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Topology of the Stable Serpin-Protease Complexes Revealed by an Autoantibody That Fails to React with the Monomeric Conformers of Antithrombin

1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês

10.1074/jbc.274.8.4586

1083-351X

Véronique Picard, Pierre-Emmanuel Marque, F Paolucci, Martine Aiach, Bernard Le Bonniec,

Blood Coagulation and Thrombosis Mechanisms

Solving the structure of the stable complex between a serine protease inhibitor (serpin) and its target has been a long standing goal. We describe herein the characterization of a monoclonal antibody that selectively recognizes antithrombin in complex with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P14 to P9 of the serpin's reactive center loop (RCL, ultimately cleaved between the P1 and P′1 residues). Accordingly, this antibody reacts with none of the monomeric conformers of antithrombin (native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge stabilizing system has been swapped for alanine). The neoepitope encompasses the motif DAFHK, located in native antithrombin on strand 4 of β-sheet A, which becomes strand 5 of β-sheet A in the RCL-cleaved and latent conformers. The inferences on the structure of the antithrombin-protease stable complex are that either a major remodeling of antithrombin accompanies the final elaboration of the complex or that, within the complex, at the most residues P14 to P6 of the RCL are inserted into β-sheet A. These conclusions limit drastically the possible locations of the defeated protease within the complex. Solving the structure of the stable complex between a serine protease inhibitor (serpin) and its target has been a long standing goal. We describe herein the characterization of a monoclonal antibody that selectively recognizes antithrombin in complex with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P14 to P9 of the serpin's reactive center loop (RCL, ultimately cleaved between the P1 and P′1 residues). Accordingly, this antibody reacts with none of the monomeric conformers of antithrombin (native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge stabilizing system has been swapped for alanine). The neoepitope encompasses the motif DAFHK, located in native antithrombin on strand 4 of β-sheet A, which becomes strand 5 of β-sheet A in the RCL-cleaved and latent conformers. The inferences on the structure of the antithrombin-protease stable complex are that either a major remodeling of antithrombin accompanies the final elaboration of the complex or that, within the complex, at the most residues P14 to P6 of the RCL are inserted into β-sheet A. These conclusions limit drastically the possible locations of the defeated protease within the complex. Serine protease inhibitors (serpins) 1The abbreviations serpinserine protease inhibitorATantithrombin (previously called antithrombin III)RCLreactive center loop (also called reactive site loop)BSAbovine serum albuminPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assay 1The abbreviations serpinserine protease inhibitorATantithrombin (previously called antithrombin III)RCLreactive center loop (also called reactive site loop)BSAbovine serum albuminPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assayare mainly composed of three β-sheets (A, B, and C) united by nine α-helices (A–I); indeed, many are inhibitors that neutralize their target(s) by forming a, stoichiometric, stable complex (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (828) Google Scholar, 2Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar, 3Engh R.A. Huber R. Bode W. Schulze A.J. Trends Biotechnol. 1995; 13: 503-510Abstract Full Text PDF PubMed Scopus (73) Google Scholar). Formation of the stable complex involves the charge stabilizing system of the target protease (4Lawrence D.A. Olson S.T. Palaniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: 27657-27662Abstract Full Text PDF PubMed Google Scholar, 5Schulze A.J. Huber R. Bode W. Engh R.A. FEBS Lett. 1994; 344: 117-124Crossref PubMed Scopus (56) Google Scholar, 6Olson S.T. Bock P.E. Kvassman J. Shore J.D. Lawrence D.A. Ginsburg D. Björk I. J. Biol. Chem. 1995; 270: 30007-30017Crossref PubMed Scopus (88) Google Scholar, 7Fa M. Karolin J. Aleshkov S. Strandberg L. Johansson L.B.-Å. Ny T. Biochemistry. 1995; 34: 13833-13840Crossref PubMed Scopus (69) Google Scholar, 8Stone S.R. Le Bonniec B.F. J. Mol. Biol. 1997; 265: 344-362Crossref PubMed Scopus (51) Google Scholar) and a surface loop of the inhibitor called the reactive center loop (RCL). The RCL connects strand 4 of β-sheet A to strand 1 of β-sheet C; it is exposed to solvent in the inhibitory serpins. By analogy with protease substrates, the 20 amino acids constituting the RCL are numbered Pn- … -P1-P′1- … -P′n, where P1-P′1 is ultimately cleaved. The mechanism of protease inhibition involves multiple steps that initiate by the formation of a reversible association, converting to a stable complex, ultimately split into regenerated enzyme and RCL-cleaved (consumed) serpin (9Patston P.A. Gettins P. Beechem J. Schapira M. Biochemistry. 1991; 30: 8876-8882Crossref PubMed Scopus (171) Google Scholar, 10Stone S.R. Hermans J.M. Biochemistry. 1995; 34: 5164-5172Crossref PubMed Scopus (41) Google Scholar, 11Hopkins P.C.R. Chang W.-S.W. Wardell M.R. Stone S.R. J. Biol. Chem. 1997; 272: 3905-3909Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 12O'Malley K.M. Nair S.A. Rubin H. Cooperman B.S. J. Biol. Chem. 1997; 272: 5354-5359Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The RCL sustains a variety of conformations (13Loebermann H. Tukuoka R. Deisenhofer J. Huber R. J. Mol. Biol. 1984; 177: 531-557Crossref PubMed Scopus (603) Google Scholar, 14Baumann U. Huber R. Bode W. Grosse D. Lesjak M. Laurell C.B. J. Mol. Biol. 1991; 218: 595-606Crossref PubMed Scopus (156) Google Scholar, 15Baumann U. Bode W. Huber R. J. Mol. Biol. 1992; 226: 1207-1218Crossref PubMed Scopus (64) Google Scholar, 16Mottonen J. Strand A. Symersky J. Sweet R.M. Danley D.E. Geoghegan K.F. Gerard R.D. Goldsmith E.J. Nature. 1992; 355: 270-273Crossref PubMed Scopus (520) Google Scholar). In antithrombin (AT) that is heparin-activated (17Huntington J.A. Olson S.T. Fan B. Gettins P.G.W. Biochemistry. 1996; 35: 8495-8503Crossref PubMed Scopus (126) Google Scholar, 18Jin L. Abrahams J.P. Skinner R. Petitou M. Pike R.N. Carrell R.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14683-14688Crossref PubMed Scopus (629) Google Scholar) and other inhibitory serpins such as α1-antitrypsin (also called α1-proteinase inhibitor; 19–21) or α1-antichymotrypsin (22Wei A. Rubin H. Cooperman B.S. Christianson D.W. Nat. Struct. Biol. 1994; 1: 251-258Crossref PubMed Scopus (165) Google Scholar), the RCL is wholly exposed, whereas in the AT monomer, residue P14 of the RCL disrupts β-sheet A (23Schreuder H.A. de Boer B. Dijkema R. Mulders J. Theunissen H.J.M. Grootenhuis P.D.J. Hol W.G. Nat. Struct. Biol. 1994; 1: 48-54Crossref PubMed Scopus (267) Google Scholar, 24Carrell R.W. Stein P.E. Fermi G. Wardell M.R. Structure. 1994; 2: 257-270Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 25Skinner R. Abrahams J.-P. Whisstock J.C. Lesk A.M. Carrell R.W. Wardell M.R. J. Mol. Biol. 1997; 266: 601-609Crossref PubMed Scopus (187) Google Scholar). In latent AT, an intact but non-inhibitory conformer (25Skinner R. Abrahams J.-P. Whisstock J.C. Lesk A.M. Carrell R.W. Wardell M.R. J. Mol. Biol. 1997; 266: 601-609Crossref PubMed Scopus (187) Google Scholar), and in latent type-1 plasminogen activator inhibitor (16Mottonen J. Strand A. Symersky J. Sweet R.M. Danley D.E. Geoghegan K.F. Gerard R.D. Goldsmith E.J. Nature. 1992; 355: 270-273Crossref PubMed Scopus (520) Google Scholar), residues P14 to P3 of the RCL are completely inserted into β-sheet A, constituting an additional, sixth, strand. The same conversion from a five- to six-stranded β-sheet A occurs in the inhibitory serpins, following cleavage of the RCL (14Baumann U. Huber R. Bode W. Grosse D. Lesjak M. Laurell C.B. J. Mol. Biol. 1991; 218: 595-606Crossref PubMed Scopus (156) Google Scholar, 26Mourey L. Samama J.P. Delarue M. Petitou M. Choay J. Moras D. J. Mol. Biol. 1993; 232: 223-241Crossref PubMed Scopus (96) Google Scholar). serine protease inhibitor antithrombin (previously called antithrombin III) reactive center loop (also called reactive site loop) bovine serum albumin phosphate-buffered saline enzyme-linked immunosorbent assay serine protease inhibitor antithrombin (previously called antithrombin III) reactive center loop (also called reactive site loop) bovine serum albumin phosphate-buffered saline enzyme-linked immunosorbent assay To date, no x-ray analysis of a serpin-protease complex has been reported; thus, its structure remains largely hypothetical. Based on functional studies of serpin variants and immunochemical investigations, a number of reports nevertheless suggest that, in the stable complex, the RCL is inserted into β-sheet A. Antibodies have been characterized that fail to react with native serpin, but recognize binary complexes with a synthetic tetradecapeptide corresponding to residues P14 to P1 of the RCL, as well as consumed inhibitors (27Skriver K. Wikoff W.R. Patston P.A. Tausk F. Schapira M. Kaplan A.P. Bock S.C. J. Biol. Chem. 1991; 266: 9216-9221Abstract Full Text PDF PubMed Google Scholar, 28Schulze A.J. Baumann U. Knof S. Jaeger E. Huber R. Laurell C.-B. Eur. J. Biochem. 1990; 194: 51-56Crossref PubMed Scopus (174) Google Scholar, 29Schulze A.J. Fronhert P.W. Engh R.A. Huber R. Biochemistry. 1992; 31: 7560-7565Crossref PubMed Scopus (72) Google Scholar, 30Björk I. Nordling K. Larsson I. Olson S.T. J. Biol. Chem. 1992; 267: 19047-19050Abstract Full Text PDF PubMed Google Scholar, 31Björk I. Ylinenjärvi K. Olson S.T. Bock P.E. J. Biol. Chem. 1992; 267: 1976-1982Abstract Full Text PDF PubMed Google Scholar, 32Debrock S. Declerck P.J. FEBS Lett. 1995; 376: 243-246Crossref PubMed Scopus (31) Google Scholar, 33Nordling K. Björk I. Biochemistry. 1996; 35: 10436-10440Crossref PubMed Scopus (6) Google Scholar). Thus, antibodies revealed that insertion of the RCL in β-sheet A exposes neoepitopes that are not present in the intact inhibitor. The same neoepitopes being exposed in stable serpin-protease complexes led to the conclusion that, during trapping of the protease, the RCL inserts at least in part into β-sheet A. Convincing evidence also suggests that the protease translocates away from the site at which initial attack occurs. Wright and Scarsdale (34Wright H.T. Scarsdale J.N. Proteins: Struct. Funct. Genet. 1995; 22: 210-225Crossref PubMed Scopus (156) Google Scholar) even proposed that the enzyme ends in a location almost opposite to that of the RCL in intact serpins, i.e. that, in the stable serpin-protease complexes, β-sheet A is a six-stranded sheet. Stratikos and Gettins (35Stratikos E. Gettins P.G.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 453-458Crossref PubMed Scopus (137) Google Scholar) demonstrated that, at least 21 Å separates position of the protease in the initial (reversible) Michaelis complex from that in the (virtually irreversible) final complex. Modeling considerations suggest a range of plausible stable structures: from full insertion of the RCL with upside-down translocation of the defeated protease, to limited RCL insertion (up to the P9residue) with concomitant stacking of the target on the F-helix. Following cross linking experiments with stable complexes of type-1 plasminogen activator inhibitor, Wilczynska et al. (36Wilczynska M. Fa M. Karolin J. Ohlsson P.-I. Johansson L.B.-Å. Ny T. Nat. Struct. Biol. 1997; 4: 354-357Crossref PubMed Scopus (137) Google Scholar) favored stacking on the F-helix, and partial rather than complete insertion of the cleaved RCL. We report herein characterization of a monoclonal antibody (12A5) that provides definite insight into the structure of the stable AT-protease complex; unless AT was unfolded, the antibody failed to react with any AT monomers, while endorsing stable complexes with thrombin or factor Xa and binary complex with a short peptide derived from the RCL. The unexpected location of the neoepitope, on the distal part of β-sheet A, has several fundamental implications. The conformations of β-sheet A and/or of the F-helix in the stable complex must simultaneously differ from the five- and six-stranded structures of native and RCL-cleaved AT, respectively. Steric hindrance considerations also limit drastically the possible topologies of the complex. Human thrombin and its S195A variant were prepared as described previously (8Stone S.R. Le Bonniec B.F. J. Mol. Biol. 1997; 265: 344-362Crossref PubMed Scopus (51) Google Scholar). Factor Xa was purchased from ERL (South Bend, IL). Porcine pancreatic elastase (type IV) and bovine serum albumin (BSA, protease-free) were from Sigma (St-Quentin Fallavier, France), as well as mouse AT and bovine, sheep, porcine, rabbit, and chicken plasma. Standard, unfractionated heparin (Heparin Choay) and pentasaccharide with high affinity for AT (M, 1714) were from Sanofi-Winthrop (Gentilly, France). Peptide P14-P9 derived from the RCL of AT (Ac-SEAAAS) was synthesized by Altergen (Schiltigheim, France). Human AT was purified from citrated frozen plasma essentially according to McKay (37McKay E.J. Thromb. Res. 1981; 21: 375-382Abstract Full Text PDF PubMed Scopus (80) Google Scholar) by affinity chromatography on heparin-Sepharose (Pharmacia, St-Quentin-en-Yvelines, France), followed by anion-exchange chromatography on a Mono-Q column (Pharmacia). AT cleaved by porcine pancreatic elastase was prepared by incubating AT (5 μm) with 50 nm enzyme for 4 h at 37 °C in 50 mm Tris-HCl, pH 8.5, containing 0.15m NaCl and 0.1% polyethylene glycol (M r 8000; w/v). The resulting C-terminal fragment was isolated by transfer onto polyvinylidene difluoride membrane after denaturing polyacrylamide gel electrophoresis (15% acrylamide), and analyzed by N-terminal sequencing (Biotechnology Department, Institut Pasteur, Paris, France). Two sites of cleavage were identified: Val389-Ile390 and Ile390-Ala391, corresponding to the P5-P4 and P4-P3residues of the RCL, respectively. Identical cleavages are obtained following incubation of AT with human neutrophil elastase (38Gettins P. Harten B. Biochemistry. 1988; 27: 3634-3639Crossref PubMed Scopus (51) Google Scholar). Thrombin-cleaved AT was prepared by incubating AT (7 μm) with thrombin (0.1 μm) for 3 h at 37 °C in 50 mm Tris-HCl, pH 7.5, containing 0.15 m NaCl and trace amounts of SDS (0.02%, w/v; Refs. 39Urano T. Strandberg L. Johansson L.B.-Å. Ny T. Eur. J. Biochem. 1992; 209: 985-992Crossref PubMed Scopus (101) Google Scholar and 40Munch M. Heegaard C.W. Andreasen P.A. Biochim. Biophys. Acta. 1993; 1202: 29-37Crossref PubMed Scopus (97) Google Scholar). Thrombin was neutralized by the addition of 1 mm phenylmethylsulfonyl fluoride (Sigma). Traces of SDS and excess phenylmethylsulfonyl fluoride were removed by extensive dialysis against 50 mmTris-HCl, pH 7.5, containing 0.15 m NaCl. Denaturing polyacrylamide gel electrophoresis (Fig. 1) suggested that in these conditions AT was fully cleaved by thrombin, at a single site. In addition to the N terminus of AT, N-terminal sequencing of the reaction mixture revealed a single sequence starting with the P′1residue of the RCL, indicating that thrombin cleaved a single bond in AT, between the P1 and P′1 residues of the RCL. AT cleaved by porcine pancreatic elastase or by thrombin were indistinguishable by polyacrylamide gel electrophoresis analysis (Fig.1) and had a similar affinity for heparin (i.e. reduced compared with native AT); both eluted at about 0.3 m NaCl on heparin-Sepharose column. Latent AT was prepared according to Wardell et al. (41Wardell M.R. Chang W.-S.W. Bruce D. Skinner R. Lesk A.M. Carrell R.W. Biochemistry. 1997; 36: 13133-13142Crossref PubMed Scopus (76) Google Scholar). Briefly, AT (1.7 μm) was incubated for 18 h at 60 °C in 10 mm Tris-HCl, pH 7.5, containing 0.35 m sodium citrate. Following incubation, the mixture was dialyzed against 10 mmTris-HCl, pH 7.5, and latent AT was recovered by heparin-Sepharose chromatography and anion-exchange chromatography on Mono Q column, both developed with a linear gradient from 0.0 to 0.5 m NaCl; latent AT eluted at 0.3 and 0.2 m NaCl, respectively. The concentration of all AT monomers was estimated assuming an absorption coefficient of 3.8 × 104m−1cm−1 at 280 nm (ε%=6.5 cm−1). Stable complexes of AT with thrombin or factor Xa were prepared by incubating AT (5 μm) for 2 h at room temperature with thrombin (5 μm) or for 4 h with factor Xa (5 μm) in 50 mm Tris-HCl, pH 7.5, containing 0.15 m NaCl and 0.1% polyethylene glycol (M r 8000; w/v). Virtually no monomer of AT (either native or RCL-cleaved) could be seen by polyacrylamide gel electrophoresis analysis in the presence of SDS (Fig. 1); therefore, concentrations of the complexes were assumed to be that of the added AT (5 μm). Reversible complex of AT with S195A thrombin was prepared by mixing AT (2 μm) with S195A thrombin (2 μm) in the presence of heparin (1 unit/ml); association was presumed to be almost instantaneous (8Stone S.R. Le Bonniec B.F. J. Mol. Biol. 1997; 265: 344-362Crossref PubMed Scopus (51) Google Scholar), and concentration of the reversible complex was assumed to be that of the added AT. Formation of a binary complex with the P14-P9 peptide derived from the RCL was carried out according to Schulze et al. (28Schulze A.J. Baumann U. Knof S. Jaeger E. Huber R. Laurell C.-B. Eur. J. Biochem. 1990; 194: 51-56Crossref PubMed Scopus (174) Google Scholar) and Chang et al. (42Chang W.-S.W. Whisstock J. Hopkins P.C.R. Lesk A.M. Carrell R.W. Wardell M.R. Protein Sci. 1997; 6: 89-98Crossref PubMed Scopus (72) Google Scholar), by incubation of AT (20 μm) and peptide (2 mm) in 50 mmTris, pH 8.0, containing 50 mm NaCl. Binary complex was used immediately after a 60-min incubation at 37 °C: when less than 50% AT formed a complex and no polymerization of AT had yet occurred (Fig. 1). Concentration of the binary complex was assumed to be 40% that of the total amount of AT. Murine monoclonal antibodies directed against human AT were selected, isolated and characterized by standard procedures (43Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Plainview, NY1988: 139-282Google Scholar). Four 6-week-old BALB/c females were immunized by subcutaneous injection of 20 μg of AT in complete Freund's adjuvant followed 3 weeks later by a further subcutaneous injection of 20 μg of AT in incomplete Freund's adjuvant. One hundred days later, mouse received 8 μg of AT subcutaneously and 12 μg of AT intravenously. One day prior to the AT injection, and for 5 days, mice also received a daily intraperitoneal injection of 20 μg of pentasaccharide. Three days after the final injection, spleen cells were fused with the mouse myeloma cell line P3-X63-Ag.8.653 by using 50% polyethylene glycol 1540, and cultured in hypoxanthine/aminopterin/thymidine media. Positive clones were detected by enzyme-linked immunosorbent assay (ELISA); Maxisorp microplates from Nunc (Polylabo, Strasbourg, France) were coated overnight at 4 °C with 5 μg/ml AT in 0.1 mNa2HCO3/NaH2CO3, pH 9.5, and washed three times in 20 mmNa2HPO4/NaH2PO4, pH 7.4, containing 0.15 m NaCl (PBS) and 0.5% Tween 20 (v/v). Residual sites were blocked for 1 h with 1 mg/ml BSA in PBS, and microplates were washed as above. Each hybridoma supernatant to be tested was added to one of the wells of the microplate and allowed to bind for 1 h at room temperature. After washing, a peroxidase-conjugated goat anti-mouse antibody (Bio-Rad, Ivry-sur-Seine, France) was added and allowed to bind for 1 h at room temperature. Following a final wash, ELISA was developed by adding 150 μl of orthophenylenediamine (0.5 mg/ml; Bio-Rad) in 0.1m sodium citrate, pH 5.5, containing 0.3% H2O2 (v/v). The reaction was stopped after 30 min by the addition of 50 μl of H2SO4(12.5%, v/v), and the absorbance at 490 nm was recorded. Selected clones were grown in pristane-primed BALB/c mice, and antibodies purified from the ascitic fluid by affinity chromatography on protein A-Sepharose (Pharmacia). IgG appeared pure by denaturing polyacrylamide gel electrophoresis analysis and were stored at −80 °C after dialysis against PBS. The subclass was determined with a commercial kit (Amersham, Les Ulis, France): monoclonal 12A5 described in this study was typed as IgG2a. To identify the conformers of AT recognized by the monoclonal antibodies, a sandwich ELISA was designed in which microplates were coated with purified IgG (5 μg/ml). Each AT conformer, diluted in PBS containing 0.5% Tween 20 (v/v) and 1 mg/ml BSA, was added to one of the wells of the microplate and allowed to bind for 2 h at room temperature. Sandwich ELISA was developed with a peroxidase-conjugated goat IgG directed against human AT (Sanofi Research, Montpellier, France). In assays with S195A-AT reversible complexes, 1 unit/ml heparin was present during the binding step, and all buffers included 0.1 unit/ml heparin to prevent release throughout the washing steps. Ability of the S195A-AT complexes to be retained on a microtiter plate was assessed by the use of a commercial kit designed to detect plasma thrombin-AT complexes (Enzygnost TAT micro; Behring, Marburg, Germany). A microplate, coated with a polyclonal antibody directed against thrombin was incubated with various amounts of S195A-AT complexes and developed with a polyclonal antibody directed against AT, according to the manufacturer's instructions except that 0.1 unit/ml heparin was added in all buffers. Customized AT peptide sets were prepared by Chiron Mimotopes Peptide Systems (Clayton, Victoria, Australia) as biotinylated peptides linked to a cleavable polyethylene pin (44Geysen H.M. Rodda S.J. Mason T.J. Tribbick G. Schoofs P.G. J. Immunol. Methods. 1987; 102: 259-274Crossref PubMed Scopus (718) Google Scholar). A four-amino acid spacer (GSGS) separated the N terminus of each peptide from the pin, except for the peptide encompassing the first 14 amino acids of AT where the spacer was coupled to the C terminus. Binding assays were performed according to the supplier's instructions. Briefly, 100 μl of streptavidin (5 μg/ml in water) was added to each well of a microplate and evaporated to dryness at 37 °C. Biotinylated peptides (about 1 μm in PBS containing 1 mg/ml BSA) were added and incubated for 1 h at room temperature. Monoclonal antibody (0.1 μg/ml) in PBS containing 0.5% Tween 20 (v/v) and 1 mg/ml BSA was incubated for 2 h at room temperature. Washing and development were otherwise performed as described above for the ELISA, using the peroxidase-conjugated goat anti-mouse IgG. Immunoblotting of the various AT conformers (after polyacrylamide gel electrophoresis in denaturing conditions) was performed on nitrocellulose membrane (Bio-Rad) essentially as described previously (45Picard V. Ersdal-Badju E. Bock S.C. Biochemistry. 1995; 34: 8433-8440Crossref PubMed Scopus (80) Google Scholar). Incubation and washing were all completed at room temperature in 50 mm Tris-HCl, pH 8.0, containing 0.15m NaCl and 0.5% Tween 20 (v/v). The membrane was saturated with nonfat dry milk (3% w/v), washed, and incubated for 1 h with monoclonal antibody 12A5 (0.5 μg/ml). After washing, the membrane was incubated for 1 h with an alkaline phosphatase-conjugated goat anti-mouse IgG (Bio-Rad). The immunoblot was further washed, and developed by adding a phosphatase substrate solution consisting of 0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphate and 0.3 mg/ml nitro blue tetrazolium in 0.1 m Tris, pH 9.5, containing 0.5 mm MgCl2. The influence of monoclonal antibody 12A5 on thrombin and factor Xa inhibition by AT was studied in the absence and in the presence of heparin, essentially as described previously (46Le Bonniec B.F. Guinto E.R. Stone S.R. Biochemistry. 1995; 34: 12241-12248Crossref PubMed Scopus (48) Google Scholar, 47Djie M.Z. Le Bonniec B.F. Hopkins P.C.R. Hipler K. Stone S.R. Biochemistry. 1996; 35: 11461-11469Crossref PubMed Scopus (40) Google Scholar). Assays were performed at 37 °C in 50 mm Tris-HCl, pH 7.6, containing 0.15 m NaCl, and 0.1% polyethylene glycol (M r 8000; w/v); 1 mg/ml BSA was included in the assay of factor Xa inhibition. Without heparin, estimates of the association rate constant (k on), in the presence of 1 μm12A5 (or control IgG), were obtained from kinetic experiments performed in microplates, under pseudo first-order conditions (20–100 nm AT with 2 nm thrombin, and 150–300 nm AT with 15 nm factor Xa). First order rate constants were estimated by non-linear regression analysis of the residual activities versus time (up to 90 min), andk on values deduced from the linear plot of the first order rate constants as a function of AT. Thek on values in presence of heparin (1.4 units/ml), were estimated by analysis of data from progress-curve kinetics also completed in pseudo first-order conditions (0.2–0.6 nm AT with 10 pm thrombin, and 1–2 nm AT with 100 pm factor Xa). Inhibition of thrombin was initiated by its addition to a microtiter well containing 0.2 μm 12A5 (or control IgG), AT at various concentration, and 100 μmH-d-Phe-pipecolyl-Arg-p-nitroanilide (S-2238, Biogenic, Montpellier, France). Before initiating factor Xa inhibition, the release of p-nitroaniline from 400 μmbenzyl-CO-Ile-Glu-(γ-OR)-Gly-Arg-p-nitroanilide (S-2222, Biogenic) by factor Xa was monitored for 10–15 min, until a steady state velocity of about 0.2 μm min−1 was reached. The inhibition reaction was initiated by the addition of a mixture of 0.2 μm 12A5 (or control IgG) and AT at various concentrations. The release of p-nitroaniline was monitored for up to 3 h using a Lambda 14 Perkin Elmer spectrophotometer, but only data corresponding to less than 10% substrate hydrolysis were used in the analysis. Estimates of the k onvalues were obtained by fitting data to the equation for slow-binding inhibition and corrected for the competition introduced by the substrate. AT may adopt at least four conformations: native, heparin-activated, latent, and RCL-cleaved. A fifth conformation is likely to occur when AT is trapped in a complex with one of its targets (thrombin or factor Xa), but the precise structure of AT within this complex remains largely unknown. In an attempt to probe the elusive conformation of trapped AT, we prepared a panel of monoclonal antibodies; 12 were selected, because they recognized AT that had been coated on a microplate. To determine which conformer of AT was recognized, the monoclonal antibodies were coated onto a microplate, then AT (native, RCL-cleaved, or in complex with thrombin) was added, and sandwich ELISA developed with a polyclonal antibody directed against AT. Two monoclonal antibodies retained our attention, because they did not react with native or RCL-cleaved AT, while giving a strong signal with complexes; the other monoclonal antibodies reacted with all three forms of AT, indicating that selectivity of the former was not due to an experimental artifact. Thus, even though AT-protease complexes were not included in the immunization mixture, all the antibodies reacted with AT in complex. Although surprising at first, we reasoned that complexes might have formed with endogenous mouse proteases, and that a neoepitope inaccessible in the native protein might be exposed in partially denatured AT. To delineate more precisely the specificity of one of the monoclonal antibodies (12A5), we evaluated its affinities for native, latent, and heparin-activated AT, for AT with the RCL cleaved by thrombin or porcine pancreatic elastase, for AT in stable complex with thrombin or factor Xa, and for AT in complex with a peptide derived from the P14-P9 residues of its RCL. AT in complex with either thrombin or factor Xa bound to 12A5 in a dose-dependent fashion, as did the binary complex of AT with the P14-P9 peptide, although to a lesser extent, but binding of the other AT conformers remained undetected at concentrations as high as 1 μm (Fig.2). In contrast, 12A5 recognized all forms of AT following polyacrylamide gel electrophoresis in the presence of SDS and immunoblotting, even after reduction of the disulfide bridges with β-mercaptoethanol. Thus, a motif recognized by 12A5, inaccessible in native, heparin-activated, latent, and RCL-cleaved AT, was exposed in denatured and target-complexed AT. Taken together, these data suggested that steric hindrance, rather than a need for a specific conformation, limited the ability of 12A5 to interact with native or RCL-cleaved AT. As 12A5 recognized unfolded AT after reduction of the disulfide bonds, it was reasonable to expect that the neoepitope was a contiguous sequence of amino acids. We scanned two sets of biotinylated peptides, covering the entire sequence of AT. The first set simply divided AT sequence into 31 peptides that each were 14 amino acids in length, starting with His1 of AT; the second set divided AT sequence into 30 peptides (each also 14 amino acids in length) but starting with Cys8 of AT. Thus, each peptide overlapped 2 peptides of the alternate set by 7 amino acids (except peptides 1 and 31 of the first set). Peptides were individually linked to the well of a microtitration plate, and incubated with 12A5. ELISA was developed with a peroxidase-conjugated goat anti-mouse antibody. Only two peptides of the whole bank were able to bind 12A5: GRDDLYVSDAFHKA and SDAFHKAFLEVNEE, suggesting that the neoepitope included the shared motif SDAFHKA. Consistent with this hypothesis, 0.5 μmthrombin-AT displaced 50% of the binding of 10 nm 12A5 to peptide SDAFHKA coated on a microplate. Similarly, 0.2 μmpeptide SDAFHK displaced 50% of the binding of 2 nmthrombin-AT to 12A5 c