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Activation by the Thrombin-Thrombomodulin Complex DEBORAH J Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10212-10216, September 1996 Cell Biology The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex DEBORAH J. STEARNS-KUROSAWA*, SHINICHIRO KUROSAWA*, JEFFERY S. MOLLICA*, GARY L. FERRELLt, AND CHARLES T. ESMON*tt§ *Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, *Departments of Pathology and Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, and tHoward Hughes Medical Institute, Oklahoma City, OK 73104 Communicated by Kenneth M. Brinkhous, University of North Carolina, Chapel Hill, NC, July 8, 1996 (received for review May 28, 1996) ABSTRACT Protein C activation on the surface of the protein C and the derivative devoid of the protein C Gla endothelium is critical to the negative regulation of blood domain (10, 11). Taken together, these data supported the coagulation. We now demonstrate that monoclonal antibodies possibility that an additional endothelial cell protein might that block protein C binding to the endothelial cell protein C facilitate protein C activation. receptor (EPCR) reduce protein C activation rates by the In this study, we describe monoclonal antibodies (mAbs) thrombin-thrombomodulin complex on endothelium, but that that bind to EPCR and block both protein C and APC binding antibodies that bind to EPCR without blocking protein C to cells stably transfected with EPCR (E7 cells), to human binding have no effect. The kinetic result of blocking the umbilical endothelial cells (HUVECs), and to EA.hy926 cells EPCR-protein C interaction is an increased apparent Km for (a transformed endothelial cell line). On those cells that the activation without altering the affinity of thrombin for express both TM and EPCR, these anti-EPCR antibodies thrombomodulin. Activation rates of the protein C derivative inhibit thrombin-dependent protein C activation, but not the lacking the y-carboxyglutamic acid domain, which is required activation of Gla-domainless protein C. This is, to our knowl- for binding to EPCR, are not altered by the anti-EPCR edge, the first evidence that EPCR participates in the process antibodies. These data indicate that the protein C activation of protein C activation and the natural anticoagulant pathway. complex involves protein C, thrombin, thrombomodulin, and EPCR. These observations open new questions about the control of coagulation reactions on vascular endothelium. EXPERIMENTAL PROCEDURES Reagents. Suppliers were as follows: fluorescein thiosemi- Our current concepts of the mechanisms of the protein C carbazide, Molecular Probes; biotinamidocaproate N- anticoagulant pathway are that the pathway is triggered when hydroxysuccinimide ester, avidin, and bovine serum albumin thrombin binds to the endothelial cell receptor, thrombo- (BSA), Sigma; streptavidin-phycoerythrin, Becton Dickinson; modulin (TM) (reviewed in refs. 1-4). This complex activates biotin-LC-hydrazide, Pierce; and Spectrozyme PCa, American protein C to generate the anticoagulant enzyme activated Diagnostica. All other reagents were of the highest quality protein C (APC), which, in complex with protein S, inhibits commercially available. coagulation by inactivating two critical regulatory proteins, Protein Preparation. sEPCR-HPC4, the recombinant solu- factors Va and VIIIa. This pathway plays a critical role in the ble form of human EPCR, was prepared as described previ- negative regulation of blood coagulation, as evidenced by the ously (7). The construct codes for the extracellular domain of fact that total deficiencies of protein C or protein S are EPCR truncated immediately above the transmembrane do- associated with severe and life-threatening thrombotic com- main at residue 210. An HPC4 epitope tag was attached to the plications (reviewed in ref. 2), and deletion of the TM gene in carboxyl terminus for calcium-dependent affinity purification mice results in embryonic lethality (5). (12). Recombinant protein C lacking the Gla domain, Gla- We recently identified and cloned the endothelial cell domainless protein C, was prepared as described (12). protein C/APC receptor (EPCR) (6). EPCR is a type 1 Bovine thrombin (13) and human protein C (14) and APC transmembrane protein that binds both its ligands with similar (14) were prepared as described. The extinction coefficients affinity (Kd 30 nM) (7) and inhibits the ability of APC to [(mg/ml)-1-cm-1] and molecular weights used for this study inactivate its natural substrate, factor Va (8). Like TM, EPCR were as follows: bovine thrombin (2.1, 37,000) (15), human is down-regulated by tumor necrosis factor a on endothelium APC (1.45, 60,000) (13), human protein C (1.45, 62,000) (13), in vitro, and the down-regulation of the two receptors follows sEPCR-HPC4 (1, 42,000) (7), and Gla-domainless protein C the same time course, suggesting that these two receptors (1.45, 58,000) (12). might function in a common pathway (6). The active site of APC was labeled with fluorescein to Several of the requirements for rapid protein C activation by generate fl-APC as described (6). Protein C was directly the endothelium overlap the requirements for ligand binding labeled with fluorescein by using methods described to selec- to EPCR. In particular, proteolytic removal of the vitamin K-dependent y-carboxyglutamic acid (Gla) domain of protein Abbreviations: TM, thrombomodulin; APC, activated protein C; C prevents binding to EPCR and reduces the activation rate of EPCR, endothelial protein C/APC receptor; sEPCR-HPC4, recom- protein C over endothelium at least 5-fold (9). In contrast, binant soluble human EPCR with the HPC4 epitope inserted in place to same of the transmembrane domain and cytosolic tail; fl-APC, APC with purified TM accelerates protein C activation the fluorescein attached to the active site; biotin-PC, protein C biotin- extent in solution irrespective of the presence of the protein C ylated on primary amino groups; fl-cho-protein C, protein C selec- Gla domain (9). Furthermore, a subgroup ofrabbit endothelial tively labeled with fluorescein on sialic acids; biotin-cho-sEPCR- cell lines were identified in earlier studies that, unlike primary HPC4, soluble EPCR selectively labeled with biotin on sialic acids; human or bovine endothelium, failed to distinguish between Gla, y-carboxyglutamic acid; FACS, fluorescence-activated cell sorter; mAb, monoclonal antibody; HUVECs, human umbilical vein endo- thelial cells. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be addressed at: Howard Hughes payment. This article must therefore be hereby marked "advertisement" in Medical Institute Research Laboratories, Acree-Woodworth Re- accordance with 18 U.S.C. §1734 solely to indicate this fact. search Building, 820 NE 15, Room A205, Oklahoma City, OK 73104. 10212 Downloaded by guest on September 28, 2021 Cell Biology: Stearns-Kurosawa et al. Proc. Natl. Acad. Sci. USA 93 (1996) 10213 tively label terminal sialic acid groups (fl-cho-protein C) (16). intensities with CTM1009 (a TM marker) and JRK-1 (an Protein C (0.8 mg/ml in 0.1 M NaCl/0.1 M sodium acetate, pH EPCR marker) at optimal antibody concentration on 5.5) was incubated with 1 mM sodium metaperiodate for 20 EA.hy926 cells were within a factor of 2. APC binding to E7 min in the dark on ice. Sodium metabisulfite was added to 0.4 cells is about 2-4 times greater than to EA.hy926 cells or mM, the mixture was incubated for 5 min at room temperature, HUVECs, which bind similar amounts. Greater than 90% of and fluorescein thiosemicarbazide was added to a final con- the APC binding is blocked by 1496 mAb on all cell types. centration of 0.5 mM. The solution was incubated for 60 min Thus, the TM and EPCR surface densities appear to be similar. on ice in the dark and the fl-cho-protein C was dialyzed Direct Binding Assay. sEPCR-HPC4 was labeled with bi- extensively at 4°C against Hanks balanced salt solution otin-LC-hydrazide by using the method described above for (HBSS). For some experiments, protein C (0.8 mg/ml at pH selective modification of sialic acid residues on protein C. 8.5) was labeled with biotin on primary amino groups by Microtiter plates (96 wells) were coated overnight at 4°C with incubation with biotinamidocaproate N-hydroxysuccinimide 50 ,ul of 10 ,ug/ml avidin in 0.1 M sodium carbonate, pH 9.6. ester (final 0.2 mM) for 2 hr on ice. The reaction was quenched The following steps were performed at room temperature. The with 2 M Tris HCl, pH 7.5, and the biotin-PC was dialyzed wells were washed and then blocked for 1 hr with 0.1% extensively against HBSS to remove free biotin. (wt/vol) gelatin in 0.02 M Tris.HCl/0.1 M NaCl, pH 7.5. The Cell Culture. All human cell lines were maintained as wells were washed and incubated for 1 hr with 50 pI of the described previously (7). E7 cells are human kidney 293 cells biotin-cho-sEPCR-HPC4 (70 nM). This concentration of la- stably transfected with full-length human EPCR and have been beled EPCR was chosen from preliminary titration curves of characterized previously (7). HUVECs were kindly provided increasing APC concentration (0-1 ,utM) added to wells coated by Craig Carson (Oklahoma City Clinic). EA.hy926 cells, a with labeled EPCR at increasing concentrations (0-2.38 ,uM). transformed human endothelial cell line (17), were kindly The wells were washed with the same buffer containing 3 mM provided by Cora-Jean Edgell (University of North Carolina at CaCl2, 0.6 mM MgCl2. 0.05% Tween 20, and 0.02% sodium Chapel Hill). azide. mAbs were added (50 ,ul of 10 ,ug/ml), the mixture was mAbs and Screening. mAbs against sEPCR-HPC4 were incubated for 1 hr, and wells were washed with the same buffer. obtained as described previously for other proteins (14).
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