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• W2067229110 abstract "The binding and assembly of the coagulation proteases on the endothelial cell surface are important steps not only in the generation of thrombin and thrombogenesis, but also in vascular cell signaling. Effector cell protease receptor (EPR-1) was identified as a novel leukocyte cell surface receptor recognizing the coagulation serine protease Factor Xa but not the precursor Factor X. We now demonstrate that EPR-1 is expressed on vascular endothelial cells and smooth muscle cells. Northern blots of endothelial and smooth muscle cells demonstrated three abundant mRNA bands of 3.0, 1.8, and 1.3 kDa. 125I-Labeled Factor Xa bound to endothelial cells in a dose-dependent saturable manner, and the binding was inhibited by antibody to EPR-1. No specific binding was observed with a recombinant mutant Factor X in which the activation site was substituted by Arg196→ Gln to prevent the proteolytic conversion to Xa. EPR-1 was identified immunohistochemically on microvascular endothelial and smooth muscle cells. Functionally, exposure of smooth muscle cells or endothelial cells to Factor Xa induced a 3-fold and a 2-fold increase in [3H]thymidine uptake, respectively. However, receptor occupancy alone is insufficient for mitogenic signaling because the active site of the enzyme is required for mitogenesis. Thus, EPR-1 represents a site of specific protease-receptor complex assembly, which during local initiation of the coagulation cascade could mediate cellular signaling and responses of the vessel wall. The binding and assembly of the coagulation proteases on the endothelial cell surface are important steps not only in the generation of thrombin and thrombogenesis, but also in vascular cell signaling. Effector cell protease receptor (EPR-1) was identified as a novel leukocyte cell surface receptor recognizing the coagulation serine protease Factor Xa but not the precursor Factor X. We now demonstrate that EPR-1 is expressed on vascular endothelial cells and smooth muscle cells. Northern blots of endothelial and smooth muscle cells demonstrated three abundant mRNA bands of 3.0, 1.8, and 1.3 kDa. 125I-Labeled Factor Xa bound to endothelial cells in a dose-dependent saturable manner, and the binding was inhibited by antibody to EPR-1. No specific binding was observed with a recombinant mutant Factor X in which the activation site was substituted by Arg196→ Gln to prevent the proteolytic conversion to Xa. EPR-1 was identified immunohistochemically on microvascular endothelial and smooth muscle cells. Functionally, exposure of smooth muscle cells or endothelial cells to Factor Xa induced a 3-fold and a 2-fold increase in [3H]thymidine uptake, respectively. However, receptor occupancy alone is insufficient for mitogenic signaling because the active site of the enzyme is required for mitogenesis. Thus, EPR-1 represents a site of specific protease-receptor complex assembly, which during local initiation of the coagulation cascade could mediate cellular signaling and responses of the vessel wall. The ordered assembly of proteins of the coagulation cascade on cell surfaces results in greatly enhanced kinetic efficiency of protease function and protease generation as well as protection of proteases from their extracellular inhibitors. Endothelial cells, which constitute the cellular barrier between blood and the smooth muscle cells of the vessel wall, may initiate and assemble functional thrombin-generating cascades following vessel injury. In addition to thrombus generation, proteins in the cascade can directly initiate receptor-coupled cellular signaling and functional responses. In addition to its role in clotting and in the anticoagulation pathways mediated by specific proteolytic conversion of fibrinogen to fibrin and thrombomodulin-dependent activation of protein C, thrombin binds to a G-protein coupled cell surface receptor and initiates intracellular signal transduction and cell activation events. In contrast to the thrombin receptor, which is activated by cleavage, EPR-1, 1The abbreviations used are: EPR-1effector protease receptor 1TFtissue factorPAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered salinemAbmonoclonal antibodyECendothelial cellTAPtick anticoagulant proteinBSAbovine serum albuminPECAMplatelet endothelial cell adhesion molecule. the receptor recognizing the protease Factor Xa, possesses high affinity (1Altieri D. Edgington T. J. Immunol. 1990; 145: 246-253PubMed Google Scholar), is not cleaved (1Altieri D. Edgington T. J. Immunol. 1990; 145: 246-253PubMed Google Scholar), can elicit responses in lymphocytes (2Altieri D. Stamnes S. Cell. Immunol. 1994; 155: 372-383Crossref PubMed Scopus (36) Google Scholar), and can serve as a functional cofactor for the bound protease (1Altieri D. Edgington T. J. Immunol. 1990; 145: 246-253PubMed Google Scholar). As a result, the association of the generated proteases with cell surface receptors can initiate such diverse cellular functions as proliferation (3Chen L. Teng N. Buchanan J. Exp. Cell Res. 1976; 101: 41-46Crossref PubMed Scopus (83) Google Scholar, 4Chen L. Buchanan J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 131-135Crossref PubMed Scopus (393) Google Scholar), chemotaxis (5Bar-Shavit R. Kahn A. Wilner G. Fenton J. Science. 1983; 220: 728-731Crossref PubMed Scopus (260) Google Scholar), growth factor gene expression and synthesis (6Gajdusek C. Carbon S. Ross R. Nawroth P. Stern D. J. Cell Biol. 1986; 103: 419-428Crossref PubMed Scopus (75) Google Scholar, 7Daniel T. Gibbs V. Milfray D. Garovoy M. Williams L. J. Biol. Chem. 1986; 261: 9579-9582Abstract Full Text PDF PubMed Google Scholar), and adhesion molecule expression (8Doi T. Higashino K. Kurihari Y. Wada Y. Miyazaki T. Nakamura H. Uesugi S. Imanishi T. Kawabe Y. Itakura H. Yazaki Y. Matsumoto A. Kodama T. J. Biol. Chem. 1993; 268: 2126-2133Abstract Full Text PDF PubMed Google Scholar). effector protease receptor 1 tissue factor polyacrylamide gel electrophoresis phosphate-buffered saline monoclonal antibody endothelial cell tick anticoagulant protein bovine serum albumin platelet endothelial cell adhesion molecule. EPR-1 was identified as a novel leukocyte cell surface receptor for the coagulation protease Factor Xa but not the precursor zymogen Factor X (1Altieri D. Edgington T. J. Immunol. 1990; 145: 246-253PubMed Google Scholar). The cDNA of this cell surface transmembrane receptor has been cloned and found to encode a novel protein (9Altieri D. J. Biol. Chem. 1994; 269: 3139-3142Abstract Full Text PDF PubMed Google Scholar). Occupancy of EPR-1 on B and T cell lymphocyte subsets by Xa or surrogate monoclonal antibody ligands increased cytosolic free [Ca2+]i and enhanced CD3-dependent mononuclear cell proliferation (2Altieri D. Stamnes S. Cell. Immunol. 1994; 155: 372-383Crossref PubMed Scopus (36) Google Scholar). Whether this is the result of direct receptor occupancy has not been clearly delineated. In the present study we addressed the hypothesis that EPR-1 may be expressed on vascular cells and if so might participate in vascular cell signaling following receptor occupancy by Factor Xa generated locally as a consequence of vascular injury, tissue factor expression, and the local thrombogenic response (10Ruf W. Edgington T. FASEB J. 1994; 8: 385-390Crossref PubMed Scopus (217) Google Scholar, 11Marmur J. Rossikhina M. Guha A. Fyfe B. Friedrich V. Meddlowitz M. Nemerson Y. Taubman M. J. Clin. Invest. 1993; 91: 2253-2259Crossref PubMed Scopus (224) Google Scholar). Tissue culture reagents were obtained from Life Technologies, Inc. The porcine heparin used to culture human umbilical vein endothelial cells was from Sigma. Endothelial cell growth supplement was from Organon Technika Corp. (Rockville, MD). The human arterial smooth muscle cells were from Clonetics Corporation (San Diego, CA). THP-1 cells were from American Type Culture Collection (Rockville, MD). ITS (insulin, transferrin, and selenious acid) was from Collaborative Biomedical Products (Bedford, MA). Recombinant tick anticoagulant protein (TAP) was kindly provided by Dr. George Valsuk (Corvas International, San Diego, CA). Factor Xa, which was inactivated at the active site by reacting with the covalent inhibitor glutamyl-glycyl-arginyl-chloromethylketone, was obtained from Hematologic Technologies Inc. (Essex Junction, VT). The radioisotopes 125I and [32P]dCTP were obtained from Amersham Corp. The [3H]thymidine was from ICN (Costa Mesa, CA). Biospin chromatography columns and Zetaprobe membranes were from Bio-Rad, the random priming kit was from Boehringer Mannheim, and the UV cross-linker was from Stratagene (La Jolla, CA). The IODO-BEADs, disuccinimidyl suberate ultralink hydrazide, and normal mouse serum were purchased from Pierce, the phycoerythrin-conjugated goat anti-mouse IgG was from Tago Immunologicals (Burlingame, CA), and the first generation anti-EPR-1 mAbs (9D4, B6, and 12H1) were generated and characterized in a previous study (1Altieri D. Edgington T. J. Immunol. 1990; 145: 246-253PubMed Google Scholar). The second generation anti-EPR-1 mAb (2E1) was characterized in a previous study (9Altieri D. J. Biol. Chem. 1994; 269: 3139-3142Abstract Full Text PDF PubMed Google Scholar). An antibody to annexin II (12Cesarman G. Guevara C. Hajjar K. J. Biol. Chem. 1994; 269: 21198-21203Abstract Full Text PDF PubMed Google Scholar, 13Hajjar K. Jacovina A. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar) was provided by Dr. Katherine Hajjar. Anti-tissue factor (TF) mAbs TF8-5G9, TF9-10H10, and TF9-6B4 to independent epitopes (14Morrissey J. Fair D. Edgington T. Thromb. Res. 1988; 52: 247-261Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 15Ruf W. Edgington T.S. Thromb. Haemostasis. 1991; 66: 529-533Crossref PubMed Scopus (85) Google Scholar) were used as an equimolar mixture of the three. Anti-Mac-1 (CD11b/CD18) mAb OKM1, which inhibits Factor X binding to monocytes, was prepared as described previously (16Altieri D.C. Edgington T.S. J. Biol. Chem. 1988; 263: 7007-7015Abstract Full Text PDF PubMed Google Scholar). Anti-platelet endothelial cell adhesion molecule (PECAM) antibody, HEC-7, was previously characterized by Muller et al. (17Muller W. Berman M. Newman P. De Lisser H. Albelda S. J. Exp. Med. 1992; 175: 1401-1404Crossref PubMed Scopus (139) Google Scholar). The endotoxin content of materials were determined by chromogenic limulus amoebocyte assay QCL-1000 (BioWhittaker, Inc., Walkersville, MD). Factor X was highly purified from human plasma as described (18Fair D. Plow E. Edgington T. J. Clin. Invest. 1979; 64: 884-894Crossref PubMed Scopus (50) Google Scholar) followed by an additional final ultra purification using a Factor X-specific monoclonal antibody as follows. It was considered necessary to remove traces of Factor VII/VIIa, which were uniformly found in all conventionally purified Factor X preparations. The contamination was readily detected when the rate of Factor Xa generation was analyzed in the presence of relipidated recombinant human TF. Typically, 10-50 pM Factor VII was detected in 100 nM Factor X, corresponding to contamination of 1-5:10,000. When further purified, as follows, the contamination with Factor VII was <1:50,000. The pool of Factor X was bound to anti-Factor X monoclonal antibody f21-4.2 immobilized on Ultralink Hydrazide, and the column was washed extensively with 1 M NaCl, 5 mM EDTA, pH 8.0. The bound Factor X was eluted with 2 M guanidine hydrochloride and immediately dialyzed against 10 mM Tris, 140 mM NaCl, pH 7.4. Affinity purified Factor X was homogeneous by SDS-PAGE. Factor Xa was produced from the highly purified Factor X by limited proteolytic activation with Russel's viper venom Factor X activator. The Factor Xa product was again purified on benzamidine-Sepharose according to Krishnaswamy et al. (19Krishnaswamy S. Church W. Nesheim M. Mann K. J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar). The initial full-length cDNA for Factor X was kindly provided by Dr. W. Church (20Messier T. Pittman D. Long G. Kaufman R. Church W. Gene (Amst.). 1991; 99: 291-294Crossref PubMed Scopus (40) Google Scholar). The mutation was introduced by oligonucleotide directed mutagenesis according to Kunkel (21Kunkel T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4903) Google Scholar). The coding sequence DNA was subcloned into pED4 (22Kaufman R. Davies M. Wasley L. Michnick D. Nucleic Acids Res. 1991; 19: 4485-4490Crossref PubMed Scopus (231) Google Scholar) and sequenced in its entirety to detect potential unrecognized mutations in the expression plasmid. The plasmid was transfected into the Chinese hamster ovary cell line DG44. Stable transfectants were selected and grown in large scale serum-free cultures as described previously in detail for the expression of the homologous protein factor VII (23Ruf W. Biochemistry. 1994; 33: 11631-11636Crossref PubMed Scopus (51) Google Scholar). Factor X Arg196→ Gln was isolated from the serum-free culture supernatant by affinity chromatography on immobilized monoclonal antibody f21-4.2, described above. The protein obtained from this purification was homogenous when analyzed by SDS-PAGE under nonreducing conditions, but under reducing conditions the preparation showed a small amount (5-10%) of unprocessed single chain Factor X. Amino-terminal sequencing of the light chain yielded the sequence predicted from the cDNA, demonstrating proper processing of the signal peptide. The mutant Factor X, upon prolonged incubation with the enzymatic complex of Factor VIIa and TF (TF·VIIa), was not converted to Factor Xa, as determined by chromogenic substrate hydrolysis. The mutant Factor X (Arg196→ Gln) protein competitively inhibited the activation of wild-type plasma derived Factor X consistent with recognition by the enzymatically active TF·VIIa complex. Rat smooth muscle cells were isolated from thoracic aorta explants. Cells were cultivated for up to 12 passages only and were routinely grown in medium 199, 10% fetal calf serum, 50 μg/ml each of penicillin and streptomycin, 2.5 μg/ml Fungizone® (Amphotericin B), and 2 mM glutamine. Human vascular smooth muscle cells were cultivated up to the third passage only and grown in medium 199, 20% fetal calf serum, 50 μg/ml each of penicillin and streptomycin, 2 mM glutamine, and 20 μg/ml endothelial cell growth supplement. Primary cultures of human umbilical vein endothelial cells were prepared by standard methods (24Jaffe E. Nachman R. Becker C. Minick C. J. Clin. Invest. 1973; 52: 2745-2756Crossref PubMed Scopus (6017) Google Scholar). Cells were cultivated up to passage 3 only and grown in medium 199, 20% fetal calf serum, penicillin/streptomycin, 2 mM glutamine, Fungizone®, 90 μg/ml heparin, and 20 μg/ml endothelial cell growth supplement. Human umbilical vein endothelial cells were plated into gelatinized 96-well plates at a density of 60 × 104 cells/well and cultured for 24 h to allow for maximum adherence. The cell monolayers were washed twice with PBS (0.14 M NaCl, 0.01 M sodium phosphate, pH 7.3), 5 mM EDTA at 4°C. 125I-Labeled Factor Xa (IODO-BEAD method of labeling) (1Altieri D. Edgington T. J. Immunol. 1990; 145: 246-253PubMed Google Scholar) was added to cells at serial concentrations from 15 to 120 μg/ml in the presence of medium 199, 0.5% BSA at 4°C for 1 h. The cell monolayers were then washed four times in PBS, 0.2% BSA, solubilized in 0.2 N NaOH, and counted in a γ counter. For the antibody inhibition of binding experiments, a similar protocol was followed with the respective antibodies, which were added in various dilutions for 1.5 h at 4°C at the same time as the addition of the labeled Factor Xa. Endothelial and smooth muscle cell monolayers were washed, scraped into PBS, and homogenized in lysis buffer (1% Triton, 0.1% Nonidet P-40, 20 mM Tris, pH 7.4, 150 mM NaCl, 20 μg/ml aprotinin, 10 μg/ml soybean trypsin inhibitor, 2 mM phenylmethylsulfonyl fluoride, 10 mM benzamidine). Lysis continued at 4°C for 30 min, after which the lysate was clarified by centrifugation and protein concentrations were determined. 5 × sample buffer was diluted to 1 ×; samples were run on 7% SDS-PAGE gels applying 220 μg of protein/lane. The protein bands were transferred to Immobilon-P and EPR-1 protein was detected using the ECL protocol and reagents from Amersham Corp. with anti-EPR-1 mAb 9D4 at 4 μg/ml. Cell monolayers were washed once in PBS and lysed in guanidinium isothiocyanate. Total RNA was isolated using ultracentrifugation in a cesium chloride gradient. RNA was quantified spectrophotometrically and loaded on a 1% agarose-formaldehyde gel at 20 μg/lane. RNA was transferred to Zetaprobe membranes in 10 × SSC buffer (1.5 M NaCl, 0.1 M NaH2PO4, 0.01 M EDTA) by capillary action. RNA was UV cross-linked to the membrane. The cDNA for EPR-1 was labeled with [32P]dCTP using random priming and then purified by centrifugation through a Sephadex G-50 spin column. Blots were prehybridized for 10 min and then hybridized for 16 h at 43°C in 50% formamide, 7% SDS, 120 mM Na2HPO4, 250 mM NaCl, and 1 mM EDTA. Blots were washed at room temperature for 20 min in 2 × SSC, 0.1% SDS, then for 20 min at 65°C in 0.5 × SSC, 0.1% SDS, and finally for 20 min at 65°C in 0.1 × SSC, 0.1% SDS. The blots were analyzed by exposure to film for 24 h. Fresh human tonsils obtained at surgery were snap frozen in optimum cutting temperature medium (Miles Laboratories, Naperville, IL). 5-m sections were made on a cryostat, fixed in acetone, mounted on polylysine coated glass slides, and reacted with anti-EPR-1 mAb 9D4 diluted 1:1000. Controls included normal mouse IgG1 and monoclonal anti-human Factor VIII (Dako Corp., Carpenteria, CA) diluted 1:500. The antibodies were detected with ABC-peroxidase (Vector Laboratories, Burlingame, CA) and 3-amino-9-ethylcarbazole chromagen (Dako). Hematoxylin was used as a counterstain. Smooth muscle cells were plated overnight in 96-well plates in medium with 10% serum. The cells were then washed in PBS and allowed to become quiescent in medium 199, 1% ITS for 24 h. Cells were then incubated with serum-free medium with or without reagents. After 18 h, 1 μCi of [3H]thymidine was added to each well. After 4 h, cells were washed twice in PBS, trypsinized, harvested, and counted in a scintillation counter. Endothelial cell proliferation assays were similar with the following exceptions. Cells were plated in 5% serum for 24 h. The following day, they were placed in medium with 1% serum, with or without reagents. Human endothelial cells were grown to confluence in T-75 cm2 tissue culture flasks, washed once in PBS, and then aliquoted in 2 × 106 cells/100-ml aliquot. Cells were incubated with anti-EPR-1 mAb B6, 2E1, or normal mouse serum for 30 min at 4°C. Cells were then pelleted by centrifugation, washed twice in PBS, 1% BSA, and resuspended in PBS, 1% BSA. The fluorescein isothiocyanate-labeled second antibody was then added (1:100, v/v), and cells were covered in aluminum foil and rotated at 4°C for 30 min The cells were washed twice in PBS, 1% BSA and then resuspended in 2% paraformaldehyde, PBS. The samples were reconstituted to 1 ml with PBS, 1% BSA, and analyzed on an EPICS XL flow cytometer. Sizing gates were set to include all nucleated cells, and for each sample, at least 104 cells were analyzed. The data were analyzed by ANOVA. Differences with p < 0.05 were considered to be significant. Radiolabeled Factor Xa was reproducibly (n = 6) observed to bind to human endothelial cells in a dose-dependent, saturable, and specific manner (Fig. 1). Zero-order kinetics were achieved at 20 nM Factor Xa. The Kd for the binding was 12.5 nM, and the number of binding sites were calculated (Bmax) to be ∼220,000/endothelial cell (Fig. 1, inset). Binding of Factor Xa was also observed on smooth muscle cells with a Kd of 32 nM and 143,000 binding sites/cell (not shown). A Factor X mutant (Argl96→ Gln), which is not converted to the active serine protease Factor Xa by TF·VIIa, did not exhibit significant binding to endothelial or smooth muscle cells (Fig. 1), thereby providing supplemental data to support the specificity for Factor Xa. In order to identify the endothelial cell receptor for Factor Xa, monoclonal antibodies recognizing the Factor Xa receptor previously identified on leukocytes, namely EPR-1 (1Altieri D. Edgington T. J. Immunol. 1990; 145: 246-253PubMed Google Scholar), were tested for their ability to inhibit binding of 125I-Factor Xa to endothelial cell monolayers (Fig. 2). Anti-EPR-1 monoclonal antibody B6 diminished specific binding to endothelial cells by approximately 60%, whereas characterized antibodies to annexin II (12Cesarman G. Guevara C. Hajjar K. J. Biol. Chem. 1994; 269: 21198-21203Abstract Full Text PDF PubMed Google Scholar, 13Hajjar K. Jacovina A. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar), TF (14Morrissey J. Fair D. Edgington T. Thromb. Res. 1988; 52: 247-261Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 15Ruf W. Edgington T.S. Thromb. Haemostasis. 1991; 66: 529-533Crossref PubMed Scopus (85) Google Scholar), and PECAM (17Muller W. Berman M. Newman P. De Lisser H. Albelda S. J. Exp. Med. 1992; 175: 1401-1404Crossref PubMed Scopus (139) Google Scholar) failed to block binding of Factor Xa (not shown). In addition, no significant inhibition of Xa binding to endothelial cells was observed with monoclonal antibody OKM1 to Mac-1, which blocks binding of Factor X to leukocyte surface Mac-1 (16Altieri D.C. Edgington T.S. J. Biol. Chem. 1988; 263: 7007-7015Abstract Full Text PDF PubMed Google Scholar) (not shown). To further substantiate endothelial cell surface expression, we demonstrated mAb B6 reactivity with the endothelial cell surface by flow cytometry (Fig. 3).Fig. 3Flow cytometric analysis of EPR-1 surface expression. EC were washed with PBS, 5 mM EDTA, then twice with PBS, 1% BSA, and resuspended (30 min at 4°C) in PBS, 1% BSA with either normal mouse serum or anti-EPR-1 monoclonal antibody B6. The cells were then washed twice in PBS, 1% BSA and resuspended in PBS, 1% BSA with second antibody (anti-mouse IgG) coupled with fluorescein isothiocyanate. The cells were washed, fixed in 2% paraformaldehyde, and analyzed by flow cytometry.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether vascular cells can synthesize and express EPR-1, we first analyzed mRNA transcript levels for EPR-1 in endothelial and smooth muscle cells by Northern blot hybridization (Fig. 4). Three EPR-1 transcripts of approximately 1.3, 1.9, and 3.0 kilobases, in agreement with previous observations in THP-1 cells (9Altieri D. J. Biol. Chem. 1994; 269: 3139-3142Abstract Full Text PDF PubMed Google Scholar), were identified in both cell types. These are in accord with the known differentially spliced transcripts of EPR-1 mRNA (25Altieri D. Biochemistry. 1994; 33: 13848-13855Crossref PubMed Scopus (33) Google Scholar). Western blot analysis demonstrated a ∼65-kDa protein present in arterial smooth muscle cell lysates (using anti-EPR-1 mAb B6). In endothelial cells a doublet of ∼55 kDa (Fig. 5) was found.Fig. 5Western analysis of EPR-1 in human EC and smooth muscle cells. Endothelial and smooth muscle cells were washed, solubilized in homogenization (lysis) buffer, and centrifuged. Supernatants (220 μg of protein/lane) were analyzed on 7% SDS-PAGE gels. Following transfer to Immobilon-P, the membrane was reacted with anti-EPR-1 monoclonal antibody B6 at 4 μg/ml. Samples were visualized using the ECL detection kit.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further substantiate the functional expression of EPR-1 by these vascular cells in vivo, immunohistochemical analysis for EPR-1 was carried out (Fig. 6). Anti-EPR antibody strongly reacted with blood vessels in frozen sections of human tonsil (Fig. 6A). Both vascular smooth muscle cells and endothelial cells were immunoreactive. In contrast control mouse IgG1 did not react (data not shown). To explore the potential functional impact of docking of Factor Xa with vascular cell EPR-1, in vitro mitogenesis experiments were performed. These experiments were designed to exclude proliferative responses that might result from local generation of thrombin. In Fig. 7, we show a representative experiment that demonstrates the ability of Factor Xa at 50 nM, slightly in excess of the 32 nM Kd, to induce 3-fold increased proliferation of arterial smooth muscle cells relative to cells grown in medium alone in an 18-h assay. Factor Xa-induced proliferation was inhibited by a monoclonal antibody (B6) against EPR-1. Notably, addition of the thrombin inhibitor, hirudin, did not diminish Factor Xa-induced mitogenesis, thereby excluding cellular signaling by thrombin via the thrombin receptor. Also, neither very highly repurified Factor X, with Factor VII contamination at <1:50,000, nor a nonactivable mutant Factor X, neither of which bind or react with EPR-1, influenced these cell proliferation assays. To determine if the active site of Factor Xa was required for Factor Xa-induced smooth muscle cell mitogenesis, we performed proliferation assays in the presence of recombinant TAP, which blocks Xa conversion of prothrombin to thrombin, or with Factor Xa, which was inactivated at the active site by reacting with the covalent inhibitor glutamyl-glycyl-arginyl-chloromethylketone. In Fig. 8 we show that Factor Xa (50 nM) increased smooth muscle cell mitogenesis by more than 4-fold relative to untreated cells. However, Factor Xa in the presence of TAP (50 μg/ml) or glutamyl-glycyl-arginyl-chloromethylketone (50 nM) did not significantly increase smooth muscle cell mitogenesis, implying that the intact catalytic active site of Factor Xa is required for EPR-1·Xa vascular cell signaling. The macromolecular assembly of the proteins of the coagulation cascade on cell surfaces can result in a diverse set of effects including conversion of zymogens to highly specific proteases, generation of proteolytic fragments of various substrate proteins, platelet and endothelial cell activation via the thrombin receptor, and formation of the fibrin gel. Assembly of these proteases on cell surface receptors results in receptor-protease complexes with greatly enhanced function such as for assembly of the TF·VIIa initiation complex (10Ruf W. Edgington T. FASEB J. 1994; 8: 385-390Crossref PubMed Scopus (217) Google Scholar), the thrombin-thrombomodulin complex (26Le Bonniec B.F. Esmon C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7371-7375Crossref PubMed Scopus (146) Google Scholar), the prothrombinase complex Factor Xa-Factor Va (27Tracy P. Nesheim M. Mann K. Methods Enzymol. 1992; 215: 329-360Crossref PubMed Scopus (45) Google Scholar), and the intrinsic complex factor IXa-Factor VIIIa (28Duffy E.J. Parker E.T. Mutucumarana V.P. Johnson A. Lollar P. J. Biol. Chem. 1992; 267: 17006-17011Abstract Full Text PDF PubMed Google Scholar). Less well appreciated has been the docking of proteases on cognate cell surface receptors, a molecular mechanism well recognized for the adhesive proteins and their receptors but also evidenced by the docking of Factor X on the integrin Mac-1 (16Altieri D.C. Edgington T.S. J. Biol. Chem. 1988; 263: 7007-7015Abstract Full Text PDF PubMed Google Scholar), the assembly of protein S and homologous proteins on Tyro 3 (29Stitt T. Conn G. Gore M. Lal C. Bruno J. Radziejewski C. Mattsson K. Fisher J. Gies D. Jones P. Masiakowski P. Ryan T. Tobkes N. Chen D. DiStefano P. Long G. Basilico C. Goldfarb M. Lemke G. Glass D. Yancopoulos G. Cell. 1995; 80: 661-670Abstract Full Text PDF PubMed Scopus (613) Google Scholar), and the identification and cloning of EPR-1, a novel high affinity receptor for Factor Xa (1Altieri D. Edgington T. J. Immunol. 1990; 145: 246-253PubMed Google Scholar, 9Altieri D. J. Biol. Chem. 1994; 269: 3139-3142Abstract Full Text PDF PubMed Google Scholar). These receptors as well as the assembly of Factor VIIa on tissue factor not only mediate functional molecular assemblies on the cell surface but possess the capability of cellular signaling via their cytoplasmic domains (2Altieri D. Stamnes S. Cell. Immunol. 1994; 155: 372-383Crossref PubMed Scopus (36) Google Scholar, 29Stitt T. Conn G. Gore M. Lal C. Bruno J. Radziejewski C. Mattsson K. Fisher J. Gies D. Jones P. Masiakowski P. Ryan T. Tobkes N. Chen D. DiStefano P. Long G. Basilico C. Goldfarb M. Lemke G. Glass D. Yancopoulos G. Cell. 1995; 80: 661-670Abstract Full Text PDF PubMed Scopus (613) Google Scholar, 30Rottingen J.-A. Enden T. Camerer E. Iversen J.-G. Prydz H. J. Biol. Chem. 1995; 270: 4650-4660Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Here, we demonstrate that vascular endothelial cells and smooth muscle cells express EPR-1, a Factor Xa receptor previously identified only on leukocytes. Further, Factor Xa binding to EPR-1 induced arterial smooth muscle cell activation, analogous to previously described engagement of EPR-1 on lymphocytes (2Altieri D. Stamnes S. Cell. Immunol. 1994; 155: 372-383Crossref PubMed Scopus (36) Google Scholar), but only if the active site of the enzyme is intact and functional. This implies that receptor occupancy alone is not sufficient for cellular activation. Quantitative characterization of the binding of Factor Xa to endothelial cells and smooth muscle cells demonstrated that it is a receptor-mediated event with a Kd of ∼12.5 n" @default.
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• W2067229110 date "1996-11-01" @default.
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• W2067229110 title "Effector Cell Protease Receptor-1 Is a Vascular Receptor for Coagulation Factor Xa" @default.
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