Eur J Biochem 270, 4059–4069 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03793.x Probing plasma clearance of the thrombin–antithrombin complex with a monoclonal antibody against the putative serpin–enzyme complex receptor-binding site George L Long1,*, Margareta Kjellberg2, Bruno O Villoutreix3 and Johan Stenflo2 Department of Biochemistry, University of Vermont, Burlington, VT, USA; 2Department of Clinical Chemistry, Lund University, University Hospital Malmoă, Malmoă, Sweden; 3INSERM U428, University of Paris V, France A high-affinity monoclonal antibody (M27), raised against the human thrombin–antithrombin complex, has been identified and characterized The epitope recognized by M27 was located to the linear sequence FIREVP (residues 411– 416), located in the C-terminal cleavage peptide of antithrombin This region overlaps, by two residues, the putative binding site of antithrombin for the serpin–enzyme complex receptor Studies in rats and with HepG2 cells in culture indicated that the Fab fragment of M27 does not block binding and uptake of the thrombin–antithrombin complex, suggesting that this region does not play a major role in the recognition and clearance of the thrombin–antithrombin complex M27 blocked the ability of antithrombin to inhibit thrombin as well as antithrombin cleavage, both in the presence and absence of heparin Antithrombin (AT), a member of the serine protease inhibitor family (serpin), is a 58-kDa molecular mass glycoprotein that circulates in human plasma at a concentration of  lM [1–5] AT modulates blood coagulation by inhibiting thrombin, active factor X (factor Xa) and active factor IX (factor IXa), and thereby prevents inappropriate clot formation and thrombosis The rate of AT-mediated inhibition of thrombin and factor Xa is increased several thousand-fold by binding of the sulfated polysaccharide heparin or heparin-like molecules Individuals with AT deficiency are at a significantly increased risk of venous thrombosis [6,7] AT and other serpins inhibit their serine protease cognates by the formation of a long-lived, covalent acyl intermediate upon specific protease cleavage [1–5] In AT, cleavage is at Arg393 in a so-called reactive center loop (RCL) with the formation of a C-terminal, 39 amino-acid residues long, disulfide-bonded (Cys247 to Cys430) peptide [8] Prompt insertion of residues P1–P17 of the RCL, with attached protease, as an additional strand into b-sheet A of the inhibitor, causes a dramatic conformational change in the serpin [9] Recent X-ray crystallographic diffraction analysis of the trypsin–antitrypsin covalent complex has shown that insertion of the RCL also leads to a critical distortion of the structure of the protease [10] As a result, the canonical active site Ser in position 195 is reoriented at a ˚ distance of more than A from His57, which is too far to form the critical hydrogen bond of the catalytic triad – a bond that is a prerequisite for cleavage of the acyl intermediate that links the protease to the serpin Moreover, the distortion of the complexed thrombin molecule renders it susceptible to proteolytic degradation A further consequence of the complexation-induced conformational change in the serpin is exposure of structure(s) that are recognized by serpin–enzyme complex (SEC) receptors on the surface of hepatocytes Receptor binding followed by endocytosis results in rapid clearance of the protease–serpin complexes from the circulatory system [11] In addition to native and complexed AT, two forms of AT have been characterized: a cleaved uncomplexed form with the RCL inserted into b-sheet A, and a so-called latent uncleaved, loop-inserted form The cleaved, loopinserted inhibitor is formed if loop insertion, with the acyl-linked protease, is not sufficiently rapid to compete with deacylation, which leads to release of the active protease Human elastase cleaves AT at Ile390 without complex formation [12] The elastase-cleaved form has properties that are indistinguishable from those of the thrombin-cleaved inhibitor The latent form is a conformational isomer of the native form in which the RCL has been inserted into b-sheet A without prior complex formation/cleavage Neither the cleaved uncomplexed nor the uncleaved latent form exhibit inhibitory activity Correspondence to J Stenflo, Department of Clinical Chemistry, Lund University, University Hospital, Malmo, S-205 02 Malmo, ă ă Sweden Fax: + 46 40 929023, Tel.: + 46 40 331421, E-mail: johan.stenflo@klkemi.mas.lu.se Abbreviations: AT, antithrombin; LRP, low-density lipoprotein receptor-related protein; PVDF, poly(vinylidene difluoride); RCL, reactive center loop; SEC, serpin–enzyme complex; SECR, serpin– enzyme complex receptor; T–AT, covalent thrombin-antithrombin complex *This work was carried out during the sabbatical of G L Long to the Department of Clinical Chemistry, Lund University, University Hospital Malmo, S-20502 Malmo, Sweden ¨ ¨ (Received July 2003, revised 30 July 2003, accepted 15 August 2003) Keywords: antithrombin; thrombin; thrombin–antithrombin complex; monoclonal antibody; serpin–enzyme complex receptor Ó FEBS 2003 4060 G L Long et al (Eur J Biochem 270) [9,13,14] Latent AT forms spontaneously under mild conditions [13,14], including storage of plasma at 37 °C [15] A naturally occurring genetic variant, AT Rouen-VI (Asn187 fi Asp), readily forms the latent form and is associated with fever-induced thromboembolic disease [16] In commercial concentrates of AT, up to 40% exists as the latent form [17] Recently, it was reported that AT has potent antiangiogenic activity and inhibits tumor growth [18,19] Native AT has little effect, cleaved AT has an intermediate effect, and latent AT is the most potent antiangiogenic agent reported to date [19] In this communication we report the characterization of a murine mAb, M27, against human AT that binds to the native, complexed, cleaved, and latent forms of AT M27 blocks the thrombin-inhibiting capacity of AT and protects it from cleavage by thrombin M27 binds to a linear epitope (residues 411–416; FIREVP) that partly overlaps a region (residues 408–412; FLVFI) implicated in the recognition of certain serpin–protease complexes by the SEC receptor (SECR), first identified on the surface of human hepatoma HepG2 cells [20] M27 binds the thrombin–AT (T–AT) complex with a picomolar dissociation constant The rate of clearance of the ternary T–AT–Fab complex in rats was only slightly slower than the rate of clearance of T–AT Studies with cultured HepG2 cells indicated that M27 does not block the binding of T–AT in an epitope-dependent manner These findings are consistent with recent results that cast doubt on the notion that the sequence FLVFI in AT, and homologous regions in certain other serpins, is crucial for receptor-mediated elimination of protease–serpin complexes from blood plasma [21,22] Materials and methods Preparation of proteins Native AT was purified from human, bovine, rabbit, and mouse plasma, as previously described [23], and stored at )20 °C Cleaved human AT was produced essentially as described previously [24] Native AT was incubated with porcine elastase (Sigma-Aldrich, Stockholm, Sweden), at a 100 : molar ratio, in 50 mM Tris/HCl, 0.15 M NaCl and 0.1% (w/v) PEG 8000 for h at 37 °C After addition of phenylmethanesulfonyl fluoride to a final concentration of mM, the solution was dialyzed against 50 mM Tris/HCl, 0.1 M NaCl (pH 7.5) and purified by heparin–Sepharose chromatography Elution with a NaCl gradient (0.1–1.0 M) gave one peak at 0.3 M NaCl Sequence analyses revealed cleavage at positions 389, 390 and 393 The material was aliquoted and frozen at )70 °C Latent human AT was prepared as described previously [13] Native AT was incubated in 10 mM Tris/HCl, 0.25 M sodium citrate, pH 7.4, for 70 h at 60 °C and purified on a heparin–Sepharose column, as described above A portion of the AT did not bind to the heparin– Sepharose, and a second major peak, which eluted at 0.3 M NaCl, showed a sequence corresponding to native AT, but migration by SDS/PAGE corresponding to that of latent AT The material eluting at 0.3 M NaCl was concentrated, stored at )70 °C, and used in further experiments Human prothrombin was purified by slight modification of a standard procedure, including precipitation with barium chloride and ammonium sulfate, followed by DEAE Sephacel chromatography [25] Prothrombin was activated with venom from Oxyuranus scutellatus (ICN Biomedicals Inc., Irvine, CA) and purified employing Q-Sepharose followed by SP–Sepharose column chromatography [26] Production of mAb M27 Murine mAbs were produced as described previously [27] The mice were immunized with human T–AT complexes These complexes were also used to test the clones in an ELISA Conditioned media were collected from cloned mouse myeloma cells cultured in a TECNOMOUSE (Integra Biosciences, Wallisellen, Switzerland) hollow fiber chamber, and stored at )20 °C until used Thawed media were centrifuged at low speed to remove cellular debris and the supernatant was diluted with an equal volume of column equilibration buffer (1 M glycine, 150 mM NaCl, pH 8.0) and purified by Protein A–Sepharose chromatography [28] mAb M27 was stored in aliquots at )20 °C Its concentration was estimated from the absorbance at 280 nm, assuming an absorbance of 1.34 for a mgỈmL)1 solution [28] M27 was determined to be of the IgG2b isotype by a standard procedure using a commercial kit (Miles Laboratories, Elkhart, IN) Production of the M27 Fab fragment The general procedure for generating Fab was that described by Parham [29] Purified M27 was dialyzed against 100 mM sodium acetate, pH 5.5, and D,L-cysteine and EDTA were added to final concentrations of 45 and 0.9 mM, respectively After of preincubation at 37 °C, digestion was performed for 30 with 0.5% (w/w) fresh papain (Sigma) After incubation with iodoacetamide (final concentration 70 mM) for 30 at room temperature, the digest was dialyzed at °C against M glycine, 150 mM NaCl, pH 8.0 The digest was then purified by protein A– Sepharose chromatography to remove traces of undigested IgG and Fc fragment The material in the flow-through peak (unbound Fab fragment) was dialyzed against 20 mM Tris/HCl, pH 8.5, and subjected to chromatography on a Q-Sepharose Fast Flow column that was eluted with a linear NaCl gradient (0–250 mM) The Fab fragment eluted at  70 mM NaCl It was homogenous, as judged by SDS/ PAGE, and was able to bind Eu3+ chelate-labeled native antithrombin in a DELPHIA assay [30] The Fab fragment was dialyzed against NaCl/Tris buffer, pH 7.5, aliquoted and stored at )20 °C About 20% of the material eluted at  160 mM NaCl; it possessed no ability to bind AT and presumAbly consisted of Fab fragments with an aberrant j chain [31] SDS/PAGE and Western blotting SDS/PAGE and Western blotting were performed using standard methods For Western blotting, poly(vinylidene difluoride) (PVDF; Immobilon P, 0.45-lm pore size) membranes (Millipore, Bedford, MA) were used Proteins Ó FEBS 2003 T–AT complexes and the SEC receptor (Eur J Biochem 270) 4061 were stained with GelCode Blue Stain Reagent (Pierce, Rockford, IL) according to the supplier’s instructions Peptide synthesis and epitope mapping Peptides were synthesized on a Milligen 9050 Plus instrument (Perkin-Elmer, Stockholm, Sweden), using Fmoc chemistry, and purified by reverse-phase HPLC followed by lyophilization and storage at )20 °C until required for use All peptides were readily dissolved in deionized water, to a nominal concentration of 0.5 mM, and stored frozen The exact concentrations were determined by amino acid analysis after acid hydrolysis Binding of the synthetic peptides to M27 was studied by an ELISA-based method The peptides and native AT, 1.5 nmol and 15 pmol, respectively, in 100 lL of coating buffer (100 mM sodium bicarbonate, pH 9.6), were delivered to wells of high-binding, polystyrene microtitre plates (Costar, Corning, NY) After incubation for 15 h at °C, the wells were rinsed several times with 10 mM sodium phosphate buffer containing 0.5 M NaCl and 0.1% Tween 20, pH 8.0 (NaCl/Pi/Tween), followed by blocking for 15 with 1% (w/v) BSA (Sigma Fraction V) in NaCl/ Pi/Tween Wells were rinsed again, and different amounts of M27, diluted in NaCl/Pi/Tween containing 0.1% BSA, were added to the wells followed by incubation on a platform shaker for h at room temperature Wells were rinsed again and then incubated, as described above, with a horseradish peroxidase-conjugated rabbit anti-mouse IgG (DAKOPATTS AB, Alvsjo, Sweden) diluted : 1000 in NaCl/Pi After washing, the chromogenic enzyme substrate 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid) (ABTS) was added and the absorbance at 405 nm was recorded as a function of time Binding of M27 to the peptides (all containing a single cysteine residue, see Fig 3A) was also determined after coating the peptides, dissolved in NaCl/Pi, pH 7.4, to a maleimide-activated microtitre plate (Pierce) for 15 h at room temperature Following coating and subsequent rinsing with NaCl/Pi, pH 7.4, wells were blocked by incubation for 90 with 150 lL of D,L-cysteine in 10 lgỈmL)1 NaCl/Pi, pH 7.4 Wells were then rinsed with NaCl/Pi/Tween, followed by antibody binding and enzymatic color development, as described above Effect of AT and M27 on thrombin activity A two-stage assay was used to determine the effect of M27 on the inhibition of thrombin by AT The first stage consists of a-thrombin incubation with native AT at 37 °C for different lengths of time in the presence or absence of M27 Freshly diluted thrombin (0.5 lg in buffer comprising 50 lL of 50 mM Tris/HCl, 150 mM NaCl, 0.1% BSA, pH 7.5; Tris/HCl/NaCl/BSA) was delivered into wells of a low-affinity microtitre plate (Bibby Sterilin, Ltd, Staffs., UK) and allowed to equilibrate for at 37 °C Native AT (0.8 lg in 10 lL of Tris/HCl/NaCl/BSA), M27 (4 lg in 10 lL of Tris/HCl/NaCl/BSA) or AT preincubated with M27 (same concentrations and volume) were added to the thrombin solution, mixed and incubated at 37 °C At different time-points, 10-lL aliquots were removed for SDS/PAGE or mixed into 90 lL of Tris/HCl/NaCl/BSA (at room temperature) Duplicate aliquots (10 lL) of the latter were immediately transferred into clean wells containing 190 lL of freshly prepared thrombin substrate (400 lM S-2238; Chromogenix, Gothenburg, Sweden) in 50 mM Tris/HCl, 0.1% BSA, pH 8.4 The samples were briefly mixed, and the increase in absorbance at 405 nm was recorded as a function of time A similar procedure was used to measure the effect of heparin on the above system Native AT (90 lgỈmL)1), M27 (435 lgỈmL)1), or AT plus M27 (same concentrations) were preincubated (for h at 37 °C) in Tris/HCl/ NaCl/BSA containing 100 mL)1 heparin (average MW, 15 kDa; Lovens Kemiske Fabrik, Ballerup, Denmark) ă Aliquots (10 lL) were then added to an equal volume of Tris/HCl/NaCl/BSA containing 20 lgỈmL)1 thrombin, followed by brief mixing and incubation at 25 °C for The interaction with thrombin was stopped by the addition of 180 lL of ÔquenchÕ solution: 110 lg of protamine sulfate (Lovens Kemiske Fabrik) per ml of ă Tris/HCl/NaCl/BSA Duplicate 10-lL quenched aliquots were then used in the measurement of thrombin amidolytic activity, as described above The experiments, with and without heparin, were performed on three separate occasions, with essentially identical results They were also performed once with a molar equivalent (antigen-binding sites) of purified M27 Fab fragment, and gave results identical to those obtained with the intact mAb Surface plasmon resonance spectroscopy Binding of M27 IgG and the Fab fragment to different forms of AT was studied using a BIACORE 2000 biosensor (Biacore AB, Uppsala, Sweden) Purified M27 or Fab fragments were diluted into 10 mM Hepes, 150 mM NaCl, mM EDTA, 0.005% Polysorbate 20, pH 7.4 (Hepes/NaCl/EDTA/Polysorbate 20) They were immobilized with NH2-coupling to a CM5 sensor chip (Biacore) to levels of 1700 and 740 response units (RU) for the intact mAb and the corresponding Fab fragment, respectively Analytes were diluted into Hepes/NaCl/ EDTA/Polysorbate 20 and used to measure binding to the immobilized IgG and Fab using a programmed protocol with 20 s preinjection delay, 180 s association time and 600 s dissociation time The experiments were performed at room temperature and the proteins were pumped at 30 lLỈmin)1 The chip was regenerated with two pulses of lL 0.1 M glycine/HCl, 0.5 M NaCl, pH 2.75, at a flow rate of lLỈmin)1 The analyte concentrations ranged from 0.1 to 100 nM (based on amino acid analysis) Runs were performed three times and with five different concentrations for each analyte Data were analyzed using the BIAEVALUATION 3.0 software package (Biacore), assuming noninteracting antibody-binding sites and a : stoichiometry of binding In vivo clearance of the T–AT complex in rats Thrombin, native AT, and elastase-cleaved AT were labeled with 125I (Amersham Pharmacia AB, Uppsala, Sweden) using the chloramine T method, according to the supplier’s instructions 125I-labeled thrombin was covalently Ó FEBS 2003 4062 G L Long et al (Eur J Biochem 270) complexed with unlabeled AT at a : molar ratio for h at room temperature, then applied to a heparin–Sepharose column and eluted The concentrations of two fractions containing the T–AT complex were estimated by comparing the absorbance at 280 nm with a nonradioactive T–AT complex, for which the concentration had been determined by amino acid analysis The proteins were stored in aliquots at )70 °C Sprague-Dawley rats (350 g) were anesthesized (2 mLỈkg)1) with a : : (v/v/v) mixture of Hypnorm (JANSSEN-CILAG Ltd, High Wycombe, UK), Dormicum (Pharma hameln GmbH, Hamelm, Germany) and deionized water 125I-labeled T–AT (73 pmol in 500 lL), with and without 2.9 nmol M27 Fab, were injected in a tail vein, and blood samples (200 lL) were drawn from a jugular vein into tubes containing 10 lL of 0.5 M EDTA after 1, 3, 5, 10, 15 and 20 Radioactivity was measured in 50 lL aliquots of the plasma Blood samples drawn after were considered to represent the amount of injected radioactivity after equilibration in the circulation 125Ilabeled thrombin, native AT, and cleaved AT alone were also injected into control animals Binding of 125 I-labeled T–AT complex to HepG2 cells The binding studies were performed, as previously described, with minor modifications [21] HepG2 cells were cultured at 37 °C, 5% CO2 in 75-cm2 flasks (Nunc, Roskilde, Denmark) containing Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Corp.) supplemented with 10% (v/v) fetal bovine serum (HyCloneÒ), mM L-glutamine (Invitrogen Corp.), and penicillin/ streptomycin (100 unitsỈmL)1/100 lgỈmL)1) (Invitrogen Corp.) Cells were transferred to 24-well plates, at a concentration of 2–3 · 105 cells per well, and grown for days before the experiments The cells were washed twice with DMEM containing 0.2% BSA, 0.5 lM PPACK and 10 mM Hepes, pH 7.4 (binding buffer) Radiolabeled T–AT complex (20 nM) in binding buffer, with or without unlabelled T–AT (2 lM) or M27 (0.2 lM) or M38 (0.2 lM), or cleaved AT (1 lM), were each added to four wells After incubation at °C for h, the wells were washed three times with 10 mM Hepes, pH 7.4, containing 0.15 M NaCl, mM CaCl2, mM MgCl2 and 0.2% BSA Cells were lysed in 0.4 mL of M NaOH overnight at room temperature and the radioactivity connected to the cells was measured The results are expressed as the mean of triplicate samples Results Identification of the M27 epitope Western blotting was used in the initial characterization of the epitope of mAb M27 (Fig 1) Bands corresponding to all forms of nonreduced AT were rapidly visible However, when the inhibitors were analyzed after reduction of the disulfide bonds, only the native and latent forms of AT (i.e not cleaved at Arg393) were observed The 39-residue C-terminal peptide (residues 394–432), formed upon complex formation with thrombin, is linked by means of Cys430 to Cys247 in the main body of the inhibitor The results suggest that this peptide, which is not visible owing to its small size, harbors the eptiope that is recognized by M27 Binding of the mAb to reduced and nonreduced native and latent AT indicates that the mAb recognizes a linear sequence These results warranted a test of the reactivity of mAb M27 in Western blotting with AT from mouse, rabbit and bovine blood plasma ATs from these species all differ from their human counterpart at three positions in the C-terminal peptide: residues 411, 416 and 432 (Fig 2A) [37] M27 did not react with AT from any of the three species (Fig 2B), whereas a control rabbit polyclonal antiserum against human AT gave positive results As mAb M27 is not sensitive to reduction of the Cys residue at position 430 of AT, it is unlikely that the epitope is at the very C-terminus of the peptide The results therefore suggested that residues 411 and 416 are part of the epitope of M27 Synthetic peptides were used to localize the epitope of M27 more precisely (Fig 3A) Wells of microtitre plates were coated with the peptides for ELISA-binding studies A peptide including residues 404–420 (P-74) was found to bind M27 (Fig 3B) In contrast, a corresponding peptide, with substitutions at positions 411 (P-80), 416 (P-81), or both (P-79), showed a very weak reaction with M27 Native AT competed less well with the synthetic peptides than with native immobilized AT for binding to M27 A control peptide with the same composition as the 404–420 peptide, but with the sequence scrambled, did not react with M27 Identical results were obtained when the peptides were covalently linked to the wells of microtitre plates by a maleimide reaction with C-terminal Cys residues (data not Molecular modeling X-ray structures of human native AT [32], latent AT [9], heparin–AT complex [33], cleaved bovine AT [34], and trypsin–antichymotrypsin covalent complex [10], were analyzed using the ACCELRYS molecular modeling package (San Diego, CA, USA), running on a Silicon Graphics workstation 02 or Fuel Solvent accessibility was computed using the method of Lee & Richards [35] The packing density was calculated with the method of Kurochking & Privalov [36], with the MOLE molecular graphics software package kindly provided by R Tarr (Applied Thermodynamics, Inc., Hunt Valley, MD, USA) Fig Western blotting of AT with mAb M27 Different forms of AT were electrophoresed by SDS/PAGE (12% gels), transferred to poly(vinylidene difluoride) (PVDF) membrane, and detected with M27 Nonreduced native, latent, elastase-cleaved, or thrombin–AT complex (1.7 pmol each) were electrophoresed in lanes 2–5, respectively The same amounts of dithiothreitol-reduced proteins were electrophoresed in lanes 6–9 The arrow points to the position of the 62 kDa molecular mass protein marker in lane Ó FEBS 2003 T–AT complexes and the SEC receptor (Eur J Biochem 270) 4063 Fig Western blotting of AT from different species with M27 (A) C-terminal sequences of AT from different species are compared (human AT numbering) Amino acids identical to that of the human are shown with dashes (–) The arrow indicates the thrombin cleavage site in AT that forms the C-terminal 39 residue peptide Cys430, which is disulfide bonded to Cys247, is underlined (B) Nonreduced native human AT (0.34 pmol) was included in lanes and Equal amounts of purified, nonreduced native mouse, rabbit or bovine AT were included in lanes 3–5 and 7–9, respectively Protein markers were run in lane Membrane-bound proteins from lanes 1–5 were incubated with M27, followed by incubation with rabbit anti-mouse IgG alkaline phosphatase conjugate and enzyme color development Membranebound proteins from lanes 6–9 were incubated in the same manner, except with a primary polyclonal rabbit anti-(human AT) Ig followed by secondary goat anti-rabbit IgG alkaline phosphatase conjugate (DAKOPATTS) shown) The results of the ELISA-binding studies are consistent with the results of the Western blotting experiments and establish that the sequence including FIREVP (residues 411–416) constitutes a critical part of the linear epitope of mAb M27 Fig Binding of M27 to immobilized synthetic peptides (A) Synthetic peptide sequences The vertical arrow designates the thrombin-cleavage site in antithrombin (AT) Peptide P-78 corresponds to the 42 Cterminal residues of human AT Bold, underlined residues are changes from the wild-type sequence Peptide P-82 is a ÔscrambledÕ sequence, whose composition is the same as that of the wild-type plus a Cterminal cysteine residue (B) Peptides were immobilized on the surface of polystyrene microtitre plate wells Binding of M27 was determined using an ELISA, with a final development of 405 nm absorption vs time Bars represent average values of duplicates at the 10-min timepoint Error bars indicate the range Color development in the assay is still linear, with respect to time, at the 10-min time-point Key: black bars, 200 ng of M27 added; white bars, 1000 ng of M27 added; stippled bars, 200 ng of M27 + lg of native AT added Effect of M27 on inhibition of thrombin by AT The effect of mAb M27 on the formation of covalent complexes between thrombin and AT was studied in a twostage assay First, thrombin and AT were incubated in microtitre plate wells, with and without M27 Aliquots were then removed at different time-points for SDS/PAGE and for measurements of the amidolytic activity M27 inhibited AT both in the presence and absence of heparin (Table 1) In the absence of heparin the M27-mediated inhibition was complete, whereas in the presence of heparin it was partial The inhibition in the presence of heparin was not influenced by increasing the mAb concentration from a four- to a 40-fold molar excess over the AT concentration, suggesting that the heparin and mAb binding sites on AT are independent of one another (data not presented) Solution binding studies with Eu3+ chelate-labeled native AT have also indicated that there is no competition between heparin and M27 for binding to AT (data not presented) As shown in Fig 4, Western blotting of aliquots removed after the first stage of the assay also revealed that the presence of M27 blocked the T–AT formation Moreover, at most, a minute amount of AT could have been cleaved by thrombin when M27 was bound to AT A very weak band above the bands of native AT in lanes and is probably a small amount of cleaved AT, which is an impurity of our native AT The bands correspond to the mobility of the cleaved inhibitor during SDS/PAGE Measurement of AT binding to M27 by surface plasmon resonance The binding of different forms of AT to immobilized intact IgG and the Fab fragment of mAb M27 was studied in real time by surface plasmon resonance using a BIACORE biosensor Representative binding curves and binding Ó FEBS 2003 4064 G L Long et al (Eur J Biochem 270) Table Effect of M27 on the inhibition of thrombin by AT Concentration and molar ratios of proteins in the second stage amidolytic assay are shown in parentheses Vmax is the rate of substrate hydrolysis, as measured by the change in absorbance at 405 nm over a period of 20 In all cases the change was linear during this time-period Values represent the average and range of duplicate measurements In part A, first-stage components were combined and incubated for 60 at 37 °C prior to addition to the second stage In part B, first-stage components were combined and incubated for at 25 °C prior to addition to the second stage Other details of the assay are described in the Materials and methods Additions Vmax (mODỈmin)1) Part A None (TBSB buffer alone) Thrombin (50 ngỈmL)1) Thrombin (50 ngỈmL)1) + Thrombin (50 ngỈmL)1) + Thrombin (50 ngỈmL)1) + Thrombin (50 ngỈmL)1) + Thrombin (50 ngỈmL)1) + AT (1 : 1.04) AT (1 : 1.04) + M27 (1 : 1.04 : 3.7) M27 (1 : 3.7) AT (1 : 1.04) + Fab (1 : 1.04 : 3.7) Fab (1 : 3.7) 0.02 36.42 3.52 35.88 35.19 33.30 35.22 ± ± ± ± ± ± ± 0.01 1.50 0.12 1.30 1.53 1.47 1.36 Part B Thrombin Thrombin Thrombin Thrombin Thrombin Thrombin Thrombin Heparin Heparin Heparin Heparin Heparin Heparin 33.60 31.28 0.13 19.90 32.04 19.44 32.19 ± ± ± ± ± ± ± 0.90 1.20 0.00 0.77 1.28 0.83 0.86 (50 (50 (50 (50 (50 (50 (50 ngỈmL)1) ngỈmL)1) ngỈmL)1) ngỈmL)1) ngỈmL)1) ngỈmL)1) ngỈmL)1) + + + + + + (0.25 (0.25 (0.25 (0.25 (0.25 (0.25 unitsỈmL)1) unitsỈmL)1) unitsỈmL)1) unitsỈmL)1) unitsỈmL)1) unitsỈmL)1) + + + + + AT (1 : 2.6) AT (1 : 2.6) + M27 (1 : 2.6 : 9.8) M27 (1 : 9.8) AT (1 : 2.6) + Fab (1 : 2.6 : 9.8) Fab (1 : 9.8) Clearance of the T–AT complex in rats Fig M27 protection of antithrombin (AT) from thrombin cleavage Samples of native AT incubated for 40 in the presence of thrombin, with or without M27, were submitted to SDS/PAGE and Western blotting with M27, as described in the legend to Fig Lanes 1–5 contain nonreduced samples, and lanes 6–9 contain reduced samples Lane 1, size markers; lane 2, 80 ng of AT before incubation; lane 3, 60 ng of AT + thrombin; lane 4, 60 ng of AT + thrombin + M27; lane 5, thrombin + M27; lane 6, 64 ng of AT before incubation; lane 7, 48 ng of AT + thrombin; lane 8, 48 ng of AT + thrombin + M27; lane 9, thrombin + M27 The intense bands at the top of lanes and are caused by reaction of M27 in the loaded samples with the secondary antibody–enzyme conjugate The arrow denotes the position of the 62 kDa molecular mass marker protein constants are presented in Fig and Table Results obtained with immobilized intact mAb and the corresponding Fab fragment were identical within experimental error The derived dissociation constants ranged from 20 pM to nM, i.e indicating that M27 has a high affinity for all forms of AT The Kd for native and elastase-cleaved AT were almost identical The highest Kd, for latent AT, was the result of both a decrease in the association rate constant (ka) and an increase in the dissociation rate constant (kd) relative to native AT, each about one order of magnitude M27 had the highest affinity for the T–AT complex (KD  · 10)11 M) This can be attributed almost totally to a very slow dissociation rate (Fig 5C) SECs are removed from the circulation by cellular receptors that recognize receptor-binding site(s) on the complex [11] A pentapeptide (residues 408–412; FLVFI in AT) in the C-terminal fragment of the cleaved inhibitor has been implicated in receptor binding and internalization [38] As the M27-binding site on complexed AT (residues 411–416; FIREVP) partially overlaps the proposed SECR-binding site, a clearance study in rats was performed to determine whether the antibody would inhibit removal of the complex from the circulation The results obtained for the complex, in the absence of the Fab fragment, and for the native and cleaved AT, were similar to that reported previously [39] Although the Fab fragment of M27 caused a small, but significant (P < 0.0001, two-factorial ANOVA), reduction in the rate of clearance of the complex, from a half-life (t½) of  in the absence of the Fab fragment to a t½ of  in its presence, it did not block the uptake to the liver (Fig 6) Considering the very high affinity of the M27 Fab fragment for the T–AT complex, this result is not consistent with a model where the putative SECR-binding site on the T–AT complexed AT is important for complex clearance [11] To determine whether the M27 could inhibit the binding of T–AT to the SEC receptor in a less complex system, HepG2 cells were incubated with radiolabeled T–AT in the presence and absence of the antibody The binding of radiolabeled T–AT was reduced to 33 and 16% when 50fold (not shown) and 100-fold molar excess of unlabeled T– AT complex was added, respectively, which is in accordance with a previous study [38] The binding was 69 and 79% in the presence of a 10-fold molar excess of M27 and Fab M27, respectively The high affinity of M27 to T–AT Ó FEBS 2003 T–AT complexes and the SEC receptor (Eur J Biochem 270) 4065 Discussion Fig BIACORE binding curves for M27 Fab interaction with AT Data obtained by surface plasmon resonance experiments were analyzed using the BIAEVALUATION 3.0 software package Symbols represent actual data points and solid lines are simulation curves, assuming univalent, noninteracting binding sites (A) Concentrations of latent AT are 24.1, 40.2, 56.2, 64.3, and 80.3 nM (B) Concentrations of elastase-cleaved AT are 0.9, 2.3, 4.6, 9.2 and 18.4 nM (C) Concentrations of the T–AT complex are 0.9, 2.3, 4.3, 8.7 and 17.4 nM (D) Concentrations of native AT are 0.8, 2.0, 4.0, 8.0 and 16.0 nM (KD 3.7 · 10)11 M), ensured that > 99.9% of the T–AT was complexed with M27 in these experiments Similar results were obtained with a 50-fold molar excess of the antibodies (not shown) As a control, HepG2 cells were incubated with labeled T–AT in the presence of M38, which is an anti-AT mAb that does not compete with M27 for binding to AT The binding of labeled T–AT was decreased to 54% A 50-fold molar excess of cleaved AT decreased the binding to 79% These results also argue against a major role for the FIREVP sequence in receptor-mediated complex binding We have generated and characterized a murine mAb, M27, possessing high affinity for human AT This antibody reacts with all naturally occurring forms of AT, with KD values ranging from to 0.02 nM The epitope for M27 was identified by comparison of Western blotting results for reduced with nonreduced cleaved and noncleaved forms of AT The results indicate that the epitope resides in the C-terminal 39-amino acid residue peptide generated by T–AT complex formation and cleavage Synthetic peptides helped define the epitope to the linear sequence, FIREVP (residues 411–416), although native AT could not compete with the synthetic immobilized peptides for the binding to M27, as well as with the immobilized AT One explanation could be that the antibody only binds with of one of its binding sites to AT and therefore could bind to a small immobilized peptide with the other It is clear that the binding of M27 to immobilized peptides is not saturated [100-fold more peptides (1.5 nmol) than AT (15 pmol) were added to the wells] Yet, the same amount of antibody gives only  50% of the signal in peptide-coated wells compared with AT-coated wells We consider that only a small fraction of the peptides immobilized on the plastic surface (without spacer arm) bind the mAb Another possibility is that our antibody is, to some extent, conformation dependent (Fig 1), but with its crucial binding pointing at the hexapeptide FIREVP Several mAbs against human AT have been reported that map to the C-terminal region of the molecule Asakura et al described a murine mAb (JITAT-16) raised to human AT that recognizes the T–AT complex as well as cleaved AT, but not native AT [40] The epitope of JITAT-16 (AAAST; residues 382–386) is just upstream of the Arg393 cleavage site [41] JITAT-16 destroyed the ability of AT to inhibit thrombin, as does M27 However, the mechanism of inhibition is different for the two antibodies JITAT-16 acts by enhancing the hydrolysis of the T–AT acyl intermediate to free, cleaved AT and active thrombin (normally a slow process) relative to the formation of a stable covalent complex Presumably, this results from delayed insertion of the RCL into b-sheet A In contrast, M27 seems to inhibit formation of the acyl intermediate quantitatively and hence complex formation and subsequent hydrolysis to the cleaved form of AT (Fig 4) Picard et al described a mAb (12A5) recognizing the linear sequence, DAFHK (residues 366–370), in the C-terminal region of AT [24] Antibody 12A5 also differs from M27 in that the former recognizes AT when it exists as a binary complex with thrombin, factor Xa and, to some exent, the P14-P9 synthetic peptide, but not native, latent or cleaved forms of AT Dawes et al have reported a conformationally sensitive mAb (ESAH 1) that recognizes native, thrombin-complexed, and cleaved AT [42] The authors also observed that binding of heparin to AT counteracts the ability of ESAH to neutralize AT inhibition of thrombin, but heparin binding had no effect on ESAH binding to AT These observations are different from those (seen by us) for M27, where the effects of heparin and antibody on AT inhibition of thrombin are independent of one another The epitope recognized by ESAH 1, involves residues 402–407 and 429 (FKANRP/P), all in the C-terminal cleavage peptide, but Ó FEBS 2003 4066 G L Long et al (Eur J Biochem 270) Table Binding constants for M27 IgG and Fab interaction with AT Measurements were made with surface plasmon resonance on a BIAcore instrument and analyzed using the software package BIAEVALUATION 3.0 See the Materials and methods for details Values represent the mean (± standard deviation) of three independent determinations, except for AT native/Fab where only two determinations were made Analyte/ligand AT native/IgG AT native/Fab AT latent/IgG AT latent/Fab AT cleaved/IgG AT cleaved/Fab T–AT complex/IgG T–AT complex/Fab ka (M)1Ỉs)1) 3.85 4.64 4.20 3.81 3.14 3.45 1.80 2.28 · · · · · · · · 10 105 104 104 105 105 105 105 (± (± (± (± (± (± (± (± kDa (s)1) 0.04) 0.04) 0.36) 0.46) 0.00) 0.02) 0.11) 0.14) 5.64 6.28 2.87 3.01 5.18 5.23 6.53 4.37 · · · · · · · · KD (M) )5 10 10)5 10)4 10)4 10)5 10)5 10)6 10)6 (± (± (± (± (± (± (± (± 0.20) 0.03) 0.11) 0.10) 0.16) 0.17) 4.37) 3.51) 1.46 1.36 6.83 8.02 1.65 1.51 3.73 1.98 · · · · · · · · 10)10 (± 0.07) 10)10 (± 0.02) 10)9 (± 0.85) 10)9 (± 1.06) 10)10 (± 0.05) 10)10 (± 0.06) 10)11 (± 2.57) 10)11 (± 1.66) Fig Effect of mAb M27 on T–AT clearance in vivo and in vitro (A) 125I-T–AT complex (73 pmol in 500 lL) was injected into the tail vein of rats in the absence (h) and presence (d) of M27 Fab fragment (2.9 nmol) Blood samples were collected at the time-points indicated and the radioactivity was measured As controls, 125I-labeled diisopropylphosphoryl (DIP)-thrombin (j), native AT (n) and elastase-cleaved AT (s) were injected in the same manner as the complex The complexes with and without M27 Fab fragment were injected into two rats each The error bars represent the range (B) HepG2 cells were incubated with 200 lL of 20 nM 125I-T–AT in the absence or presence of lM unlabeled T–AT, 0.2 lM Fab M27, 0.2 lM mAb M27, 0.2 lM mAb M38, and lM cleaved AT Each bar represents the percentage of binding ± 2SD Binding of radiolabeled T–AT without competitor was set to 100% distinct from the region recognized by M27 (residues 411– 416).Three-dimensional molecular models for native AT are presented in Fig Examination of the model reveals that the epitope recognized by M27 resides in strand of b-sheet B of AT, on the face opposite b-sheet A into which the reactive-center loop inserts Analyses of X-ray-derived structures for the latent and cleaved forms of AT suggest that the epitope is in the same general location for all forms of AT and at least partially surface exposed (Fig 7) The model derived from the crystal structures indicates that only the side-chains of residues 413–416 are exposed to the surface, in agreement with residue 416 playing an important role in M27 binding However, X-ray structures showing that residue 411 is buried is in apparent contradiction with our peptide epitope mapping that implicates residue 411 in M27 binding Examination of molecular models for native, cleaved, and latent AT, based upon X-ray crystallography, does not lead to a clear explanation of the differences in the equilibrium-binding constants of M27 for the different forms The antibody-binding site shown in Fig is sufficiently ˚ close to the RCL of AT (40–50 A) to allow antibodymediated blocking of the formation of the Michaelis-like complex with thrombin, even in the case of the Fab fragment, which typically has a length from the antigen˚ binding site to the papain cleavage site of  45 A However, this explanation for the neutralization of AT by M27 would require that the bound antibody has very little free movement relative to the RCL, and is always in an orientation that blocks the binding of thrombin to AT An alternative explanation is that binding of M27 distorts the conformation of the RCL in a manner that prevents Ó FEBS 2003 T–AT complexes and the SEC receptor (Eur J Biochem 270) 4067 Fig Molecular models of the M27 epitope on antithrombin recognition by thrombin A precedent for such a subtle, yet significant, conformational change is offered by heparin binding to AT, resulting in a conformational change in the RCL [33] Also consistent with this proposal is the ease with which AT can adopt alternative conformations (e.g the latent form) This explanation may also, in part, explain the only partial neutralization by M27 observed in the presence of heparin In the presence of high-molecular-weight heparin, the M27-mediated block of AT inhibition was not complete and was not influenced by a 10-fold increase in antibody concentration (data not presented) This suggests that the binding sites on AT for antibody and heparin are independent of one another Second-order association rate constants of 8.9 · 10)3 and 3.7 · 10)7ỈM)1Ỉs)1 have been reported for T–AT in the absence and presence of heparin, Ó FEBS 2003 4068 G L Long et al (Eur J Biochem 270) respectively [43] Dissociation of M27 from AT (the Kd value for the M27–AT complex is 5.6 Ã 10)5ặs)1; tẵ, 3.4 h) cannot account for the T–AT complex formation in the presence of heparin We propose that heparin induces a conformational change in the RCL of AT that allows slow complex formation with thrombin, even with M27 bound Lollar & Owen were the first to demonstrate that the AT–125I-thrombin complex is rapidly cleared by the liver in rabbits [44] Subsequent studies by others, involving competitive clearance studies, indicated that a common pathway exists for several SECs, including AT–thrombin, heparin cofactor II–thrombin, a1-antitrypsin–trypsin, and a1-antitrypsin–elastase [45,46] Mast et al demonstrated that the rate of SEC clearance is 10–50 times faster than that of the corresponding free inhibitor [47] In the case of T–AT, the t½ for the elimination of the complex from the circulation is in the order of several minutes [45,47] The first receptor identified as being involved in SEC binding and clearance, termed SECR, was implicated by Perlmutter and co-workers based on in vitro studies of HepG2 and monocyte stimulation of a1-antitrypsin biosynthesis [20] Subsequently, SECR was reported to recognize a minimal pentapeptide sequence (FVFLM) in a1-antitrypsin, based upon synthetic peptide competitive-binding studies [38] The authors also proposed that the corresponding pentapeptide (FLVFI in antithrombin) in the homologous serpin portion of SEC complexes, is similarly and competitively recognized by SECR In contrast to Perlmutter and co-workers, Maekawa et al showed that five recombinant heparin cofactor II–thrombin complexes, each with different single mutations in the proposed receptor-binding pentapeptide, were not prevented from binding and uptake to HepG2 cells [22] In recent years, conjugates of poly Lys-peptides, containing the FVFLM sequence, have been used to effectively transfer DNA into hepatic cells both in vitro and in vivo [48–50] Kounnas et al demonstrated the importance of the low-density lipoprotein receptor-related protein (LRP) in the clearance of SEC complexes in vivo and in vitro [39] Their conclusions were based on the ability of the receptor-associated protein, an inhibitor of LRP activity, to prevent uptake of SEC complexes to liver in rats and to HepG2 cells We have demonstrated that mAb M27 recognizes a hexapeptide segment (FIREVP), which overlaps the last two residues of the pentapeptide (FLVFI) reported to be recognized by the SECR We found that clearance of 125IT–AT from the blood circulation of rats was only slightly reduced by bound M27 Fab fragment Furthermore, the Fab fragment only marginally reduced binding of the complex to HepG2 cells The effect was similar to that obtained with a control antibody against AT The limited effect obtained with Fab M27 bound to T–AT is presumably nonspecific and caused by to an increase in size and/or change of charge Our interpretation is that the pentapeptide site is, at most, marginally involved in complex clearance Acknowledgements We acknowledge the technical assistance of Bjorn Hambe in purifying antithrombin from bovine, rabbit and mouse plasma, and of Ulla Persson in production of conditioned media containing the mAb, M27 Financial support to G L L., while on sabbatical stay at the Department of Clinical Chemistry, Lund University Hospital, Malmo, ă was provided, in part, by the Wenner-Gren Foundation This work was supported by grants from the Swedish Medical 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slightly slower than the rate of clearance of T–AT Studies with cultured HepG2 cells indicated that M27... stoichiometry of binding In vivo clearance of the T–AT complex in rats Thrombin, native AT, and elastase-cleaved AT were labeled with 125I (Amersham Pharmacia AB, Uppsala, Sweden) using the chloramine... Although the Fab fragment of M27 caused a small, but significant (P < 0.0001, two-factorial ANOVA), reduction in the rate of clearance of the complex, from a half-life (t½) of  in the absence of the