Endothelial Cell Protein C Receptor Cellular Localization and Trafficking

Abstract Endothelial cell protein C receptor (EPCR) is the cellular receptor for protein C (PC) and activated protein C (APC). Recent studies from others and us showed that EPCR also acts as a cellular binding site for factor VII (FVII) and activated factor VII (FVIIa). Although much is know about the biochemistry and pathophysiological importance of EPCR, little is know how EPCR interaction with its ligands on cell surfaces affects its expression and facilitate internalization of the ligands. The present study was undertaken to characterize cellular localization and trafficking of EPCR and investigate how FVIIa or APC binding to EPCR influences these processes. The studies employed two cell model systems - primary cultured human umbilical endothelial cells (HUVEC) and CHO cells stably transfected with EPCR. Cellular localization of EPCR and its trafficking was analyzed by immunofluorescence confocal microscopy or monitoring the expression of EPCR tagged with green fluorescence protein (GFP). EPCR endocytosis was evaluated by biotinylation of cell surface proteins with NHS-SS- biotin, followed by monitoring the protection of biotinylated EPCR from a membrane-impermeable reducing agent. FVIIa and APC internalization and recycling was evaluated by monitoring the uptake/release of 125I-labeled ligands or following the intracellular routing of fluorescent dye (AF488)-conjugated ligands added to EPCR expressing cells. Data from these studies showed that a majority of EPCR is localized on the cell surface and distributed in a patchy and punctuate manner. Immunostaining of HUVEC and CHO-EPCR cells with EPCR mAb and caveolin-1 antibodies showed a high degree of co-localization of EPCR and caveolin-1. Depletion of cholesterol from the plasma membrane, which disrupts caveolae, by ß methyl cyclodextrin reduced the extent EPCR and caveloin-1 colocalization. These data indicate EPCR on the cell surface predominantly localizes in caveolae. Inside the cell, EPCR is mainly localized in a small perinuclear structure, which is the site of centrosome. Colocalization of EPCR with tubulin (a marker for centrosome) and rab 11 (a marker of recycling compartment, REC) revealed that EPCR is localized actually in the REC and not in the centrosome. A small fraction of EPCR is also localized in endosomes as evident from colocalization of EPCR with EEA1 and Rab5, early endosome markers. Chasing the cell surface biotinylated proteins showed no significant increase in the biotinylated EPCR in the intracellular pool of proteins, which indicate that EPCR is not actively endocytosed constitutively or that the internalized EPCR is immediately recycled back to the cell surface. FVIIa or APC binding to EPCR promoted the EPCR endocytosis. The endocytosed receptors were first observed in proximity of the plasma membrane after 10 min and by 30 to 60 min most of the endocytosed EPCR was accumulated in the REC. The internalized FVIIa or APC appeared to follow the same route of the endocytosed EPCR. Proteolytically inactive FVIIa or APC behaved same as FVIIa or APC in promoting EPCR endocytosis and its trafficking. EPCR-dependent FVIIa or APC internalization is a dynamin-dependent process as the inhibition of the GTPase activity of dynamin by a specific inhibitor (Dynasore) completely abrogated their internalization and accumulation in the REC. Additional studies revealed that disruption of coated-pit pathway by potassium depletion blocked the endocytosis of transferrin, a classic marker for endocytosis via clathrin-dependent coated-pit pathway, but had no effect on EPCR-dependent FVIIa or APC internalization. In contrast, disruption of caveolae by cholesterol depletion blocked the internalization of FVIIa but not transferrin. Rab11 dominant negative mutant form (S25N) prevented the passage of the endocytosed EPCR or the internalized FVIIa or APC into the REC. After peaking at 30 min, the amount of both the ligands and the receptor in the REC gradually decreased, and some of the internalized ligand re-appeared at the cell surface. Overall the data provided herein suggest that FVIIa or APC binding to EPCR, independent of their protease activity, promotes EPCR endocytosis via the dynamin-dependent caveolar pathway and the activation of rab11 by GTP is required for exit of the endocytosed receptor or the ligands from sorting endosomes to the recycling compartment.

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Endothelial cell protein C receptor cellular localization and trafficking: potential functional implications

Blood ◽

10.1182/blood-2009-03-208900 ◽

2009 ◽

Vol 114 (9) ◽

pp. 1974-1986 ◽

Cited By ~ 39

Author(s):

Ramesh C. Nayak ◽

Prosenjit Sen ◽

Samit Ghosh ◽

Ramakrishnan Gopalakrishnan ◽

Charles T. Esmon ◽

...

Keyword(s):

Endothelial Cell ◽

Cell Surface ◽

Protein C ◽

Cellular Localization ◽

Factor Viia ◽

Expression System ◽

Activated Protein C ◽

In Vivo Studies ◽

Cell Protein ◽

Membrane Microdomains

Although the binding of endothelial cell protein C receptor (EPCR) to its ligands is well characterized at the biochemical level, it remains unclear how EPCR interaction with its ligands at the cell surface impacts its cellular trafficking. We characterized the cellular localization and trafficking of EPCR in endothelial cells and a heterologous expression system. Immunofluorescence confocal microscopy studies revealed that a majority of EPCR is localized on the cell surface in membrane microdomains that are positive for caveolin-1. A small fraction of EPCR is also localized intracellularly in the recycling compartment. Factor VIIa (FVIIa) or activated protein C binding to EPCR promoted the internalization of EPCR. EPCR and EPCR-bound ligands were endocytosed rapidly via a dynamin- and caveolar-dependent pathway. The endocytosed receptor-ligand complexes were accumulated in a recycling compartment before being targeted back to the cell surface. EPCR-mediated FVIIa endocytosis/recycling also resulted in transport of FVIIa from the apical to the basal side. In vivo studies in mice showed that blockade of EPCR with EPCR-blocking antibodies impaired the early phase of FVIIa clearance. Overall, our results show that FVIIa or activated protein C binding to EPCR promotes EPCR endocytosis, and EPCR-mediated endocytosis may facilitate the transcytosis of FVIIa and its clearance from the circulation.

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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 ◽

10.1182/blood.v116.21.346.346 ◽

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.

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Interaction of Factor VIIa with Endothelial Cell Protein C Receptor: Differences Between the Human and Mouse Systems

Blood ◽

10.1182/blood.v118.21.1181.1181 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1181-1181

Author(s):

Prosenjit Sen ◽

Curtis A Clark ◽

Ramakrishnan Gopalakrishnan ◽

Ulla Hedner ◽

Charles T Esmon ◽

...

Keyword(s):

Endothelial Cell ◽

Mouse Model ◽

Plasma Levels ◽

Protein C ◽

Model Systems ◽

Cell Protein ◽

Wild Type ◽

Binding Studies ◽

Human And Mouse ◽

Deficient Mice

Abstract Abstract 1181 Recent studies from our laboratory and others have shown that both the zymogen and activated form of FVII bind to endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C (APC). Although at present the pathophysiological significance of this interaction is unclear, recent studies indicate that FVIIa binding to EPCR may facilitate FVIIa transport from blood to extravasculature, mediate FVIIa-induced cell signaling and provide endothelial barrier protection. However, at present, there is no direct evidence demonstrating FVIIa actually associates with EPCR in vivo. Further, the Gla region involved in the EPCR binding is not fully conserved in mouse FVII, which raises questions on the ability of mouse FVIIa to associate with EPCR and the validity of mouse model systems in investigating the importance of the interaction between FVIIa and EPCR. Thus, it is important to first characterize the interaction of mouse FVIIa with mouse EPCR before employing murine model systems to investigate the importance of FVIIa interaction with EPCR in hemostasis and inflammation. Measurement of plasma levels of FVII, using both clotting and antigen assays, in wild-type, EPCR-deficient and EPCR-over expressing mice showed a small but insignificant increase (∼10%) in the circulating levels of FVII in EPCR-deficient mice and a ∼15% reduction in EPCR-over expressing mice in comparison to plasma FVII levels in wild-type mice. In comparison, the plasma level of protein C was decreased by more than 60% in EPCR-over expressing mice. Infusion of high concentrations of either human APCi or human FVIIai (400 μg/mice) to EPCR-over expressing mice failed to displace and increase the plasma levels of endogenous mouse FVII, while they increased plasma levels of endogenous mouse protein C by 2–3-fold. In additional studies, either mouse FVIIa or human FVIIa (120 μg/kg, tagged with AF488 probe) was administered exogenously via tail-vein to the wild-type, EPCR-deficient and EPCR-over expressing mice and FVIIa association with EPCR was evaluated by immunohistochemistry using anti-AF488 antibodies. In the case of mice injected with human FVIIa, the staining of FVIIa was undetectable or negligible in EPCR-deficient mice, whereas visible staining for FVIIa was clearly observed on the endothelium lining of the vessel walls in the wild-type mice. Furthermore, very intense staining (more so than wild-type) of FVIIa was observed on the endothelium of EPCR-over expressing mice. Although exogenously infused mouse FVIIa also appears to associate with the endothelium, there are no discernable differences in the staining intensity among wild-type, EPCR-deficient and EPCR-over expressing mice, suggesting that mouse FVIIa association with the endothelium is unaffected by EPCR. Next, in vitro binding studies were performed using mouse and human ligands and EPCR to further evaluate differences between binding of human and mouse ligands to EPCR. Surface Plasmon Resonance binding studies showed negligible binding of mouse FVIIa to either soluble human EPCR or mouse EPCR immobilized on the sensor chip. Under identical experimental conditions, human FVIIa was shown to bind both human and mouse EPCR. Experiments conducted with mouse and human endothelial cells or CHO cells transfected to express mouse or human EPCR also showed that mouse FVIIa does not bind, in any appreciable amount, to mouse EPCR and binds very poorly to human EPCR. Conversely, human FVIIa was found to bind to both human and mouse EPCR with a similar efficiency, both of which were approximately equivalent to that of human APC binding to EPCR. Interestingly, the binding of human FVIIa and APC to mouse EPCR is more dependent on Mg2+ ions as compared to their binding to human EPCR, indicating that differences between human and murine EPCR may also contribute to the differential binding of human and mouse ligands to EPCR. In summary, our data indicate that significant differences exist between human and mouse FVIIa in their ability to interact with EPCR. Given that human but not mouse FVIIa binds to EPCR, it may be more appropriate to use human FVII/FVIIa in mouse model systems to properly investigate the importance of FVIIa interaction with EPCR in hemostasis and inflammation. Disclosures: Hedner: Novo Nordisk: Consultancy. Rao:Novo Nordisk: Research Funding.

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Activation Mechanism of Anticoagulant Protein C in Large Blood Vessels Involving the Endothelial Cell Protein C Receptor

Journal of Experimental Medicine ◽

10.1084/jem.187.7.1029 ◽

1998 ◽

Vol 187 (7) ◽

pp. 1029-1035 ◽

Cited By ~ 71

Author(s):

Kenji Fukudome ◽

Xiaofen Ye ◽

Naoko Tsuneyoshi ◽

Osamu Tokunaga ◽

Keishin Sugawara ◽

...

Keyword(s):

Endothelial Cell ◽

Cell Surface ◽

Blood Coagulation ◽

Protein C ◽

Cell Surface Receptor ◽

Activation Mechanism ◽

Cell Protein ◽

Endothelial Cell Surface ◽

Surface Receptor ◽

Large Vessels

Protein C is an important regulatory mechanism of blood coagulation. Protein C functions as an anticoagulant when converted to the active serine protease form on the endothelial cell surface. Thrombomodulin (TM), an endothelial cell surface receptor specific for thrombin, has been identified as an essential component for protein C activation. Although protein C can be activated directly by the thrombin–TM complex, the conversion is known as a relatively low-affinity reaction. Therefore, protein C activation has been believed to occur only in microcirculation. On the other hand, we have identified and cloned a novel endothelial cell surface receptor (EPCR) that is capable of high-affinity binding of protein C and activated protein C. In this study, we demonstrate the constitutive, endothelial cell–specific expression of EPCR in vivo. Abundant expression was particularly detected in the aorta and large arteries. In vitro cultured, arterial endothelial cells were also found to express abundant EPCR and were capable of promoting significant levels of protein C activation. EPCR was found to greatly accelerate protein C activation by examining functional activity in transfected cell lines expressing EPCR and/or TM. EPCR decreased the dissociation constant and increased the maximum velocity for protein C activation mediated by the thrombin–TM complex. By these mechanisms, EPCR appears to enable significant levels of protein C activation in large vessels. These results suggest that the protein C anticoagulation pathway is important for the regulation of blood coagulation not only in microvessels but also in large vessels.

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The endothelial cell protein C receptor: cell surface conductor of cytoprotective coagulation factor signaling

Cellular and Molecular Life Sciences ◽

10.1007/s00018-011-0825-0 ◽

2011 ◽

Vol 69 (5) ◽

pp. 717-726 ◽

Cited By ~ 34

Author(s):

Eimear M. Gleeson ◽

James S. O’Donnell ◽

Roger J. S. Preston

Keyword(s):

Endothelial Cell ◽

Cell Surface ◽

Protein C ◽

Receptor Cell ◽

Coagulation Factor ◽

Cell Protein

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

Journal of Biological Chemistry ◽

10.1074/jbc.m010572200 ◽

2000 ◽

Vol 276 (11) ◽

pp. 8364-8370 ◽

Cited By ~ 56

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

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Cellular localization and trafficking of tissue factor

Blood ◽

10.1182/blood-2005-11-4674 ◽

2006 ◽

Vol 107 (12) ◽

pp. 4746-4753 ◽

Cited By ~ 53

Author(s):

Samir K. Mandal ◽

Usha R. Pendurthi ◽

L. Vijaya Mohan Rao

Keyword(s):

Cell Surface ◽

Tissue Factor ◽

Cellular Localization ◽

Factor Viia ◽

Coagulation Cascade ◽

Clotting Factor ◽

Cell Surfaces ◽

Caveolin 1 ◽

Intracellular Compartments ◽

Resultant Increase

AbstractTissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa). The formation of TF-FVIIa complexes on cell surfaces triggers the activation of coagulation cascade and cell signaling. In the present study, we characterized the subcellular distribution of TF and its transport in fibroblasts by dual immunofluorescence confocal microscopy and biochemical methods. Our data show that a majority of TF resides in various intracellular compartments, predominantly in the Golgi. Tissue factor at the cell surface is localized in cholesterol-rich lipid rafts and extensively colocalized with caveolin-1. FVIIa binding to TF induces the internalization of TF. Of interest, we found that TF-FVIIa complex formation at the cell surface leads to TF mobilization from the Golgi with a resultant increase in TF expression at the cell surface. This process is dependent on FVIIa protease activity. Overall, the present data suggest a novel mechanism for TF expression at the cell surface by FVIIa. This mechanism could play an important role in hemostasis in response to vascular injury by increasing TF activity where and when it is needed.

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

Biochemical Journal ◽

10.1042/bj20030341 ◽

2003 ◽

Vol 373 (1) ◽

pp. 65-70 ◽

Cited By ~ 163

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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