Type‐1 inhibitor of plasminogen activators (original) (raw)
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Journal of Biological Chemistry, 1999
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 -sheet A. A murine monoclonal antibody (MA-33B8), raised against the human plasminogen activator (tPA)⅐PAI-1 complex, rapidly inactivates PAI-1. Results presented here indicate that MA-33B8 induces acceleration of the active-to-latent conversion. The antibodyinduced inactivation of PAI-1 labeled with the fluorescent probe N,N-dimethyl-N-(acetyl)-N-(7-nitrobenz-2oxa-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 P9⅐NBD 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 ؋ 10 4 M ؊1 s ؊1), resulting in a 4000fold faster inactivation at 4.2 M 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.
Detection of specific forms of plasminogen activator inhibitor type 1-by monoclonal antibodies
Fibrinolysis, 1991
Monoclonal antibodies to plasminogen activator inhibitor type-l (PAI-1) were generated and characterised for their ability to inhibit PAI-interaction with tissue plasminogen activator (t-PA) and urokinase (u-PA) and detection of the various forms of PAI-(native, complexed, and degraded) by immunoblotting. Mabl7 inhibited both complex formation between PAI-and t-PA/u-PA and PAL-1 activity in a dose dependent manner by 90%. Mab 25 was much less effective, blocking complex formation less than 30% and PAI-activity less than 20%. The Kds of Mab17 and Mab25 were 2.8~ 10-l' M and 2.6~ lo-" M, respectively. Following SDS-PAGE and immunoblotting, Mab17 detected native PAI-only; PAI-in complex and the t-PA/u-PA degraded form of PAI-(M,=42000) did not react with this antibody. In contrast, Mab25 detected all three forms of PAI-although the affinity for the native form appeared to be greater than Mabl7 or the PAI-polyclonal employed. Despite these differences, both monoclonal antibodies immunoprecipitated native and degraded PAIequally as well. These results suggest that the epitope of Mab17 is associated with the reactive site of PAI-and that this region is either missing or not accessible in the cleaved form or in complex.
Journal of Biological Chemistry, 1998
The physiological roles of plasminogen activator inhibitor-2 (PAI-2) are not yet well understood. Kinetic studies suggest a role in the regulation of plasminogen activator-driven proteolysis in many cell types. This study describes a monoclonal antibody (2H5), which uniquely recognizes neoepitope determinants on PAI-2 appearing after thermodynamic relaxation of the molecule. Enzyme-linked immunosorbent assays and native polyacrylamide gel electrophoresis immunoblotting confirmed the specificity of 2H5 for urokinase type plasminogen activator⅐PAI-2 complexes. Examination of the affinity of 2H5 for complexes formed between PAI-2 and a synthetic 14-mer reactive site loop peptide, PAI-2 treated with tissue plasminogen activator, or thrombin suggests that the 2H5 epitope is determined exclusively by sequences found only on PAI-2 following proteolytic cleavage of the Arg 380-Thr 381 bond and insertion of the reactive site loop into -sheet A. Peptides lacking both the P13 (Glu 368) and P14 (Thr 367) residues did not induce a conformational change or affect the inhibitory activity of PAI-2, indicating that one or both of these residues are critical for PAI-2 function. To our knowledge, this is the first description of a monoclonal antibody that can distinguish conformational changes in PAI-2 related specifically to its potential biological function(s).
FEBS Letters, 1986
Both the urokinase-type and tissue-type plasminogen activator can convert their -54 kDa type-l inhibitor (PAI-1) to an inactive form with a lower apparent molecular mass. We have determined the amino-terminal amino acid sequences of human native and converted PAI-1, and isolated PAI-I cDNA and determined the nucleotide sequence in regions corresponding to the amino-terminus and the cleavage site. The data show that the conversion of the inhibitor consists of cleavage of an Arg-Met bond 33 residues from the carboxy-terminus, thus localizing the reactive center of the inhibitor to that position. In addition, a heterogeneity was found at the amino-terminus, with a Ser-Ala-Val-His-His form and a two-residue shorter form (Val-His-His-) occurring in approximately equal quantities.
European Journal of Biochemistry, 1997
We have analysed the susceptibility of latent, active, reactive-centre-cleaved and plasminogen-activator-complexed type-I plasniinogen-activator inhibitor (PAI-1) to the non-target proteinases trypsin, endoproteinase Asp-N, proteinase K and subtilisin. This analysis has allowed us to detect conformational differences between the different forms of PAI-1 outside the reactive-centre loop and P-sheet A. Proteinase-hypersensitive sites were clustered in three regions. Firstly, susceptibility was observed in the region around a-helix E, p-strand IA, and the flanking loops, which are believed to form tlexible joints during movements of p-sheet A. Secondly, hypersensitive sites were observed in the loop between a-helix I and p-strand 5A. Thirdly, the gate region, encompassing ,&strands 3C and 4C, was highly susceptible to trypsin in latent PAI-1, but not in the other conformations. The digestion patterns differed among all four forms of PAT-1, indicating that each represents a unique conformation. The differential proteolytic susceptibility of the flexible-joint region may be coupled to the differential affinity to vitronectin, binding in the same region. The analysis also allowed detection of conformational differences between reactivecentre-cleaved forms produced under different solvent conditions. The digestion pattern of plasminogenactivator-complexed PAI-1 was different from that of active PAI-1, but indistinguishable from that of one of the reactive-centre-cleaved forms, as the complexed and this particular cleaved PAI-1 were completely resistant to all the non-target proteinases tested. This observation is in agreement with the notion that complex formation involves reactive-centre cleavage and a large degree of insertion of the reactive-centre loop into /?-sheet A. Our analysis has allowed the identification of some flexible regions that appear to be implicated in the conformational changes during the movements of P-sheet A and during the inhibitory reaction of serpins with their target proteinases.
Fibrinolysis, 1995
Human recombinant PAI-1, expressed in Escherichia coil, was purified and separated into its active and latent components by chromatography on heparin-and phenyl-substituted agarose under conditions which favour the stability of the active inhibitor. Two columns, with a combined volume of less than 40 ml, were used to purify and separate up to 40mg of PAI-1 in one day. Purified fractions of PAI-1 were analysed by SDS-PAGE, fluorescence spectroscopy and thermostability measurements. A method for concentrating the inhibitor and conditions for storage of concentrated PAl-1 were established. Since PAl-1 spontaneously refolds its reactive-centre loop in a way similar to what is believed to occur in the proteinase-serpin complexes, studies with this inhibitor may play an important role in elucidating the mechanism of serpin action. The method we are presenting yields highly purified fractions of active and latent PAI-1 with relative ease and facilitates detailed investigations of its reaction mechanism.
Biochemical Journal, 1990
The structural events taking place during the reaction between PAI-1 (plasminogen-activator inhibitor 1) and the plasminogen activators sc-tPA (single-chain tissue plasminogen activator) and tc-tPA (two-chain tissue plasminogen activator) were studied. Complexes were formed by mixing sc-tPA or tc-tPA with PAI-1 in slight excess (on an activity basis). The complexes were purified from excess PAI-1 by affinity chromatography on fibrin-Sepharose. Examination of the purified complexes by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE) and N-terminal amino acid sequence analysis demonstrated that a stoichiometric 1:1 complex is formed between PAI-1 and both forms of tPA. Data obtained from both complexes revealed the amino acid sequences of the parent molecules and, in addition, a new sequence: Met-Ala-Pro-Glu-Glu-. This sequence is found in the C-terminal portion of the intact PAI-1 molecule and thus locates the reactive centre of PAI-1 to Arg346-Met347. The proteolytic activity of sc...
Journal of Molecular Biology, 2000
The crystal structure of a constitutively active multiple site mutant of plasminogen activator inhibitor 1 (PAI-1) was determined and refined at a resolution of 2.7 A. The present structure comprises a dimer of two crystallographically independent PAI-1 molecules that pack by association of the residues P6 to P3 of the reactive centre loop of one molecule (A) with the edge of the main beta-sheet A of the other molecule (B).Thus, the reactive centre loop is ordered for molecule A by crystal packing forces, while for molecule B it is unconstrained by crystal packing contacts and is disordered. The overall structure of active PAI-1 is similar to the structures of other active inhibitory serpins exhibiting as the major secondary structural feature a five-stranded beta-sheet A and an intact proteinase-binding loop protruding from the one end of the elongated molecule. No preinsertion of the reactive centre loop is observed in this structure.A comparison of the present structure with the previously determined crystal structures of PAI-1 in its alternative conformations reveals that, upon cleavage of an intact form of PAI-1 or formation of latent PAI-1, the well-characterised rearrangements of the serpin secondary structural elements are accompanied by dramatic and partly unexpected conformational changes of helical and loop structures proximal to beta-sheet A. The present structure explains the stabilising effects of the mutated residues, reveals the structural cause for the observed spectroscopic differences between active and latent PAI-1, and provides new insights into possible mechanisms of stabilisation by its natural binding partner, vitronectin.
Evidence for a Discrete Binding Protein of Plasminogen Activator Inhibitor in Plasma
Thrombosis and Haemostasis, 1988
SummaryGel-filtration experiments of mixtures of functionally active and inactive forms of plasminogen activator inhibitor (PAI) with human plasma or bovine serum albumin have provided evidence for the existence of a discrete binding protein of PAI in plasma. Most likely it is a glycoprotein with a molecular weight of approximately 150,000. The data suggest that it forms a very stable complex with functionally active forms of PAI, but not with the inactive or “latent” PAI. However, the PAI activity seems not to be significantly altered by the interaction with the binding protein. Assuming that a stoichiometric complex is formed, titration experiments suggest that a pool of normal human plasma contains about 40–50 mg of PAI-binding protein liter.