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• W1992648023 abstract "The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential therapeutic target in cardiovascular and cancerous diseases. PAI-1 circulates in blood as a complex with vitronectin. A PAI-1 variant (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 PAI-1) with a fluorescent tag at the reactive center loop (RCL) was used to study the effects of vitronectin and monoclonal antibodies (mAbs) directed against α-helix F (Mab-2 and MA-55F4C12) on the reactions of PAI-1 with tissue-type and urokinase-type plasminogen activators. Both mAbs delay the RCL insertion and induce an increase in the stoichiometry of inhibition (SI) to 1.4-9.5. Binding of vitronectin to NBD P9 PAI-1 does not affect SI but results in a 2.0-6.5-fold decrease in the limiting rate constant (klim) of RCL insertion for urokinase-type plasminogen activator at pH 6.2-8.0 and for tissue-type plasminogen activator at pH 6.2. Binding of vitronectin to the complexes of NBD P9 PAI-1 with mAbs results in a decrease in klim and in a 1.5-22-fold increase in SI. Thus, vitronectin and mAbs demonstrated additivity in the effects on the reaction with target proteinases. The same step in the reaction mechanism remains limiting for the rate of RCL insertion in the absence and presence of Vn and mAbs. We hypothesize that vitronectin, bound to α-helix F on the side opposite to the epitopes of the mAbs, potentiates the mAb-induced delay in RCL insertion and the associated substrate behavior by selectively decreasing the rate constant for the inhibitory branch of PAI-1 reaction (ki). These results demonstrate that mAbs represent a valid approach for inactivation of vitronectin-bound PAI-1 in vivo. The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential therapeutic target in cardiovascular and cancerous diseases. PAI-1 circulates in blood as a complex with vitronectin. A PAI-1 variant (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 PAI-1) with a fluorescent tag at the reactive center loop (RCL) was used to study the effects of vitronectin and monoclonal antibodies (mAbs) directed against α-helix F (Mab-2 and MA-55F4C12) on the reactions of PAI-1 with tissue-type and urokinase-type plasminogen activators. Both mAbs delay the RCL insertion and induce an increase in the stoichiometry of inhibition (SI) to 1.4-9.5. Binding of vitronectin to NBD P9 PAI-1 does not affect SI but results in a 2.0-6.5-fold decrease in the limiting rate constant (klim) of RCL insertion for urokinase-type plasminogen activator at pH 6.2-8.0 and for tissue-type plasminogen activator at pH 6.2. Binding of vitronectin to the complexes of NBD P9 PAI-1 with mAbs results in a decrease in klim and in a 1.5-22-fold increase in SI. Thus, vitronectin and mAbs demonstrated additivity in the effects on the reaction with target proteinases. The same step in the reaction mechanism remains limiting for the rate of RCL insertion in the absence and presence of Vn and mAbs. We hypothesize that vitronectin, bound to α-helix F on the side opposite to the epitopes of the mAbs, potentiates the mAb-induced delay in RCL insertion and the associated substrate behavior by selectively decreasing the rate constant for the inhibitory branch of PAI-1 reaction (ki). These results demonstrate that mAbs represent a valid approach for inactivation of vitronectin-bound PAI-1 in vivo. Plasminogen activator inhibitor-1 (PAI-1), 1The abbreviations used are: PAI-1, plasminogen activator inhibitor-1; NBD, N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole; NBD P9 PAI-1, S338C (position P9 of RCL) mutant variant of PAI-1 with NBD group attached to the cysteine residue; RCL, reactive center loop; SI, stoichiometry of inhibition; tPA, two-chain tissue-type plasminogen activator; uPA, urokinase type plasminogen activator; Vn, vitronectin; mAb, monoclonal antibody.1The abbreviations used are: PAI-1, plasminogen activator inhibitor-1; NBD, N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole; NBD P9 PAI-1, S338C (position P9 of RCL) mutant variant of PAI-1 with NBD group attached to the cysteine residue; RCL, reactive center loop; SI, stoichiometry of inhibition; tPA, two-chain tissue-type plasminogen activator; uPA, urokinase type plasminogen activator; Vn, vitronectin; mAb, monoclonal antibody. a member of the serpin superfamily of proteinase inhibitors (1Silverman G.A. Bird P.I. Carrell R.W. Church F.C. Coughlin P.B. Gettins P.G. Irving J.A. Lomas D.A. Luke C.J. Moyer R.W. Pemberton P.A. Remold-O'Donnell E. Salvesen G.S. Travis J. Whisstock J.C. J. Biol. Chem. 2001; 276: 33293-33296Abstract Full Text Full Text PDF PubMed Scopus (1060) Google Scholar, 2Gils A. Declerck P.J. Thromb. Haemostasis. 2004; 91: 425-437Crossref PubMed Scopus (1) Google Scholar, 3Ye S. Cech A.L. Belmares R. Bergstrom R.C. Tong Y. Corey D.R. Kanost M.R. Goldsmith E.J. Nat. Struct. Biol. 2001; 8: 979-983Crossref PubMed Scopus (143) Google Scholar, 4Gettins P.G. Chem. Rev. 2002; : 4751-4804Crossref PubMed Scopus (983) Google Scholar), is involved in regulation of normal and pathological thrombolysis and fibrinolysis as well as in tumor invasion and metastasis (5Andreasen P.A. Egelund R. Petersen H.H. Cell Mol. Life Sci. 2000; 57: 25-40Crossref PubMed Scopus (836) Google Scholar, 6Wind T. Hansen M. Jensen J.K. Andreasen P.A. Biol. Chem. 2002; 383: 21-36Crossref PubMed Scopus (68) Google Scholar, 7Stefansson S. McMahon G.A. Petitclerc E. Lawrence D.A. Curr. Pharm. Des. 2003; 9: 1545-1564Crossref PubMed Scopus (155) Google Scholar). PAI-1 is a major endogenous regulator of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators (8van Mourik J.A. Lawrence D.A. Loskutoff D.J. J. Biol. Chem. 1984; 259: 14914-14921Abstract Full Text PDF PubMed Google Scholar), which produce the active serine proteinase plasmin by cleavage of plasminogen. Similar to other serpins, PAI-1 employs the difference in the free energy between the active (“stressed”; Fig. 1) and inactive (“relaxed”) conformations of the molecule to execute a unique suicide mechanism of proteinase inactivation (Scheme 1). The enzyme (E) forms a Michaelis complex (EI) with PAI-1 (I), interacting with the reactive center loop (RCL) exposed to the solution in the active conformation (Fig. 1). However, cleavage of the scissile bond and formation of the acyl-enzyme (E∼I′) triggers “stressed-to-relaxed” transition of PAI-1, resulting in fast insertion of the C-terminal part of RCL as strand 4 of β-sheet A (Fig. 1), translocation of the proteinase from the initial binding site to the opposite pole of the PAI-1 molecule, and its inactivation, due to a mechanical distortion of the enzyme's active site (9Huntington J.A. Read R.J. Carrell R.W. Nature. 2000; 407: 923-926Crossref PubMed Scopus (939) Google Scholar). An elegant hypothesis, proposing conservation of the free energy for proteinase distortion through reversible displacement of α-helix F, has been recently published (10Gettins P.G. FEBS Lett. 2002; 523: 2-6Crossref PubMed Scopus (59) Google Scholar). Since deacylation of proteinase also occurs during the reaction, the PAI-1 mechanism includes inhibitory and substrate branches (Scheme 1), which yield either the final inhibitory complex (E-I*) or a cleaved inactive PAI-1 (I*) together with regenerated enzyme. The efficiency of the inhibitory reaction (Scheme 1) could be expressed quantitatively by the stoichiometry of inhibition (SI = 1 + ks/ki) (11Chaillan-Huntington C.E. Gettins P.G.W. Huntington J.A. Patston P.A. Biochemistry. 1997; 36: 9562-9570Crossref PubMed Scopus (40) Google Scholar, 12Lawrence D.A. Olson S.T. Muhammad S. Day D.E. Kvassman J.O. Ginsburg D. Shore J.D. J. Biol. Chem. 2000; 275: 5839-5844Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Although the reaction mostly follows the inhibitory pathway (SI∼1), the thermodynamically favorable products (E and I*) finally form (Scheme 1) through slow (kd << ki) hydrolysis of the inhibitory complex (E-I*). Unlike other serpins, PAI-1 possesses an ability for spontaneous inactivation due to a “stressed-to-relaxed” transition via insertion of uncleaved RCL, resulting in the latent conformation of PAI-1 (13Shore J.D. Day D.E. Francis-Chmura A.M. Verhamme I. Kvassman J. Lawrence D.A. Ginsburg D. J. Biol. Chem. 1995; 270: 5395-5398Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). I+Ek−1large{⇄}k1EIk−2large{⇄}k2E∼I′kiamp;↗ksamp;↘kdE−I∗↓I∗+E Inhibitory pathwaySubstrate pathway(Eq. 1) However, there is a physiological mechanism of stabilization of the active conformation of PAI-1. PAI-1 circulates in blood as a complex with vitronectin (Vn) (14Wiman B. Almquist A. Sigurdardottir O. Lindahl T. FEBS Lett. 1988; 242: 125-128Crossref PubMed Scopus (130) Google Scholar, 15Declerck P.J. De Mol M. Alessi M.C. Baudner S. Paques E.P. Preissner K.T. Muller-Berghaus G. Collen D. J. Biol. Chem. 1988; 263: 15454-15461Abstract Full Text PDF PubMed Google Scholar), a cell-adhesive glyco-protein present in vivo in micromolar concentrations (16Tomasini B.R. Mosher D.F. Prog. Hemostasis Thromb. 1991; 10: 269-305PubMed Google Scholar, 17Preissner K.T. Seiffert D. Thromb. Res. 1998; 89: 1-21Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). It has been shown that Vn interacts with PAI-1 with high affinity through its somatomedin B domain, which is composed of the 44 N-terminal amino acids (18Seiffert D. Loskutoff D.J. J. Biol. Chem. 1991; 266: 2824-2830Abstract Full Text PDF PubMed Google Scholar). Binding of Vn induces conformational changes of the PAI-1 molecule (19Gibson A. Baburaj K. Day D.E. Verhamme I. Shore J.D. Peterson C.B. J. Biol. Chem. 1997; 272: 5112-5121Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), resulting in a delay in spontaneous inactivation of PAI-1 due to its conversion from the active to the latent form (15Declerck P.J. De Mol M. Alessi M.C. Baudner S. Paques E.P. Preissner K.T. Muller-Berghaus G. Collen D. J. Biol. Chem. 1988; 263: 15454-15461Abstract Full Text PDF PubMed Google Scholar, 20Preissner K.T. Grulich-Henn J. Ehrlich H.J. Declerck P. Justus C. Collen D. Pannekoek H. Muller-Berghaus G. J. Biol. Chem. 1990; 265: 18490-18498Abstract Full Text PDF PubMed Google Scholar). X-ray crystal structure analysis of the complex of PAI-1 with somatomedin B domain (21Zhou A. Huntington J.A. Pannu N.S. Carrell R.W. Read R.J. Nat. Struct. Biol. 2003; 10: 541-544Crossref PubMed Scopus (225) Google Scholar), together with random and site-directed mutagenesis (22Lawrence D.A. Berkenpas M.B. Palaniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: 15223-15228Abstract Full Text PDF PubMed Google Scholar, 23Jensen J.K. Wind T. Andreasen P.A. FEBS Lett. 2002; 521: 91-94Crossref PubMed Scopus (54) Google Scholar, 24Jensen J.K. Durand M.K. Skeldal S. Dupont D.M. Bodker J.S. Wind T. Andreasen P.A. FEBS Lett. 2004; 556: 175-179Crossref PubMed Scopus (27) Google Scholar), have demonstrated interaction of α-helices F and E as well as strand 1 of β-sheet A of PAI-1 with Vn (Fig. 1). On the other hand, binding of monoclonal antibodies (mAbs) to epitopes located on the side of α-helix F opposite to the Vn binding site side (Fig. 1) induces a decrease (Mab-2) (25Wind T. Jensen M.A. Andreasen P.A. Eur. J. Biochem. 2001; 268: 1095-1106Crossref PubMed Scopus (52) Google Scholar) or even a complete loss (mAb CLB-2C8) (26van Meijer M. Stoop A. Smilde A. Preissner K.T. van Zonneveld A.J. Pannekoek H. Thromb. Haemost. 1997; 77: 516-521Crossref PubMed Scopus (19) Google Scholar) of the ability to bind Vn and an increase in the fraction of PAI-1 following the substrate pathway (for a review, see Ref. 2Gils A. Declerck P.J. Thromb. Haemostasis. 2004; 91: 425-437Crossref PubMed Scopus (1) Google Scholar). Recent studies demonstrate that binding of Vn enhances PAI-1 neutralization by Mab-2 in reaction with uPA (25Wind T. Jensen M.A. Andreasen P.A. Eur. J. Biochem. 2001; 268: 1095-1106Crossref PubMed Scopus (52) Google Scholar, 27Schousboe S.L. Egelund R. Kirkegaard T. Preissner K.T. Rodenburg K.W. Andreasen P.A. Thromb. Haemost. 2000; 83: 742-751Crossref PubMed Scopus (28) Google Scholar). These observations strongly call for further studies of the mechanism by which Vn, alone and in combination with mAbs, affects the kinetics and stoichiometry of the reaction of PAI-1 with target proteinases. To the best of our knowledge, there are no previously reported data on effects of Vn on the kinetics of insertion of RCL during the reaction of PAI-1 and its mAb complexes with target proteinases (Scheme 1).Fig. 2Effects of Mab-2, Vn, and both ligands on the kinetics of RCL insertion for the reaction of NBD P9 PAI-1 with uPA (A) and tPA (B) in 0.05 m phosphate buffer (pH 7.4) at 25 °C. Shown is the dependence of kobs on the concentration of proteinase for the reaction with NBD P9 PAI-1 (10-20 nm), with no mAb added (•), with 80 nm Mab-2 (▪), with 20-40 nm Vn (○), and with Mab-2 with Vn (□). Values of kobs for the reactions of NBD P9 PAI-1 and its complexes with Mab-2 and Vn were calculated by fitting the time traces of the increase in the NBD fluorescence emission to a single exponential equation, as described under “Experimental Procedures.” The values of Km and klim (shown in Table I) as well as the line of best fit (r2 > 0.98) of a hyperbolic equation kobs = klim × [E]/(Km + [E]) to the experimental data were calculated using nonlinear regression employing SigmaPlot 8.0. The values of klim and Km for the reaction of uncomplexed NBD P9 PAI-1 with uPA and tPA (•) were published previously (28Komissarov A.A. Declerck P.J. Shore J.D. J. Biol. Chem. 2004; 279: 23007-23013Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 1The binding areas for Mab-2 (Fab-2) and Vn indicated on the three-dimensional structure of PAI-1.A, ribbon diagram of the three-dimensional structure of active PAI-1. Indicated are the strands of β-sheet A, including β-strand 1A (Lys122-Val124), α-helix E (hE; Met110-Phe117), α-helix F (hF; Val129-Thr142), the loop between α-helix F and β-strand 3A, including a short 310 helix (hF-s3A loop; His143-Thr161), and the RCL (Asn329-Pro349). B, surface diagram in the same orientation as the diagram in A. The binding area for Mab-2 (25Wind T. Jensen M.A. Andreasen P.A. Eur. J. Biochem. 2001; 268: 1095-1106Crossref PubMed Scopus (52) Google Scholar) is indicated in red. The epitope of MA-55F4C12 placed at the N-terminal part of α-helix F (residues 128-131 and 154) (32Bijnens A.P. Gils A. Knockaert I. Stassen J.M. Declerck P.J. J. Biol. Chem. 2000; 275: 6375-6380Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The binding area for Vn (21Zhou A. Huntington J.A. Pannu N.S. Carrell R.W. Read R.J. Nat. Struct. Biol. 2003; 10: 541-544Crossref PubMed Scopus (225) Google Scholar, 23Jensen J.K. Wind T. Andreasen P.A. FEBS Lett. 2002; 521: 91-94Crossref PubMed Scopus (54) Google Scholar, 24Jensen J.K. Durand M.K. Skeldal S. Dupont D.M. Bodker J.S. Wind T. Andreasen P.A. FEBS Lett. 2004; 556: 175-179Crossref PubMed Scopus (27) Google Scholar) is indicated in cyan. Asp138, part of both binding areas, is colored pink. The figures were prepared from the coordinates for the “stable” variant of PAI-1 (43Sharp A.M. Stein P.E. Pannu N.S. Carrell R.W. Berkenpas M.B. Ginsburg D. Lawrence D.A. Read R.J. Struct. Fold. Des. 1999; 7: 111-118Abstract Full Text Full Text PDF Scopus (162) Google Scholar) by the use of Swiss PDB Viewer version 3.7b2 (available on the World Wide Web at www.expasy.ch).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the present study, we have investigated the effects of Vn on the reactions of tPA and uPA with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 PAI-1 and its complexes with mAbs, interacting with α-helix F. Effects on kinetics of RCL insertion and stoichiometry of the reaction of NBD P9 PAI-1 with target proteinases were determined for Vn, in combination with Mab-2, its Fab fragment (Fab-2), and MA-55F4C12, which have overlapping epitopes at or close to the N-terminal part of α-helix F of PAI-1 (Fig. 1). Since protonation of a histidine residue at the PAI-1/tPA interface induces a significant increase in the limiting rate of RCL insertion for NBD P9 PAI-1 and its complex with MA-55F4C12 (28Komissarov A.A. Declerck P.J. Shore J.D. J. Biol. Chem. 2004; 279: 23007-23013Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar), the effects of Vn were studied at normal physiological pH and at pH 8.0 and 6.2. The results obtained reveal the mechanism of modulation of PAI-1 activity by Vn and demonstrate additivity in the effects of Vn and mAbs directed against α-helix F on the reactions of PAI-1 with tPA and uPA. Proteins and Reagents—Human Vn (2 mg/ml), purified from plasma, was from Promega (Madison, WI). Human recombinant tPA (Activase) was provided by Genentech (South San Francisco, CA). Human recombinant uPA was from Abbott. Analytical grade buffer reagents were from Sigma. The S338C (P9 Cys) PAI-1 mutant variant was purified, labeled with NBD, and characterized as described previously (13Shore J.D. Day D.E. Francis-Chmura A.M. Verhamme I. Kvassman J. Lawrence D.A. Ginsburg D. J. Biol. Chem. 1995; 270: 5395-5398Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The concentrations of NBD PAI-1, tPA, uPA, and antibodies were determined spectrophotometrically (29Komissarov A.A. Declerck P.J. Shore J.D. J. Biol. Chem. 2002; 277: 43858-43865Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). To minimize buffer change effects, all experiments were performed in 50 mm phosphate buffer solutions with pH 7.4, 6.2, and 8.0 at 25 °C. A buffer with a certain pH was prepared directly by mixing solutions of 50 mm KH2PO4 with 50 mm K2HPO4. The concentration of buffer was selected to provide capacity (at least 5 mm) to control pH in all of the experiments designed for this study. Stopped-flow Measurements—An SX-18MV stopped-flow reaction analyzer (Applied Photophysics Ltd.), equipped with a fluorescence detector, was used to study the effects of Vn on the rates of reaction between proteinases and complexes of NBD P9 PAI-1 (10-20 nm) with mAbs or Fab-2 (50-200 nm). RCL insertion was monitored by measuring an increase in NBD fluorescence emission through a 500-nm cut-off filter (excitation wavelength at 480 nm). Vn (30-80 nm) was added to preformed NBD P9 PAI-1·mAb complexes at least 20 min prior to data collection. During the progress of the reaction, the time-dependent increase in the fluorescence emission intensity of the NBD group has been recorded as 1000 data points. Data Analysis—Nonlinear least squares fitting of the results has been carried out employing the Levenberg-Marquardt algorithm in SigmaPlot 8.0 (SPSS Inc.) for Windows. The stopped-flow NBD fluorescence traces were analyzed using either the BioKine software (Applied Photophysics) or SigmaPlot 8.0 software. Assuming the pseudo-first-order kinetics (concentration of proteinase was at least 5-fold higher than concentration of NBD P9 PAI-1 or its complexes), the stopped flow traces were fit using the single exponential equation: Ft = A + B(1 - e-kobst), where Ft represents fluorescence emission (500-nm cut-off filter) at time t (seconds), A is an offset (the fluorescence signal at t = 0), and B is the amplitude. The values of kobs (average of 5-10 measurements; S.E. <10%) obtained at different concentrations of uPA or tPA were plotted against proteinase concentration using SigmaPlot 8.0, and the corresponding plots were fit by a hyperbolic equation kobs = klim × [E]/(Km + [E]), where klim is the limiting rate constant of RCL insertion, and Km is the proteinase concentration ([E]) at half of saturation. The quality of the fit of NBD fluorescence traces was estimated by visual analysis of plots of the residuals (deviation of the fitted function from the data). Correlation coefficients (r2) calculated from curve fittings were also used as a parameter of goodness of fit (r2 of fit exceeded 0.98 for all stopped-flow data). Measurements of Stoichiometry of Inhibition (SI)—To determine the effect of Vn, mAbs, and both ligands on the distribution between inhibitory and substrate branches of PAI-1 reaction, the SI was determined. Values of SI for NBD P9 PAI-1·mAb complexes, with and without Vn, were measured directly by titration of complexes of NBD P9 PAI-1 with tPA or uPA, as described previously (29Komissarov A.A. Declerck P.J. Shore J.D. J. Biol. Chem. 2002; 277: 43858-43865Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Binary complexes of NBD P9 PAI-1 (10-40 nm) with mAbs, Fab-2 (50-200 nm), or Vn (30-80 nm) as well as ternary Vn·NBD P9 PAI-1·mAb (Fab-2) complexes were preformed and equilibrated at room temperature for at least 30 min. A Varian Cary Eclipse fluorescence spectrophotometer (excitation wavelength of 480 nm, emission at 530 nm; slit widths 5 and 10 nm for excitation and emission, respectively) was employed for titration of NBD P9 PAI-1 and NBD P9 PAI-1·mAb, NBD P9 PAI-1·Vn, or Vn·NBD P9 PAI-1·mAb complexes. The titration was performed by monitoring an increase in NBD fluorescence emission (13Shore J.D. Day D.E. Francis-Chmura A.M. Verhamme I. Kvassman J. Lawrence D.A. Ginsburg D. J. Biol. Chem. 1995; 270: 5395-5398Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), resulting from interaction of NBD P9 PAI-1 or its complex with an aliquot of proteinase. After changes of fluorescence emission had reached the maximum, the next portion of the enzyme was added. At the end point of titration, the increase in fluorescence emission of the NBD group due to RCL insertion approached saturation, since all of the NBD P9 PAI-1 was cleaved. The SI values were calculated as the ratios between the numbers of moles of NBD P9 PAI-1 present and the numbers of moles of proteinase required for complete titration of uncomplexed NBD P9 PAI-1. The SI values were reported as averages of at least two independent experiments. Fab Fragment of Mab-2—The Fab fragment of monoclonal anti-PAI-1 clone 2 antibody (Mab-2) (30Nielsen L.S. Andreasen P.A. Grondahl-Hansen J. Huang J.Y. Kristensen P. Dano K. Thromb. Haemost. 1986; 55: 206-212Crossref PubMed Scopus (93) Google Scholar) was obtained by digesting Mab-2 with 1:100 (w/w) papain for 18 h. After digestion was stopped with E64, Fab fragments were separated from the Fc domains using a Protein A-coupled Sepharose column and affinity-purified on a PAI-1-coupled Sepharose column. The affinity of Fab-2 to NBD P9 PAI-1 and NBD P9 PAI-1·Vn was estimated from the dependence of kobs for the reaction with tPA on Fab-2 concentration, as described earlier for other anti-PAI-1 mAbs (29Komissarov A.A. Declerck P.J. Shore J.D. J. Biol. Chem. 2002; 277: 43858-43865Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Briefly, the reaction between tPA, taken at a concentration (1.5-2.0 μm) at least 2 times higher than the value of Km, and NBD P9 PAI-1 or its preformed complex with Vn were carried out using SX-18MV stopped-flow reaction analyzer. The values of kobs were calculated by fitting the changes in the fluorescence of NBD group by a single exponential process. The values of Kd were estimated from the midpoints of dependence of kobsversus the logarithm of Fab-2 concentration. The effects of Fab-2 on the reactions of NBD P9 PAI-1 with target proteinases as well as effects of Vn on SI and the kinetics of the RCL insertion, due to the reactions of NBD P9 PAI-1·Fab-2 complex with tPA and uPA at pH 6.2 and 8.0, were determined in a manner similar to the whole mAbs. Additivity in Effects of Vn on the Reaction of NBD P9 PAI-1·mAb Complexes with Target Proteinases—The effects of Vn on the kinetics of RCL insertion and stoichiometry of the reaction of NBD P9 PAI-1·mAb complexes with target proteinases was tested through comparison of changes in the free energy of activation for the limiting step of the PAI-1 reaction (ΔΔGRCL‡) and partition activation free energy (ΔΔGpartition‡). The values of ΔΔGRCL‡ were calculated from klim as ΔΔGRCL‡=−RTln(klim/klim0), where klim0 is the limiting rate of RCL insertion for the reaction of NBD P9 PAI-1 with target proteinase in the absence of ligands; klim values are the limiting rates of RCL insertion for NBD P9 PAI-1·mAb or ternary complexes Vn·NBD P9 PAI-1·mAb, T is the standard temperature 298 K, and R = 1.987 cal/mol K is the gas constant. The values of ΔΔGpartition‡(ΔΔGpartition‡=−RTln(ks/ki)=−RTln(SI−1)) were calculated from the SI values for NBD P9 PAI-1·mAb without and with Vn. The data were plotted as ΔΔGpartition‡versusΔΔGRCL‡. A line was fitted to the data, using SigmaPlot version 8.0 software. Effects of Vn and mAbs, Directed against α-helix F, on the Kinetics of RCL Insertion during the Reaction of NBD P9 PAI-1 with tPA and uPA—To determine the effects of Vn on the kinetics of RCL insertion, the dependences of the observed rate constant (kobs) of increase in the NBD-fluorescence emission on the proteinase concentration (Fig. 2) were determined at normal physiologic pH 7.4 (25 °C). The dependences of kobs on proteinase concentration always demonstrated saturation (Fig. 2) for both target proteinases. Therefore, tPA and uPA interact with complexes of NBD P9 PAI-1 with mAb or Vn in a manner similar to that described for uncomplexed NBD P9 PAI-1 (31Olson S.T. Swanson R. Day D. Verhamme I. Kvassman J. Shore J.D. Biochemistry. 2001; 40: 11742-11756Crossref PubMed Scopus (89) Google Scholar). The values of the limiting rate of RCL insertion (klim) and the concentration of proteinase at half-saturation (Km) for the reactions of NBD P9 PAI-1 and its mAb or Vn complexes with tPA and uPA were calculated (Table I). Vn, on its own, induced an almost 7-fold decrease in klim for the reaction of NBD P9 PAI-1 with uPA, but only a 1.4-fold decrease in klim for the reaction with tPA (Table I). Mab-2 alone induced a 4.5-fold decrease in klim for uPA and a 17-fold decrease in klim for tPA (Table I). The effect of Mab-2 on the reaction of NBD P9 PAI-1 with target proteinases was similar to that observed earlier for MA-55F4C12 (29Komissarov A.A. Declerck P.J. Shore J.D. J. Biol. Chem. 2002; 277: 43858-43865Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Both mAbs have epitopes at the N terminus of α-helix F (32Bijnens A.P. Gils A. Knockaert I. Stassen J.M. Declerck P.J. J. Biol. Chem. 2000; 275: 6375-6380Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Binding of Vn did not change dramatically the specificity (klim/Km) for the reaction with uPA; the decrease in klim, observed for the reaction of NBD P9 PAI-1 with uPA in the presence of Vn, was accompanied by similar changes in Km (Table I). In contrast, binding of mAbs induced significant decrease in klim for the reaction with tPA, without changes in Km (Table I), which resulted in a corresponding decrease in klim/Km. The different effects of Vn and mAbs on the kinetics of the reaction of PAI-1 with uPA and tPA could indicate different mechanisms of modulation of the PAI-1 reaction by Vn and mAbs.Table IEffects of Vn on the kinetics of RCL insertion (calculated by fitting a hyperbolic equation (kobs = klim × [E]/(Km + [E])) to plots of kobs versus proteinase concentration (Fig. 2) using SigmaPlot (SPSS, Inc.) version 8.0 for Windows) for the reactions of target proteinases with complexes of NBD P9 PAI-1 with Mab-2 and MA-55F4C12 All measurements were carried out at 25 °C in 0.05 m phosphate buffer, pH 7.4Ligand(s)tPAuPAklimKmklimKms-1μms-1μmNone2.4 ± 0.20.06 ± 0.0226.0 ± 1.83.0 ± 0.6Vn1.7 ± 0.10.09 ± 0.023.9 ± 0.10.55 ± 0.04Mab-20.14 ± 0.040.05 ± 0.025.8 ± 0.40.65 ± 0.15MA-55F4C120.18 ± 0.060.04 ± 0.016.1 ± 0.40.76 ± 0.17Mab-2 + Vn0.08 ± 0.010.04 ± 0.010.49 ± 0.030.13 ± 0.02MA-55F4C12 + Vn0.09 ± 0.030.05 ± 0.020.65 ± 0.150.10 ± 0.03 Open table in a new tab Vn affected the kinetics of RCL insertion for the reactions of NBD P9 PAI-1·mAb complexes with tPA and uPA in a manner similar to the reactions of the uncomplexed serpin (Table I). In the presence of Vn, klim for the reaction of complexes of NBD P9 PAI-1 with Mab-2 or MA-55F4C12 with uPA decreased by almost an order of magnitude (Table I). Similar to the effects on uncomplexed NBD P9 PAI-1, a decrease in klim for the reaction with uPA due to binding of Vn to NBD P9 PAI-1·mAb was accompanied by a 5.0- and 7.5-fold decrease in Km for Mab-2 and MA-55F4C12, respectively (Table I). In contrast to the reaction with uPA, Vn caused a 1.8-2.0-fold decrease in klim for the reaction of complexes with tPA with a corresponding decrease in the specificity of the reaction (Table I). The similarity in the effects of Vn on the reactions of free NBD P9 PAI-1 and its complexes with mAbs could indicate additivity in the effects of the two ligands on kinetics of RCL insertion during reaction with target proteinases. Effects of Vn and mAbs on SI for the Reactions of NBD P9 PAI-1 with Target Proteinases—To determine the effect of Vn on partitioning between inhibitory and substrate branches (Scheme 1) of the PAI-1·mAb mechanism, the stoichiometry of inhibition (SI) for the reactions of tPA and uPA with NBD P9 PAI-1·mAb complexes was measured with and without Vn (Fig. 3). Vn did not affect the SI for the reactions of NBD P9 PAI-1 with target proteinases, which agrees wit" @default.
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• W1992648023 title "Additivity in Effects of Vitronectin and Monoclonal Antibodies against α-Helix F of Plasminogen Activator Inhibitor-1 on Its Reactions with Target Proteinases" @default.
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