Accelerated Conversion of Human Plasminogen Activator Inhibitor-1 to Its Latent Form by Antibody Binding

The serpin plasminogen activator inhibitor-1 (PAI-1) slowly converts to an inactive latent form by inserting a major part of its reactive center loop (RCL) into its β-sheet A. A murine monoclonal antibody (MA-33B8), raised against the human plasminogen activator (tPA)·PAI-1 complex, rapidly inactivates PAI-1. Results presented here indicate that MA-33B8 induces acceleration of the active-to-latent conversion. The antibody-induced inactivation of PAI-1 labeled with the fluorescent probeN,N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) at P9 in the RCL caused a fluorescence enhancement and shift identical to those accompanying the spontaneous conversion of the P9·NBD PAI-1 to the latent form. Like latent PAI-1, antibody-inactivated PAI-1 was protected from cleavage by elastase. The rate constants for MA-33B8 binding, measured by NBD fluorescence or inactivation, were similar (1.3–1.8 × 104m−1 s−1), resulting in a 4000-fold faster inactivation at 4.2 μm antibody binding sites. The apparent antibody binding rate constant, at least 1000 times slower than one limited by diffusion, indicates that exposure of its epitope depends on an unfavorable equilibrium of PAI-1. Our observations are consistent with this idea and suggest that the equilibrium involves partial insertion of the RCL into sheet A: latent, RCL-cleaved, and tPA-complexed PAI-1, which are inactive loop-inserted forms, bound much faster than active PAI-1 to MA-33B8, whereas two loop-extracted forms of PAI-1, modified to prevent loop insertion, did not bind or bound much more weakly to the antibody. The serpin plasminogen activator inhibitor-1 (PAI-1) slowly converts to an inactive latent form by inserting a major part of its reactive center loop (RCL) into its β-sheet A. A murine monoclonal antibody (MA-33B8), raised against the human plasminogen activator (tPA)·PAI-1 complex, rapidly inactivates PAI-1. Results presented here indicate that MA-33B8 induces acceleration of the active-to-latent conversion. The antibody-induced inactivation of PAI-1 labeled with the fluorescent probeN,N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) at P9 in the RCL caused a fluorescence enhancement and shift identical to those accompanying the spontaneous conversion of the P9·NBD PAI-1 to the latent form. Like latent PAI-1, antibody-inactivated PAI-1 was protected from cleavage by elastase. The rate constants for MA-33B8 binding, measured by NBD fluorescence or inactivation, were similar (1.3–1.8 × 104m−1 s−1), resulting in a 4000-fold faster inactivation at 4.2 μm antibody binding sites. The apparent antibody binding rate constant, at least 1000 times slower than one limited by diffusion, indicates that exposure of its epitope depends on an unfavorable equilibrium of PAI-1. Our observations are consistent with this idea and suggest that the equilibrium involves partial insertion of the RCL into sheet A: latent, RCL-cleaved, and tPA-complexed PAI-1, which are inactive loop-inserted forms, bound much faster than active PAI-1 to MA-33B8, whereas two loop-extracted forms of PAI-1, modified to prevent loop insertion, did not bind or bound much more weakly to the antibody. Plasminogen activator inhibitor-1 (PAI-1) 1The abbreviations used are: PAI, plasminogen activator inhibitor; NBD, N,N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine; Bicine, N,N-bis(2-hydroxyethyl)glycine; PAGE, polyacrylamide gel electrophoresis; RCL, reactive center loop; tPA, human tissue-type plasminogen activator; Ab, antibody. 1The abbreviations used are: PAI, plasminogen activator inhibitor; NBD, N,N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine; Bicine, N,N-bis(2-hydroxyethyl)glycine; PAGE, polyacrylamide gel electrophoresis; RCL, reactive center loop; tPA, human tissue-type plasminogen activator; Ab, antibody. is a serine proteinase inhibitor of the serpin family, a class of inhibitory and noninhibitory proteins sharing a common tertiary structure. Its most prominent features are the major, 5-stranded β-sheet A, which can accommodate an additional strand in position 4, and a flexible reactive center loop (RCL) of about 20 residues denoted P15-P5′ (1Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4742) Google Scholar, 2Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (832) Google Scholar). Inhibitory serpins function as suicide substrate inhibitors and acylate their target proteinases (3Lawrence D.A. Ginsburg D. Day D.E. Berkenpas M.B. Verhamme I. Kvassman J.-O. Shore J.D. J. Biol. Chem. 1995; 270: 25309-25312Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 4Wilczynska M. Fa M. Ohlsson P.-I. Ny T. J. Biol. Chem. 1995; 270: 29652-29655Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The acyl-enzyme complex is stabilized by insertion of the cleaved RCL as strand 4 of β-sheet A, accompanied by a translocation of the acylated enzyme from its initial binding site to its “locking” site. If RCL insertion is hindered by mutating the P14 threonine to an arginine (5Lawrence D.A. Olson S.T. Palaniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: 27657-27662Abstract Full Text PDF PubMed Google Scholar), or by blocking position 4 of β-sheet A with a peptide analog of the P14-P7 part of the RCL (6Kvassman J.-O. Lawrence D. Shore J.D. J. Biol. Chem. 1995; 270: 27942-27947Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), PAI-1 becomes a substrate that is completely hydrolyzed at the scissile bond by its target proteinases. PAI-1 is the only serpin known to fold spontaneously to an inactive latent form (7Mottonen J. Strand A. Symerski J. Sweet R.M. Danley D.E. Geoghegan K.F. Gerard R.D. Goldsmith E.J. Nature. 1992; 355: 270-273Crossref PubMed Scopus (525) Google Scholar), and the t ½ for this conversion is 9.5 h at 25 °C and pH 7.4 (6Kvassman J.-O. Lawrence D. Shore J.D. J. Biol. Chem. 1995; 270: 27942-27947Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 8Shore 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). In this process, the P15-P3 portion of the RCL, which is normally exposed at the apex of the molecule, is inserted as strand 4 in β-sheet A, forcing the remainder of the loop, including the reactive center, to adopt an extended conformation alongside the protein scaffold. The term “latent” was introduced because PAI-1 traditionally has been isolated in the inactive form, and its activity was partly restored by transient treatment with chaotropic agents (9Lawrence D. Strandberg L. Grundström T. Ny T. Eur. J. Biochem. 1989; 186: 523-533Crossref PubMed Scopus (119) Google Scholar). In the absence of such treatment, however, formation of latent PAI-1 is a virtually irreversible process (6Kvassman J.-O. Lawrence D. Shore J.D. J. Biol. Chem. 1995; 270: 27942-27947Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Latent PAI-1 has greater thermal than PAI-1 M. Eur. J. Biochem. PubMed Scopus Google Scholar), and its reactive center is with for the conversion is that in the latent form, the RCL is protected from cleavage by T. M. M. A. 1995; PubMed Google Scholar). of murine monoclonal against the human complex been with to their on the and function of one inactivates PAI-1 forms a complex with its target is hydrolyzed at the scissile the inactivates PAI-1 by a substrate for its target proteinases S. Biophys. PubMed Scopus Google Scholar). The on one antibody of the denoted MA-33B8, which has been to PAI-1 rapidly J.D. S. Day D.E. Verhamme I. and of Ginsburg D. M. in and Scholar). a of PAI-1 or to the epitope for MA-33B8 S. and S. and and the by which inactivates PAI-1 of the antibody can the reactive center bond in PAI-1 from proteinase by one of two that antibody binding with binding of the proteinase or that antibody binding induces a that the reactive center into a which the the active-to-latent conversion was by the antibody. that the PAI-1, with the at P9 in the RCL, and labeled with NBD at the to the spontaneous conversion to the latent form by a and a shift of the NBD fluorescence and that are caused by loop insertion 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 fluorescence enhancement is consistent with a translocation of the NBD from a exposed of the RCL of active PAI-1 to the more position in the latent form in the of the loop and β-sheet A, fluorescence were P9·NBD PAI-1 with tPA, plasminogen and as as with of which the RCL and insertion of the RCL into β-sheet A, whereas from P9·NBD PAI-1 with and been by their serine residues to and 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 S.T. Kvassman J.-O. Shore J.D. Lawrence D.A. Ginsburg D. I. J. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). J.-O. D. and J. D. J.-O. D. and J. D. In the that the inactivation of P9·NBD PAI-1 with in the NBD fluorescence that were identical to those the spontaneous inactivation of the labeled that the active and latent PAI-1 in to by are by the antibody-induced inactivation of PAI-1. the fluorescence of P9·NBD PAI-1 or the inactivation of PAI-1 as a measured rate constants for MA-33B8 binding of the of 104m−1 The rate constants for the antibody the J. T. J. Biophys. PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar), the by The much rate for binding MA-33B8 to PAI-1 an in the binding of MA-33B8 to PAI-1, an unfavorable in PAI-1. this involves loop insertion, loop-inserted forms of PAI-1, as as two with a rate of loop insertion, were for their to with P9·NBD PAI-1 for binding to a in consistent with antibody binding to a site on PAI-1 that is exposed only the RCL is inserted into β-sheet A. the of P9·NBD PAI-1 with and indicate that binding of not with binding of If not and were at pH 7.4 and 25 °C in a were an The used for NBD fluorescence was were with to protein by was to Nature. PubMed Scopus Google Scholar). and were from was from The and was from D. of was from were from The was by the of PAI-1 was in the (9Lawrence D. Strandberg L. Grundström T. Ny T. Eur. J. Biochem. 1989; 186: 523-533Crossref PubMed Scopus (119) Google Scholar). The and PAI-1 as as a PAI-1 were by as (9Lawrence D. Strandberg L. Grundström T. Ny T. Eur. J. Biochem. 1989; 186: 523-533Crossref PubMed Scopus (119) Google Scholar). PAI-1 was and into the active and latent forms to a from this J.-O. Shore J.D. 1995; Scopus Google Scholar). PAI-1 protein were measured at an of and a of D.E. D. Biophys. PubMed Scopus Google Scholar). The of the P9 PAI-1 was labeled with NBD as 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). latent PAI-1, in or and PAI-1 were by with and P9·NBD PAI-1 were with the as (6Kvassman J.-O. Lawrence D. Shore J.D. J. Biol. Chem. 1995; 270: 27942-27947Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and peptide was by gel on a In the with PAI-1, latent PAI-1, in the process, was by on J.-O. Shore J.D. 1995; Scopus Google Scholar). of the by and of tPA, that was of active PAI-1 and than PAI-1 that was not hydrolyzed by The complex is and not the (6Kvassman J.-O. Lawrence D. Shore J.D. J. Biol. Chem. 1995; 270: 27942-27947Abstract Full Text Full Text PDF PubMed Scopus (68) Google or cleavage of the RCL by plasminogen as PAI-1 hydrolyzed at the peptide bond in the RCL was with μm PAI-1 and was by on (9Lawrence D. Strandberg L. Grundström T. Ny T. Eur. J. Biochem. 1989; 186: 523-533Crossref PubMed Scopus (119) Google Scholar), which PAI-1 not elastase. The complex was by of PAI-1 with for in the at was by treatment with was a of The enzyme was to the form by treatment with J.-O. Shore J.D. 1995; Scopus Google Scholar). The monoclonal antibody was raised against the complex as S. Biophys. PubMed Scopus Google Scholar). The was from by on protein PubMed Scopus Google Scholar). The antibody protein was at an of and a of The antibody binding site is as two and binding on and P9·NBD PAI-1 were with MA-33B8 in at a from was and to active PAI-1 was on the of the activity of the substrate and against The latent form of P9·NBD PAI-1 was by of active P9·NBD PAI-1 in a and at 25 °C for at pH in Bicine, (6Kvassman J.-O. Lawrence D. Shore J.D. J. Biol. Chem. 1995; 270: 27942-27947Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 8Shore 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 of the was for inhibitory activity and by with at pH inactivation of the labeled was by P9·NBD PAI-1 with MA-33B8 and was 20 P9·NBD PAI-1 and the spontaneously and P9·NBD PAI-1 were to μm in the and the fluorescence of was The of MA-33B8 on the NBD fluorescence of spontaneously latent P9·NBD PAI-1 as as on that of the and P14 of P9·NBD PAI-1 was by the and antibody. The P9·NBD PAI-1 used in this was from the The rate of the fluorescence by P9·NBD PAI-1 with MA-33B8 was measured at as a function of antibody to P9·NBD PAI-1 at μm to The rate constants were by least of a to the fluorescence The apparent rate for antibody binding was by the rate constants against the of P9·NBD PAI-1 and MA-33B8 were at and and the of the NBD fluorescence was at in the absence and of latent PAI-1 at from to In P9·NBD PAI-1 and MA-33B8 were and the of the NBD fluorescence was in the absence and of of the PAI-1 active PAI-1 PAI-1 in complex with PAI-1 and the complex the fluorescence of P9·NBD PAI-1 with MA-33B8 was in the absence and of P14 PAI-1 binding were on the of the MA-33B8 for protein A. Latent and PAI-1 were to a of MA-33B8 in 20 pH to with The was of protein with the pH 7.4 The was with the and that was The antibody was with a of to pH with and antibody were by and treatment with at pH The of PAI-1 forms to by was by of inhibitor and enzyme for 20 at The were with and by The were active and P9·NBD PAI-1, latent PAI-1, and the antibody-inactivated and P9·NBD PAI-1 The were by the active PAI-1 form with MA-33B8 for 20 P9·NBD PAI-1 was with MA-33B8 in the absence and of and the resulting fluorescence enhancement as a function of the fluorescence PAI-1 was with and the were by the antibody. a and P9·NBD PAI-1 were in the absence of antibody. the fluorescence enhancement caused by MA-33B8 with P9·NBD PAI-1 was in the absence and of the of on PAI-1 active and P9·NBD PAI-1 were with and the activity of the two PAI-1 forms was at The of MA-33B8 on and P9·NBD PAI-1 were by the PAI-1 form with MA-33B8 and inhibitory activity as a function of that with 4.2 MA-33B8, the antibody-induced inactivation of and P9·NBD PAI-1 at to inactivation rate enhancement of and The for P9·NBD PAI-1 from its rate of spontaneous on the of inactivation of and P9·NBD PAI-1 by MA-33B8 at the apparent rate for antibody binding to the PAI-1 form was and × the inactive forms of P9·NBD PAI-1 in the absence and of the of P9·NBD PAI-1 and spontaneous or inactivation were The in that antibody-induced inactivation of P9·NBD PAI-1 to the NBD identical to the spontaneous inactivation of P9·NBD PAI-1 a shift of the and a fluorescence The NBD fluorescence of latent P9·NBD PAI-1 was not by of The spontaneously and antibody-induced inactive forms of P9·NBD PAI-1 were with to by elastase. in that to of and with active P9·NBD PAI-1 a consistent with of the RCL peptide as for of PAI-1 by T. M. M. A. 1995; PubMed Google Scholar). inactivation by PAI-1 P9·NBD PAI-1 was hydrolyzed by The of the NBD fluorescence by μm P9 NBD PAI-1 with MA-33B8 is in The rate for a of such with antibody to the The an apparent rate for antibody-induced fluorescence enhancement of × similar to the of × 104m−1 presented and on the rate of inactivation of P9·NBD PAI-1 by the antibody. presented indicate that MA-33B8 binding to PAI-1 insertion of the RCL into β-sheet A. is a enhancement of antibody binding the RCL is into sheet A, active PAI-1 and of the inhibitor were for their to with P9·NBD PAI-1 for binding to PAI-1, P9·NBD PAI-1, and MA-33B8 were in the of the NBD fluorescence was to about of that in the absence of the inhibitor that P9·NBD and PAI-1 for binding to The loop-inserted PAI-1 forms latent, and on the the of the NBD fluorescence in a is most by the of in which a in the fluorescence enhancement of P9·NBD PAI-1 with the of latent PAI-1. is by the of the of MA-33B8 with P9·NBD PAI-1 by the loop-inserted PAI-1 forms in The indicates that binding of the loop-inserted PAI-1 forms to MA-33B8 is faster than binding of active PAI-1 by a than that that the rate constants are to or of inactivation of NBD PAI-1 by latent PAI-1. A, the fluorescence PAI-1 with MA-33B8 in the absence and of and μm latent PAI-1 in the of the in A against the of latent PAI-1. The indicates of the fluorescence enhancement at a of latent PAI-1 to that of antibody binding the against loop insertion in PAI-1 caused by mutating the threonine at P14 in the RCL to an arginine or by binding the to position 4 in sheet A, were introduced into P9·NBD PAI-1. loop insertion to by of the NBD on the of the on PAI-1, the modified P9·NBD PAI-1 forms were for and hydrolyzed completely at the scissile insertion of the RCL was with P14 P9·NBD PAI-1 and to measured with the P9·NBD form. In the RCL of inhibitory P9·NBD PAI-1 with a rate of in the with 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 binding of MA-33B8 to active PAI-1 caused by an unfavorable equilibrium in PAI-1 that the epitope in a conformation of the inhibitor with insertion of the RCL into β-sheet A. this the binding were extended to P14 PAI-1 and PAI-1 in complex with by from the presented on the of P9·NBD PAI-1, are from inserting the RCL into β-sheet A at a the P14 PAI-1, on the rate of binding MA-33B8 to P9·NBD PAI-1 at μm P14 PAI-1, a P9·NBD PAI-1 and MA-33B8 binding A on the rate of the fluorescence not on its was PAI-1 was to a of P9·NBD and MA-33B8 on this rate and the that binding of PAI-1 to MA-33B8 with the of the the binding the PAI-1 to MA-33B8 was to The absence of an of P14 PAI-1 on MA-33B8 binding to P9·NBD PAI-1 indicates that the antibody this with a that The and of MA-33B8 for latent and peptide PAI-1, were by to PAI-1 form from a of antibody binding on protein A. A the the Latent PAI-1 was not on the Latent PAI-1 and however, were completely on the and were at pH the most of the PAI-1 was from the antibody in the and in the The however, was extended at its in with the of antibody used and the as as the of are in PAI-1 has for and R. Biochemistry. PubMed Scopus Google D.A. Berkenpas M.B. Palaniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). were to binding of with binding of and on the NBD fluorescence of P9·NBD PAI-1, consistent with the binding from the exposed the rate or of the fluorescence enhancement of MA-33B8, that the binding for and on PAI-1 D.A. Berkenpas M.B. Palaniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, J. 1995; PubMed Scopus Google not with that for MA-33B8 and that the two not with the of loop binding has been to the rate by which PAI-1 becomes latent M. S. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The absence of an of on the rate of of the P9·NBD fluorescence by MA-33B8 for the by which and MA-33B8 PAI-1. the used in this was to a on PAI-1, than the of for M. S. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of on P9·NBD PAI-1 was presented that suggest that the murine monoclonal antibody MA-33B8, against the human complex, rapidly converts inhibitory PAI-1 to its latent form. inactivation of the P9 PAI-1, labeled with the fluorescent NBD at the in a enhancement and shift of the NBD identical to the for the spontaneous conversion of the labeled to the latent form. Like latent PAI-1, antibody-inactivated PAI-1 was with target as as with T. M. M. A. 1995; PubMed Google Scholar). The rate of the fluorescence enhancement of P9·NBD PAI-1 was to antibody to the with an apparent rate of × 104m−1 rate constants and × 104m−1 were on the rate of inactivation of P9·NBD PAI-1 and PAI-1 by MA-33B8, which at 4.2 antibody in of inactivation and times faster than the spontaneous inactivation of the PAI-1 form. MA-33B8 bind to an epitope on PAI-1 in the of the reactive center and the P9·NBD fluorescence and prevent proteinases from the The that antibody binding to active PAI-1 of the RCL not in that this in is that of the RCL of P9·NBD PAI-1, insertion into sheet A, in of the fluorescence of the that are identical to those resulting from formation of the latent labeled PAI-1. The that the of binding to MA-33B8 of PAI-1 forms than active P9·NBD PAI-1 additional PAI-1 at a to that of the P9·NBD the of the antibody-induced fluorescence enhancement by indicates that P9 NBD and PAI-1 bind to MA-33B8 at similar consistent with the that the antibody the two PAI-1 forms at The loop-inserted forms of PAI-1, however, including latent PAI-1, PAI-1, and the complex, bound much more rapidly to MA-33B8 than active PAI-1, that loop insertion to a greater exposure of the The that the PAI-1 and the P14 which rapidly the loop, or not at with MA-33B8 is consistent with this A of the antibody is the with which conversion to 25 the spontaneous active-to-latent conversion of and PAI-1 has a t ½ of 9.5 and h whereas in the of 4.2 MA-33B8 ½ was to for for was in the antibody of the rate of conversion to the latent form, that inactivation of PAI-1 faster at this rate antibody binding a and faster for in PAI-1, or the faster by the the of the active PAI-1 and a major for its conversion to the latent form. In the the epitope is exposed in active PAI-1 and binding is for the conformation of the inhibitor or a binding, however, a of the inhibitor the of which are from that of the and which is at the of the of thermal If the epitope were exposed in active PAI-1, as by the in MA-33B8 bind active PAI-1 as as the loop inserted PAI-1 The in however, that whereas binding of MA-33B8 to loop-inserted forms of PAI-1 at a rate to that for antibody s−1), the rate for MA-33B8 binding to active PAI-1 was for an apparent rate for the process in as as 104m−1 to that the is much slower than the of the binding If this were the however, of rate at of MA-33B8 to the constant, which is by than 4.2 μm J. T. J. Biophys. PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar). the in can In the presented in the form of active PAI-1 the epitope for The epitope becomes completely exposed in a conformation that is a for formation of the latent binding this and its to the active resulting in an rate of PAI-1 is to that used to the rate enhancement caused by binding a substrate to the of an enzyme and and Scholar). The spontaneous formation of latent PAI-1 was for the of antibody binding The binding of MA-33B8 to latent PAI-1 is in of the in not the rate one of the two to are to the rate enhancement is to the of and for the antibody and is and are the equilibrium constants for of and Our indicate that which that MA-33B8 more than times than I. The rate enhancement was not at 4.2 binding which the that at this or MA-33B8 binding to PAI-1 which the conformation of to that of in by the binding of MA-33B8 to the loop exposed PAI-1 is binding of the antibody in to an exposed to for the of and into the latent of however, the on the in In exposure of the epitope in by an unfavorable that the rate measured for formation of latent PAI-1 with that measured for in and for binding of MA-33B8 to the exposed epitope in PAI-1 in a in with is of is binding of to or I. at an antibody binding site as as 4.2 the rate of inactivation is not The equilibrium of is the of the two of and involves binding to an epitope exposed in an unfavorable equilibrium or unfavorable binding to a partly exposed to a more equilibrium for its In with that MA-33B8 at a rate to latent, and at a rate to active and not at or only weakly to the P14 and PAI-1 The is by or partial insertion of strand 4 into sheet active PAI-1 has its RCL exposed the for loop and the RCL of the substrate PAI-1 forms is exposed and at a this that the that the epitope for MA-33B8 is with insertion of the RCL into sheet A. the spontaneous formation of latent PAI-1 is virtually irreversible and because antibody binding the rate of this process by a than the epitope exposed in an in the absence of has greater to to the active loop conformation than to to form latent PAI-1, as in insertion of strand to to for additional is that insertion of only a of the residues of strand is to the A is that the epitope for MA-33B8 is exposed in an that has inserted the part of the RCL into sheet A to a such that insertion of at its Our not to the epitope for insertion can an epitope that is by the RCL, or with a that an epitope on the however, the epitope residues that are not exposed in the the residues in the portion of the RCL, which are inserted into sheet A, by the loop, or in with the are residues by the proteinase at its site. The that latent P9·NBD PAI-1 MA-33B8 in NBD fluorescence against the epitope of the on residues in the loop that which has been to the for the murine that PAI-1 to a substrate for S. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). Our indicate that the and binding on PAI-1 not the epitope for of a form of to PAI-1 has been to the rate of by a M. S. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). however, has not been with which was used in this of the inactivation of and P9·NBD PAI-1 in the of at a that was on observations and on presented in the A. Day D.E. Verhamme I. Shore J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), a on PAI-1 of only whereas on P9·NBD PAI-1 was The absence of an of on the rate of of the P9·NBD PAI-1 fluorescence by antibody binding is consistent with In that binding of MA-33B8 induces at least a 4000-fold of the of active PAI-1 and that the inactive is latent PAI-1. presented that suggest that binding of antibody depends on partial insertion of the RCL into β-sheet A of the inhibitor and that antibody binding an that or is to a on the to latent PAI-1. Our the for at the epitope for MA-33B8 and the for the as as the formation of latent PAI-1.