©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Dissection of Effector Cell Protease Receptor-1 Recognition of Factor Xa
ASSIGNMENT OF CRITICAL RESIDUES INVOLVED IN ANTIBODY REACTIVITY AND LIGAND BINDING (*)

(Received for publication, September 25, 1995; and in revised form, November 9, 1995)

Grazia Ambrosini Dario C. Altieri (§)

From the Molecular Cardiobiology Program, Boyer Center for Molecular Medicine, Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06536

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Receptor-mediated assembly of blood proteases on vascular cells maintains the hemostatic balance and initiates intracellular signal transduction. Effector cell protease receptor-1 (EPR-1) is an 62-kDa vascular cell membrane receptor for the clotting protease factor Xa, participating in thrombin formation and lymphocyte activation. Here, recombinant EPR-1 fragments were engineered in the frame of intercellular adhesion molecule-1, transfected in mammalian cells, and analyzed for antibody recognition and ligand binding. Chimeric transfectants containing the EPR-1 sequence Met^1-Arg bound the immunosuppressive anti-EPR-1 monoclonal antibody (mAb) 2E1. In contrast, transfected cells expressing the EPR-1 sequence Pro-Ala were recognized by the functionally inhibitory anti-EPR-1 mAbs 9D4 and B6, bound I-factor Xa in a reaction quantitatively indistinguishable from that of wild-type EPR-1 transfectants, and promoted factor Xa concentration-dependent prothrombin activation in the absence of exogenous factor V/Va. Chimeric transfectants expressing the COOH terminus end of the EPR-1 extracellular domain (Ala-Glu) did not bind anti-EPR-1 mAbs and did not associate with factor Xa. Mutagenesis of Asn or Lys in the EPR-1 ligand recognition domain abolished factor Xa binding by 80 ± 5.5 and 96 ± 4%, respectively, while mutation of Lys, Gly, Asn, and Asn was without effect. A synthetic peptide duplicating the EPR-1 sequence SPGKPGNQNSKNEPP dose dependently inhibited factor V/Va-independent thrombin generation of resting endothelium (IC 1 µM), while the adjacent EPR-1 sequence PPKKRERERSSHCYP was ineffective. These findings demonstrate that EPR-1 contains two spatially distinct functional domains implicated in lymphocyte activation (Met^1-Arg) or factor Xa binding and prothrombin activation (Pro-Ala). These interacting sequences may provide a novel potential target for inhibition of factor Xa-dependent vascular cell responses.


INTRODUCTION

Assembly of coagulation and anticoagulation pathways occurs on vascular cells through the regulated ligand recognition of membrane protease receptors(1, 2) . Belonging to structurally different and evolutionarily unrelated gene superfamilies, cell-surface receptors for thrombin(3) , urokinase(4) , protein S(5) , or factor Xa (6) provide a controlled microenvironment for limited proteolytic activation of the clotting and fibrinolytic cascades(1, 2) . Recent studies have also underscored the participation of protease receptors in pleiotropic mechanisms of vascular cell signal transduction, including transcription of activation-dependent genes(7) , generation of intracellular second messengers(3) , modulation of immune inflammatory responses(8) , and cell proliferation(5, 9) . Aberrations of protease-dependent signaling pathways may play a primary pathogenetic role in the establishment and progression of the atherothrombotic disease (9, 10) as well as in neoplastic transformation and tumor cell dissemination(5, 11) .

Binding of factor Xa to leukocytes (12) or endothelial cells (^1)is contributed by effector cell protease receptor-1 (EPR-1), (^2)a 337-amino acid single membrane-spanning glycoprotein lacking significant homologies to other protease receptors and expressed in a cell-type specific fashion by alternative mRNA splicing(13) . Occupancy of EPR-1 with factor Xa participates in factor V/Va-independent mechanisms of prothrombin activation(6) , generates intermediate product(s) of factor IX activation(14) , or contributes an accessory pathway of lymphocyte costimulation(8) .

To gain insights into the potential pathophysiological relevance of EPR-1-factor Xa interaction in hemostasis and vascular cell signal transduction, we sought to identify the structural requirements implicated in receptor-ligand recognition. Using a eukaryotic expression strategy of EPR-1 chimeric constructs combined with site-directed mutagenesis and synthetic peptidyl mimicry, we have identified two spatially distinct regions in the EPR-1 extracellular domain separately involved in lymphocyte activation mechanisms or factor Xa binding and prothrombin activation.


EXPERIMENTAL PROCEDURES

Construction of EPR-1-ICAM-1 Chimeras

Three nonoverlapping regions in the EPR-1 extracellular domain were amplified from the full-length EPR-1 cDNA sequence (6) by the polymerase chain reaction (PCR). The NH(2)-terminal domain, designated G2, contained the EPR-1 sequence Met^1-Arg. A second region, designated A1, encompassed residues Pro-Ala. A third region at the COOH terminus end of the EPR-1 extracellular domain, designated G3, comprised residues Ala-Glu. The following EPR-1-derived oligonucleotides were used for amplification experiments: F1 (coding sequence, bp 1-18), 5`CCATGCCATGGAATGACCTCCAGAGGTTTC-3`; R1 (bp 180-163), 5`-CTC CAGGCGCCGGTCTGTCACGTTCTCCAC-3`; F2 (bp 357-372), 5`-CCATGCCAT GGACCTCACTTCTCACCTGG-3`; R2 (bp 462-448), 5`-CTCCAGGCGCCGGAGC GGGTGCTGCTGG-3`; F3 (bp 469-483), 5`-CATCACCATGGAGCAGAAGCACCT CTGG-3`; and R3 (bp 663-647), 5`-CGTTGGGCGCCGGCTCGATGGATTGAGGCC-3`. Each oligonucleotide contained a 5`-end restriction site (underlined), NcoI (forward primer, F) or NarI (reverse primer, R), to allow directional insertion of the amplified EPR-1 PCR products into the cDNA of ICAM-1(15) , used as an unrelated frame to generate the chimeric constructs. A topographical representation of the EPR-1-ICAM-1 chimeras is schematically shown in Fig. 1A. PCR amplification was carried out as described(13) . Briefly, reaction mixtures containing 200 ng of the full-length EPR-1 cDNA were subjected to amplification in a Perkin-Elmer 480 thermocycler with denaturation at 94 °C, annealing at 50 °C, and extension at 72 °C for 1 min each in the presence of 0.5 µg/reaction of the indicated oligonucleotides and 1 unit of Vent^R DNA polymerase (New England Biolabs Inc., Beverly, MA). A full-length ICAM-1 cDNA clone (15) was inserted in pBluescript (Stratagene, La Jolla, CA), cleaved at the unique sites NcoI (bp 549) and NarI (bp 946), and ligated with each digested and gel-purified EPR-1 PCR product. Fused cDNA constructs were sequenced through the chimeric region by the dideoxy chain termination reaction method using Sequenase (U. S. Biochemical Corp.) and found to be mutation-free. In some experiments, the constructs were in vitro translated by TNT coupled reticulocyte lysate systems (Promega) in the presence of [S]methionine (DuPont NEN). Samples were separated by electrophoresis on a 10% SDS-polyacrylamide linear gel and visualized by autoradiography using Kodak X-Omat AR x-ray film and intensifying screens. Finally, chimeric constructs were directionally subcloned in the HindIII/XbaI sites of the eukaryotic expression vector pcDNA3 (Invitrogen, San Diego, CA) for transfection experiments.


Figure 1: Design and characterization of EPR-1-ICAM-1 chimeras. A, schematic diagram of EPR-1 chimeras. EPR-1 sequences G2 (Met^1-Arg), A1 (Pro-Ala), and G3 (Ala-Glu) were amplified by PCR and directionally inserted in the unique NcoI and NarI sites in the extracellular domain of ICAM-1. B, in vitro translation of EPR-1 chimeras. EPR-1-ICAM-1 chimeric constructs G2 (lane 1), A1 (lane 2), and G3 (lane 3) or control pcDNA3 vector alone (lane 4) was translated using the reticulocyte lysate method in the presence of [S]methionine, separated by electrophoresis on a 10% SDS-polyacrylamide linear gel, and visualized by autoradiography. Relative molecular weight markers are shown on the left. TM, transmembrane domain.



Site-directed Mutagenesis

Oligonucleotide-directed mutagenesis of the A1 chimera cDNA was carried out using the Altered Site II in vitro mutagenesis system (Promega) following the manufacturer's recommendations. The single-strand A1 chimera cDNA in pAlter vector (Promega) was annealed with the following EPR-1-derived synthetic oligonucleotides carrying a single base substitution (underlined): 5`-CACCTGGTATGCCCGGGAA-3` (Lys Met), 5`-GTAAGCCCGCGAATCAAAA-3` (Gly Ala), 5`-AGCCCGGGAGTCAAAACAG-3` (Asn Ser), 5`-GGAATCAAATCAGCAAAAA-3` (Asn Ile), 5`-CAAAACAGCATAAATGAGCC-3` (Lys Ile), and 5`-AACAGCAAAAGTGAGCCCCC-3` (Asn Ser). Inserted mutations were confirmed by DNA sequencing. All mutants were subcloned in pcDNA3, transfected in CHO cells by electroporation, and tested in I-factor Xa binding experiments as described below.

Transfections

Full-length ICAM-1 (15) or EPR-1(6, 13) cDNA or the EPR-1-ICAM-1 chimera G2, A1, or G3 was transfected in subconfluent cultures of CHO cells by electroporation. Aliquots of the cell suspension (20 times 10^6 cells/ml) were preparatively incubated with 15 µg of each plasmid DNA for 15 min on ice followed by a single electric pulse delivered by a Gene Pulser apparatus (Bio-Rad) at 350 V and 960 microfarads. After a 48-h culture at 37 °C, cells were harvested, phenotypically characterized for transfection efficiency with anti-ICAM-1 monoclonal antibody (mAb) 6E6 (16) by flow cytometry, and used in I-factor Xa binding experiments and prothrombin activation. Control experiments were carried out with CHO cells transfected with pcDNA3 vector alone under the same experimental conditions.

Flow Cytometry

Phenotypical characterization of the various transfectants was carried out by flow cytometry as described(6) . Briefly, transfected CHO cells were detached by treatment with 2 mM EDTA/phosphate-buffered saline, pH 7.4; washed in phosphate-buffered saline, pH 7.4; and incubated with 20 µg/ml anti-ICAM-1 mAb 6E6 (16) or anti-EPR-1 mAb 2E1 (6) or 9D4 (12) or with a 1:50 ascites dilution of mAb B6^1 for 30 min at 4 °C. Characterization of mAb B6 recognition of EPR-1 on vascular endothelium and its ability to block binding of I-factor Xa to these cells will be reported elsewhere in detail.^1 After washes, cells were stained with a 1:20 dilution of fluorescein-conjugated goat F(ab`)(2) anti-mouse IgG (BioSource, International, Camarillo, TX), washed, and immediately analyzed by flow cytometry on a Becton-Dickinson FACSort.

Protein Purification and Labeling, Binding Studies, and Thrombin Generation

The experimental procedures for the preparation and characterization of activated factor X (factor Xa) from human plasma have been described previously in detail(17) . Aliquots of human factor Xa purchased from Calbiochem or from Hematologic Technologies (Burlington, VT) gave indistinguishable results in binding assays and thrombin generation experiments. Human prothrombin was purchased from Calbiochem. Factor Xa was iodinated by the IODO-GEN method to a specific activity of 0.5-1 µCi/µg of protein, with separation of free from protein-incorporated radioactivity by gel filtration on a PD-10 Sephadex G-25 column (Pharmacia Biotech Inc.) pre-equilibrated with phosphate-buffered saline, pH 7.4, with collection of 0.5-ml fractions. For binding studies, CHO cells transfected with full-length EPR-1 cDNA(6) , control vector pcDNA3 alone, or EPR-1-ICAM-1 chimera A1 or G3 were detached by treatment with EDTA/phosphate-buffered saline, washed, diluted to 3 times 10^6/ml in serum-free RPMI 1640 (BioWhittaker, Inc.) tissue culture medium, and further incubated (0.2 ml) with increasing concentrations of I-factor Xa (0.2-2 µg/ml) in the presence of 2.5 mM CaCl(2) for 15 min at 22 °C. At the end of each incubation, free from cell-bound radioactivity was separated by centrifugation of aliquots of each reaction through a mixture of silicone oil (Dow Corning, New Bedford, MA) at 14,000 times g for 5 min with determination of cell-associated radioactivity in a -counter. Nonspecific binding was assessed in the presence of a 50-fold molar excess of unlabeled factor Xa added at the start of the incubation and was subtracted from the total binding to calculate net specific binding, as described(6) . Similar binding experiments were carried out with CHO cells transfected with wild-type or six different single-residue mutagenized A1 constructs.

For prothrombin activation studies, wild-type CHO cells or CHO cells transfected with full-length EPR-1 cDNA or the A1 or G3 chimera were seeded in 48-well plates at 4 times 10^4 cells/well 24 h prior to the experiment. Cells were incubated in phenol red-free RPMI 1640 medium (Cellgro, Mediatech, Washington, D. C.) in the presence of increasing concentrations of factor Xa (0.1-1 µg/ml), 1.25 mM CaCl(2), and 20 µg/ml prothrombin for 5 min at 22 °C. Thrombin generated under the various conditions tested was quantitated at 22 °C immediately after addition of a 50 µg/ml concentration of the thrombin-sensitive chromogenic substrate S-2238 (Chromogenix, Mölndal, Sweden) by absorbance at a 405-nm wavelength on a ThermoMax microplate reader (Molecular Devices, Menlo Park, CA). Background hydrolysis of S-2238 in the presence of factor Xa alone (8-14%) was subtracted from the absorbance determined under the various experimental conditions tested. Absorbance units were converted to units of thrombin/milliliter using a standard curve constructed with 2-fold serial increasing concentrations of bovine thrombin (Calbiochem) from 0.015 to 2 units/ml. The A1 sequence in EPR-1 (Pro-Ala) was duplicated by two partially overlapping synthetic peptides, AG1 (SPGKPGNQNSKNEPP) and AG2 (PPKKRERERSSHCYP). Authenticity and purity of the EPR-1 peptides were confirmed by amino acid composition and mass spectrometry. For peptide competition experiments, human umbilical vein endothelial cells (HUVEC) were seeded in 96-well tissue culture plates and grown to confluency for 2-4 days prior to the assay. Two µg/ml factor Xa was separately preincubated with increasing concentrations (0.1-25 µM) of peptide AG1 or AG2 for 5 min at 22 °C before addition to HUVEC monolayers and determination of prothrombin activation by S-2238 hydrolysis as described above. Functional characterization of EPR-1 expression on HUVEC by immunoblotting, Northern hybridization, and factor V/Va-independent binding of I-factor Xa will be reported elsewhere in detail.^1


RESULTS

Characterization of EPR-1 Chimeras

Three spatially separate and nonoverlapping regions of the EPR-1 extracellular domain (G2, Met^1-Arg; A1, Pro-Ala; and G3, Ala-Glu) were engineered in the frame of ICAM-1 as schematically represented in Fig. 1A. Although disruptive of the architecture of the second and third immunoglobulin-like domains of ICAM-1(15) , insertion of the EPR-1 segments did not affect the epitope(s) recognized by the recently generated anti-ICAM-1 mAb 6E6(16) . Flow cytofluorometric staining of the various EPR-1 transfectants with mAb 6E6 was carried out before each experiment and demonstrated that all the chimeric constructs used in this study were expressed at comparable levels on CHO cells. In vitro translation of the EPR-1-ICAM-1 chimeric constructs G2, A1, and G3 originated three polypeptides with the expected molecular sizes of 55.5, 45, and 56 kDa, respectively, thus confirming the existence of a continuous open reading frame throughout the ICAM-1 cDNA frame and the EPR-1 insertion (Fig. 1B).

Epitope Mapping Experiments

The epitope(s) recognized by two classes of anti-EPR-1 mAbs exhibiting different functional activities on lymphocyte activation (^3)or vascular cell binding of factor Xa and prothrombin activation (12) ^1 were characterized in flow cytofluorometric experiments. Transfection of full-length wild-type ICAM-1 cDNA in CHO cells was associated with surface expression of a recombinant molecule genuinely recognized by anti-ICAM-1 mAb 6E6 (Fig. 2), in agreement with previous observations(16) . Under these experimental conditions, transient expression of the G2 chimera, containing the EPR-1 sequence Met^1-Arg, was associated with strong reactivity of the second generation anti-EPR-1 mAb 2E1 (6) with transfected cells (Fig. 2). In contrast, A1 chimeric transfectants expressing the EPR-1 sequence Pro-Ala specifically bound the first generation of functionally inhibitory mAb 9D4(12, 17) or B6 (Fig. 2).^1 Confirming the specificity of the observed recognition, A1 or G3 transfectants failed to react with mAb 2E1, and, conversely, no reactivity of mAb B6 or 9D4 with G2 or G3 transfectants was observed under the same experimental conditions ( Fig. 2and data not shown). Differences in fluorescence intensity in mAb binding presumably reflected a lower affinity for EPR-1 recognition of the first generation of cross-reacting mAb 9D4 or B6 (12) as compared with that of mAb 2E1(6) .


Figure 2: Epitope mapping of anti-EPR-1 mAbs. CHO cells were transfected with full-length ICAM-1 cDNA, with the various EPR-1-ICAM-1 chimeras, or with control vector pcDNA3 by electroporation. Forty-eight h after transfection, cells were analyzed by flow cytometry for reactivity with anti-ICAM-1 mAb 6E6, with the second generation anti-EPR-1 mAb 2E1, or with the first generation of functionally blocking anti-EPR-1 mAb 9D4 or B6. Horizontal and vertical axes measure fluorescence intensity on a 4-log scale and the cell number, respectively. WT, wild-type.



Identification of Factor Xa-binding Site on EPR-1

CHO cells transfected with wild-type EPR-1 cDNA bound I-factor Xa in a reaction specifically inhibited by a molar excess of unlabeled factor Xa and approaching saturation when 68 ± 5 ng/ml factor Xa was specifically associated with the cell surface (Fig. 3), in agreement with previous observations(6) . In contrast, wild-type CHO cells transfected with pcDNA3 vector alone did not specifically bind I-factor Xa (Fig. 3). Under these experimental conditions, CHO cells expressing EPR-1 residues Pro-Arg in the context of the A1 chimera specifically and saturably bound I-factor Xa in a reaction quantitatively indistinguishable from the binding isotherm observed with wild-type EPR-1 transfectants (Fig. 3) and completed with maximal specific association of 74 ± 13 ng/ml I-factor Xa with the cell surface (Fig. 3). In contrast, CHO cells expressing comparable levels of the G3 chimera, comprising EPR-1 residues Ala-Glu, did not specifically bind I-factor Xa under the same experimental conditions (Fig. 3).


Figure 3: I-Factor Xa binding to EPR-1 chimeras. CHO cells transiently transfected with control vector pcDNA3, wild-type EPR-1 cDNA, or the indicated EPR-1-ICAM-1 chimeras were incubated with increasing concentrations (0.2-2 µg/ml) of I-factor Xa in the presence of 2.5 mM CaCl(2) for 15 min at 22 °C. Nonspecific binding was quantitated in the presence of a 50-fold molar excess of unlabeled factor Xa added at the start of the incubation and was subtracted from the total to calculate net specific binding. Data are the means ± S.E. of at least three independent experiments. WT, wild-type.



The role of chimeric EPR-1 sequences in thrombin formation was investigated. In agreement with previous observations(6) , wild-type EPR-1 transfectants promoted prothrombin activation in a factor Xa concentration-dependent manner, with the generation of 0.55 ± 0.06 units of thrombin/ml for saturating concentrations of factor Xa of 1 µg/ml (Fig. 4). Consistent with their inability to bind I-factor Xa (Fig. 3), control CHO cells transfected with pcDNA3 vector alone did not significantly promote prothrombin activation at comparable concentrations of factor Xa added (Fig. 4). Under these experimental conditions, A1 chimeric transfectants generated increasing amounts of thrombin in a factor Xa concentration-dependent reaction quantitatively indistinguishable from the response observed with wild-type EPR-1 transfectants (Fig. 4). In contrast, G3 transfectants did not generate thrombin under the same experimental conditions (data not shown).


Figure 4: Prothrombin activation by EPR-1 chimeras. CHO cells transfected with control vector pcDNA3 or EPR-1 constructs were incubated with the indicated increasing concentrations of factor Xa in the presence of 20 µg/ml prothrombin and 1.2 mM CaCl(2) for 5 min at 22 °C. Thrombin generation under the various conditions tested was quantitated by hydrolysis of the thrombin-sensitive chromogenic substrate S-2238 at a 405-nm wavelength and converted to units of thrombin/milliliter using a standard curve constructed with serial increasing concentrations (0.015-2 units/ml) of bovine thrombin. Data are the means ± S.E. of three independent experiments. WT, wild-type.



Identification of Critical Residues Implicated in EPR-1-Factor Xa Interaction

Molecular dissection of the A1 sequence and assignment of critical residues implicated in factor Xa recognition were carried out in site-directed mutagenesis experiments. CHO transfectants expressing six variant forms of the A1 chimera carrying the following point mutations, Lys Met, Gly Ala, Asn Ser, Asn Ile, Lys Ile, and Asn Ser, were separately tested for their ability to bind I-factor Xa. Mutagenesis of Lys, Gly, Asn, or Asn in the A1 sequence did not diminish the ability of the transfected cells to specifically bind I-factor Xa as compared with wild-type A1 transfectants (Fig. 5A). In contrast, I-factor Xa binding to A1 chimeric transfectants carrying an Asn Ile substitution or a Lys Ile substitution was decreased by 80 or 96%, respectively (Fig. 5A). Tentative prediction of secondary structure using the Chou-Fasman algorithm suggested that the Lys Ile mutation, which nearly completely abolished EPR-1-factor Xa interaction (Fig. 5A), caused a profound structural rearrangement of the A1 interacting sequence, with disruption of a discrete reverse turn structure, as compared with the predicted conformation of wild-type EPR-1 (Fig. 5B). The inhibitory Asn Ile mutation was apparently associated with a less profound structural change in the same reverse turn structure, while none of the other A1 mutations affected the conformation of the EPR-1 ligand-binding domain (data not shown).


Figure 5: Assignment of A1 critical residues implicated in factor Xa binding. A, CHO cells transfected with wild-type (WT) EPR-1 cDNA or the indicated single-residue mutagenized A1 chimeras (Pro-Ala) were incubated with 0.5 µg/ml I-factor Xa in the presence of 2.5 mM CaCl(2) for 15 min at 22 °C before quantitation of specific binding as described for Fig. 3. Data are the means ± S.E. of three independent experiments. B, shown is the secondary structure prediction of the wild-type sequence and the Lys Ile A1 mutant. Secondary structure prediction of the wild-type EPR-1 sequence Pro-Ala (solid line) and the mutant Lys Ile (broken line) was generated with the algorithm of Chou-Fasman using the Plotstructure program in a Genetics Computer Group software package. Hydrophilicity and hydrophobicity according to Kyte-Doolittle (KD) are indicated.



Participation of A1 Sequences in HUVEC Thrombin Formation

The potential contribution of EPR-1 sequences in vascular cell thrombin generation was investigated by testing the ability of two partially overlapping synthetic peptides duplicating the EPR-1 sequence Pro-Ala to decrease prothrombin activation on HUVEC monolayers in the absence of exogenous factor V/Va. Preincubation of factor Xa with increasing concentrations of the AG1 peptide containing the amino-terminal A1 sequence SPGKPGNQNSKNEPP completely inhibited prothrombin activation on HUVEC monolayers in a dose-dependent manner, with IC 1 µM (Fig. 6). Consistent with the crucial role of Asn and Lys in factor Xa recognition (Fig. 5), comparable concentrations of the AG2 peptide, duplicating the carboxyl-terminal A1 sequence PPKKRERERSSHCYP, did not diminish prothrombin activation on HUVEC monolayers under the same experimental conditions (Fig. 6).


Figure 6: Effect of A1-derived peptides on HUVEC prothrombin activation. The experimental conditions are essentially as described for Fig. 4, except that factor Xa was preincubated with the indicated increasing concentrations of the partially overlapping A1-derived synthetic peptide AG1 (SPGKPGNQNSKNEPP) or AG2 (PPKKRERERSSHCYP) for 5 min at 22 °C before addition of each incubation mixture to HUVEC monolayers and determination of thrombin formation by S-2238 hydrolysis as described for Fig. 4. Thrombin generation by HUVEC monolayers in the absence of peptide was 0.33 ± 0.03 units/ml. Data are the means ± S.E. of three independent experiments.




DISCUSSION

In this study, we have identified the ligand-binding site and functional epitope requirements of a novel vascular cell receptor for the clotting protease factor Xa, designated EPR-1(6) . Cellular receptors for coagulation and fibrinolytic proteases have recently emerged as a novel class of modulators of the hemostatic balance as well as potent signal-transducing molecules in vascular and nonvascular cell types. In addition to the pleiotropic cellular functions mediated by thrombin (18) and transduced by a G-protein-coupled seven-transmembrane domain receptor(3) , binding of urokinase to its receptor (4) has been implicated in the modulation of macrophage gene expression(19) , lymphocyte activation(20) , intercellular adhesion (21) , and monocyte chemotaxis(22) . Furthermore, the association of the anticoagulant protein S with the recently characterized Tyro-3 tyrosine kinase receptor (5) has been implicated in signal transduction and vascular smooth muscle cell proliferation(5, 9) . As a fourth vascular cell protease receptor for factor Xa(6) , EPR-1 participates in thrombin formation (12) and generation of intermediate products of factor IX activation (14) as well as in lymphocyte activation mechanisms(8) .

The first functional EPR-1 domain identified here comprised the amino-terminal sequence Met^1-Arg and contained the epitope of the second generation of anti-EPR-1 mAbs, i.e. mAb 2E1(6) . As judged from the partial overlap between this region and the sequence of a mAb 2E1-immunoreactive cDNA clone isolated from a phage T cell expression library(6) , the minimal epitope recognized by this class of anti-EPR-1 mAbs is predicted to be included between Ala and Arg. Characterized by high hydrophilicity and antigenic index by Kyte-Doolittle analysis, this region is predicted by Chou-Fasman and Robson-Garnier algorithms to be structurally organized in two reverse turn structures, localized in a highly surface-exposed loop in the molecule. The importance of the mAb 2E1 epitope in EPR-1 is underscored by the profound immunosuppressive properties of this mAb, in vitro and in vivo. In these experiments and consistent with a more general role of EPR-1 and other protease receptors in lymphocyte activation mechanisms(8) , mAb 2E1 completely inhibited lymphocyte proliferation (IC 0.1 µg/ml), suppressed cytokine release, and blocked immunoglobulin production and B cell lymphomagenesis in a human severe combined immunodeficiency mouse model.^3 The identification of this immunoregulatory epitope on EPR-1 may facilitate the rational design of advanced derivatives of mAb 2E1 or of synthetic peptidyl antagonists targeted at manipulating the immune response in vivo.

The second functional domain of EPR-1 identified here corresponded to the sequence Pro-Arg and contained a receptor-binding site for factor Xa. As judged by I-factor Xa binding parameters of Pro-Arg chimeric transfectants versus wild-type EPR-1 transfectants, most, if not all, of the EPR-1-factor Xa interaction can be recapitulated by this recognition sequence. Consistent with this scheme, Pro-Arg transfectants mediated prothrombin activation in a factor Xa concentration-dependent manner and were recognized by the first generation of anti-EPR-1 mAbs B6 and 9D4, previously characterized for their ability to block factor Xa association with leukocytes (12) or endothelium.^1 The specificity of this recognition was also independently substantiated by the inability of other expressed extracellular regions in EPR-1, i.e. Ala-Glu, to bind factor Xa, to participate in prothrombin activation, or to associate with the functionally inhibitory mAb 9D4 or B6. Molecular dissection of the EPR-1 ligand binding sequence Pro-Arg by single-amino acid mutagenesis identified Asn and Lys as crucial residues involved in factor Xa recognition. Interestingly, the Lys Ile substitution, which completely abolished EPR-1-factor Xa interaction, was apparently associated with a profound structural change in this interacting domain, with disruption of a discrete reverse turn structure, as tentatively predicted by the Chou-Fasman algorithm of wild-type versus mutated EPR-1 sequences. Altogether, these data suggest that EPR-1-factor Xa interaction depends on the integrity of a discrete ligand-binding groove. As compared with the recognition of other protease receptors, this model is reminiscent of the ligand binding requirements of the urokinase receptor (23) as opposed to the proteolytic pathway of receptor activation mediated by thrombin (3) .

The identification of the EPR-1 sequence Pro-Arg as a novel cellular binding site for factor Xa may have important pathophysiological implications for vascular cell assembly of proteolytic activity and thrombin formation. Consistent with the expression and function of EPR-1 on endothelium,^1 a synthetic peptide duplicating the interacting motif SPGKPGNQNSKNEPP dose-dependently inhibited thrombin formation on these cells in the absence of factor V/Va. That prothrombin activation on HUVEC could not be solely mediated by released factor V/Va (24) was suggested earlier by the absence of factor V/Va on normal unperturbed endothelium (25) and/or by the lack of factor V transcript in these cells(26) . Moreover, cellular receptors for factor Xa, distinct from factor V/Va, were identified on HUVEC (27, 28) and implicated in ligand processing (29) and intracellular signal transduction with release of endothelial cell mitogens(30) . The data of synthetic peptidyl mimicry reported here suggest that under experimental conditions of unperturbed endothelium (25) and in the absence of detectable membrane expression of factor V/Va,^1 most of the prothrombin activation potential of these cells may be recapitulated by the EPR-1 recognition of factor Xa and competitively inhibited by antagonists of the Pro-Arg sequence, i.e. the AG1 peptide. This leads to the speculation that, although considerably less catalytically efficient than the prothrombinase complex coordinated by factor V/Va(31) , the EPR-1-factor Xa interaction may function as a low-level thrombin-generating system in the vasculature, thus providing an essential ``first signal'' for clotting(1) , thrombomodulin-dependent anticoagulation pathways(32) , or protease-dependent vascular cell signal transduction pathways.

In summary, we have identified two spatially distinct structural domains in the factor Xa receptor (EPR-1) that are separately committed to the dual function of this receptor in the modulation of leukocyte activation (8) and factor Xa assembly on vascular cells. Elucidation of the complementary EPR-1 interacting sequence(s) in factor Xa will provide new insights into the potential pathophysiological role of this pathway in protease-dependent hemostatic and vascular cell signaling processes.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants RO1 HL54131 and HL43773. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Performed this work during the tenure of an American Heart Established Investigatorship award. To whom correspondence should be addressed: Boyer Center for Molecular Medicine, Rm. 436, Dept. of Pathology, Yale University School of Medicine, 295 Congress Ave., New Haven, CT 06536. Tel.: 203-737-2869; Fax: 203-737-2290.

(^1)
D. P. Hajjar, D. C. Altieri, B. Summers, A. Nicholson, W. Ruf, T. S. Edgington, and R. L. Nachman, submitted for publication.

(^2)
The abbreviations used are: EPR-1, effector cell protease receptor-1; ICAM-1, intercellular adhesion molecule-1; PCR, polymerase chain reaction; bp, base pairs; CHO, Chinese hamster ovary; mAb monoclonal antibody; HUVEC, human umbilical vein endothelial cell(s).

(^3)
M. A. Duchosal, A. L. Rothermel, P. McConahey, F. J. Dixon, and D. C. Altieri, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. A. Y. Jan for computer analysis.


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