APC in the absence of a cofactor. Protease activated receptor 1 (PAR-1), a common substrate for both thrombin and APC contains an acidic Asp at the P3 position. In this study we examined the ability of Glu-192 to Gln (E192Q) substitution mutants of both thrombin and APC to recognize and cleave PAR-1 in human umbilical vein endothelial cells (HUVEC) transfected with a PAR-1 cleavage reporter plasmid in which the exodomain of the receptor has been fused to a cDNA fragment encoding the soluble alkaline phosphatase. Thrombin E192Q cleaved PAR-1 with a catalytic efficiency that was approximately 5-fold higher than that of wild-type thrombin. On the other hand, the activity of APC E192Q toward PAR-1 was improved approximately 100-fold. Thus, unlike an approximately 1000-fold lower activity for APC in cleaving PAR-1 relative to thrombin, the activity of APC E192Q toward PAR-1 was only 10-fold lower than that of thrombin. These results suggest that the inhibitory interaction of Glu-192 of APC with P3-Asp of PAR-1 is responsible for its poor activity toward PAR-1. It is known that the cleavage of PAR-1 by thrombin in endothelial cells elicits a proinflammatory response. However, the cleavage of the same receptor by APC in complex with endothelial protein C receptor (EPCR) invokes a protective antiinflammatory response. The mechanism of the paradoxical effect of PAR-1 signaling by the two proteases is not known. We used these mutants to investigate the possibility that the level of PAR-1 activation by either thrombin or APC dictates the type of the response in endothelial cells. Furthermore, we used these mutants in TNF-a-stimulated endothelial cell permeability and apoptosis assays to understand the mechanism by which EPCR enables APC to activate PAR-1 in endothelial cells. We discovered that the dose of receptor activation is not responsible for the paradoxical effect of PAR-1 signaling by APC and thrombin in endothelial cells. Furthermore, the interaction of APC with EPCR is not associated with an improvement in the catalytic efficiency of the protease toward PAR-1. The possible EPCR and PAR-1 dependent recognition and signaling mechanism of APC and thrombin is discussed.

Thrombin Inhibits NF-κb and RhoA Pathways in Cytokine-Stimulated Vascular Endothelial Cells When EPCR Is Occupied by Protein C.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1888-1888
Author(s):  
Alireza R. Rezaie ◽  
Jong-Sup Bae
Keyword(s):  
Endothelial Cells ◽  
Adhesion Molecules ◽  
Protein C ◽  
Vascular Endothelial Cells ◽  
Activated Protein C ◽  
Small Gtpases ◽  
Vascular Endothelial ◽  
Endothelial Protein C Receptor ◽  
Signaling Specificity ◽  
Natural Ligand

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.

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

2011 ◽  
Vol 300 (4) ◽  
pp. C833-C842 ◽  
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.

scholarly journals Protein C system defects inflicted by the malaria parasite protein PfEMP1 can be overcome by a soluble EPCR variant

2015 ◽  
Vol 114 (11) ◽  
pp. 1038-1048 ◽  
Author(s):  
Eveline A. M. Bouwens ◽  
Ibai Tamayo ◽  
Louise Turner ◽  
Christian W. Wang ◽  
Monique Stins ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Severe Malaria ◽  
Protein C ◽  
Cellular Response ◽  
Malaria Parasite ◽  
Vascular Endothelial Cells ◽  
Protective Effects ◽  
Endothelial Protein C Receptor ◽  
Parasite Protein ◽  
Soluble Epcr

SummaryThe Endothelial Protein C receptor (EPCR) is essential for the anticoagulant and cytoprotective functions of the Protein C (PC) system. Selected variants of the malaria parasite protein, Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) associated with severe malaria, including cerebral malaria, specifically target EPCR on vascular endothelial cells. Here, we examine the cellular response to PfEMP1 engagement to elucidate its role in malaria pathogenesis. Binding of the CIDRα1.1 domain of PfEMP1 to EPCR obstructed activated PC (APC) binding to EPCR and induced a loss of cellular EPCR functions. CIDRα1.1 severely impaired endothelial PC activation and effectively blocked APC-mediated activation of protease-activated receptor-1 (PAR1) and associated barrier protective effects of APC on endothelial cells. A soluble EPCR variant (E86A-sEPCR) bound CIDRα1.1 with high affinity and did not interfere with (A)PC binding to cellular EPCR. E86A-sEPCR used as a decoy to capture PfEMP1, permitted normal PC activation on endothelial cells, normal barrier protective effects of APC, and greatly reduced cytoadhesion of infected erythrocytes to brain endothelial cells. These data imply important contributions of PfEMP1-induced protein C pathway defects in the pathogenesis of severe malaria. Furthermore, the E86A-sEPCR decoy provides a proof-of-principle strategy for the development of novel adjunct therapies for severe malaria.

Recombinant human activated protein C upregulates cyclooxygenase-2 expression in endothelial cells via binding to endothelial cell protein C receptor and activation of protease-activated receptor-1

2005 ◽  
Vol 93 (04) ◽  
pp. 743-750 ◽  
Author(s):  
Sarah Horn ◽  
Siegfried Lang ◽  
Kenji Fukudome ◽  
Adriane Nahrup ◽  
Ursula Hoffmann ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein ◽  
Mrna Levels ◽  
Endothelial Protein C Receptor ◽  
Drotrecogin Alfa Activated ◽  
Cox 2 ◽  
Vitro Effect

SummaryProstacyclin (PGI2) has beneficial cytoprotective properties, is a potent inhibitor of platelet aggregation and has been reported to improve microcirculatory blood flow during sepsis. The formation of PGI2 in response to proinflammatory cytokines is catalysed by the inducible cyclooxygenase (COX) isoform COX-2. Recombinant human activated protein C (rhAPC, drotrecogin alfa (activated)) was shown to have multiple biological activities in vitro and to promote resolution of organ dysfunction in septic patients. Whether rhAPC exerts its beneficial effects by modulating prostanoid generation is unknown up to now. It was therefore the aim of the study to examine the in vitro effect of rhAPC on COX-2-mRNA-expression and PGI2 release from human umbilical vein endothelial cells (HUVEC). We found that rhAPC, at supra-therapeutical concentrations (500ng/ml-20μg/ ml), upregulated the amount of COX-2-mRNA in HUVEC at t=3–9h and caused a time- and dose-dependent release of 6-keto PGF1α, the stable hydrolysis product of prostacyclin. RhAPC further increased the stimulating effect of tumor necrosis factor-α (TNF-α) and thrombin on COX-2-mRNA-levels. Transcript levels of cyclooxygenase-1 (COX-1) and prostagland-in I2 synthase, however, were unaffected by the stimulation with rhAPC or thrombin. The upregulatory effect on COX2-mRNA levels was specific for rhAPC since the zymogen protein C in equimolar concentrations had no effect on COX-2-mRNA-levels or 6keto PGF1α-release. Western Blot analysis revealed an increase of COX-2-protein content in HUVEC after treatment with rhAPC. As shown by experiments using monoclonal antibodies against the thrombin receptor PAR-1 (mAb=ATAP2) and against the endothelial protein C receptor (EPCR; mAb=RCR-252), the effect of rhAPC on COX-2-mRNA up-regulation was mediated by binding to the EPCR-receptor and signaling via PAR-1. These results demonstrate that induction of COX-2-expression is an important response of HUVEC to stimulation with rhAPC and may represent a new molecular mechanism, by which rhAPC promotes upregulation of prostanoid production in human endothelium.

Efficient Barrier Protective Signaling by Activated Protein C Is Mechanistically Linked to Protein C Activation on Endothelial Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 28-28
Author(s):  
Clemens Feistritzer ◽  
Laurent O. Mosnier ◽  
Enrico Di Cera ◽  
John H. Griffin ◽  
Matthias Riewald
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Activated Protein C ◽  
Endothelial Permeability ◽  
Average Concentration ◽  
Protective Effects ◽  
Activation Pathway ◽  
Protective Signaling ◽  
Cell Medium

Abstract Protein C (PC) is activated by thrombomodulin-bound thrombin on the endothelial cell surface and activated protein C (APC) inhibits blood coagulation in a negative feedback loop. Endothelial PC receptor (EPCR) can bind PC/APC and activation of EPCR-bound PC is enhanced. Exogenous APC has barrier protective effects on endothelial cells that depend on EPCR binding and protease activated receptor-1 (PAR1) cleavage and that may contribute to the anti-inflammatory effects of APC. Plasma APC concentrations in vivo are low compared to the substrate PC and in order to induce protective signaling exogenous APC has to compete with PC for EPCR binding. In this study we investigated whether the endogenous PC activation pathway may be linked to efficient protective responses analyzing endothelial barrier permeability in a dual chamber system. When endothelial EA.hy926 cells were incubated for 3 h in the presence of 80 nM purified PC and different concentrations of thrombin a dose-dependent linear increase of APC activity in the cell medium was observed over time. APC generation was detectable upon incubation with 20 pM thrombin or higher and a significant barrier protective response to 20 pM thrombin was found only in the presence of PC. 40 pM thrombin enhanced barrier integrity in the presence and absence of PC, consistent with our previous results. To exclude direct thrombin effects on endothelial permeability and to compare protective effects of exogenous and endogenously generated APC, we used the anticoagulant double mutant thrombin W215A/E217A (WE). WE was about 10 times less active than wildtype thrombin for PC activation in our system. However, PAR1-dependent induction of MAP kinase phosphorylation required more than 1000-fold higher concentrations of the thrombin mutant. Thus, 1–10 nM WE leads to APC generation without directly inducing PAR1-dependent signaling. When cells were incubated with various concentrations of exogenous APC or WE+80 nM PC, barrier protective effects of 5 nM exogenous APC and 2 nM WE+80 nM PC (1.3 nM APC generated after 3 h) were similar. Because APC is generated at a constant rate during the incubation period, the average concentration of generated APC in the cell medium was only about 0.65 nM, suggesting that signaling by endogenously generated APC was significantly more efficient. To conclusively demonstrate that protective effects in response to WE are mediated by APC generation, we used recombinant zymogen wildtype PC and a PC variant with a substitution of the active site serine with alanine (PC S360A). Cells were incubated with control or 80 nM wildtype PC and PC S360A, in the presence or absence of WE (4 nM) and exogenous APC (3.3 nM). WE induced protective signaling only in the presence of wildtype PC but not PC S360A. Barrier protective effects of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. These data demonstrate that efficient barrier enhancement by APC is indeed mechanistically coupled to the PC activation pathway. Signaling by endogenously generated APC may play an important role in the regulation of inflammation.

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