Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proteolytic modification of circulating insulin-like growth factor binding protein-3 (IGFBP-3) has been described in a number of conditions. Using Western ligand blotting and SDS-PAGE analysis of fragmentation patterns of 125I-labelled IGFBP-3 and 125-labelled IGFBP-1, we have examined conditioned media from cultured human cell lines for the presence of proteolytic activity and compared this with the action of circulating proteases and with characterized enzymes including cathepsin D, kallikrein, plasmin and tissue plasminogen activator. 125I-Labelled IGFBP-3 was incubated with serum from pregnant women, patients following heart surgery and patients with cancer of the breast, lung or head/neck. Following separation of the preincubated samples by SDS-PAGE, a distinct pattern of degradation fragments was observed which was similar in all cases. This proteolytic activity was inhibited in the presence of EDTA, phenanthroline and 4(-2-aminoethyl)-benzenesulphonylfluoride, HCl. These proteases had no detectable effect on IGFBP-1. Serum-free conditioned medium from a human dermal fibroblast cell line, a rabdomyosarcoma, a cervical, a bladder, a chorio- and two-tongue squamous cell carcinoma cell lines all contained proteolytic activity which fragmented IGFBP-3. The pattern of fragments was similar in all cell lines but different from that produced by the circulating proteases. Six out of nine cell lines produced protease(s) which degraded IGFBP-1 in addition to IGFBP-3. Whilst all the characterized enzymes tested also fragmented IGFBP-3 and plasmin cleaved IGFBP-1, none of these acted in the same way as either circulating or cell line-derived proteolytic activity. The activity associated with the characterized enzymes and cell lines was inhibited in the presence of serum from normal healthy subjects. These results demonstrate that the serum of pregnant women, post-operative patients and patients with cancer contain circulating proteases which cause fragmentation of IGFBP-3 but have little effect on IGFBP-1. Cell-derived proteases were shown to act on IGFBP-3 and IGFBP-1 in a number of instances but are not active in the presence of circulating inhibitors. These proteases may play an important role in regulating the availability of IGFs to normal and neoplastic tissues.
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PMID:Proteolytic modification of insulin-like growth factor-binding proteins: comparison of conditioned media from human cell lines, circulating proteases and characterized enzymes. 750 92

When human leukocyte elastase (HLE) activity (1.0 microgram/ml) was analysed in the presence of PAI-1 (0.15.0 micrograms/ml), HLE activity, measured with the low molecular weight paranitroanilide substrate L-pyroglutamyl-prolyl-L-valine-p-nitroanilide was increased time and dose dependently (a plateau of stimulation was reached after 30 minutes) with a simultaneous decrease in PAI-1 inhibitory activity. This effect was neither influenced by the presence of vitronectin nor heparin. When PAI-1 was converted into its latent form by incubation for 48 hours at 37 degrees C or incubated with an excess of recombinant t-PA to convert free PAI-1 into t-PA-PAI-1 complexes, the stimulatory effect of both the latent and the complexed form of PAI-1 was significantly greater than that of the active form. Analysing HLE PAI-1 interaction on a molecular level using SDS-PAGE, no SDS stable complex formation between HLE and PAI-1 could be observed but lower molecular weight cleavage products of PAI-1 were generated. The stimulatory effect of PAI-1 on HLE activity was not restricted to the low molecular weight pNA-substrate but was also revealed using a natural substrate assay (bovine neck ligament elastin solubilization). Therefore interaction of HLE and PAI-1 seems not to be restricted just to decrease PAI-1 activity but would simultaneously also increase HLE activity, thereby supporting enzymatic activity necessary for migration of leukocytes, dissolution of blood clots and tissue remodelling.
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PMID:Effect of type-1 plasminogen activator inhibitor on human leukocyte elastase. 752 Oct 69

Plasminogen activator inhibitor-1 (PAI-1) is the most important inhibitor of tissue-type plasminogen activator (t-PA) in plasma and plays a major role in the regulation of fibrinolysis. Plasma t-PA/PAI-1 complexes are cleared via a receptor-dependent mechanism in hepatocytes, while the fate of complexes formed in the extracellular matrix and in thrombi is less well understood. In this study, the degradation of t-PA/PAI-1 complexes by monocytes was examined. THP-1 monocytoid cells and freshly isolated human monocytes internalize and degrade [125I]t-PA/PAI-1 complexes at rates of 11.4 +/- 5.9 (mean +/- S.D.) and 44.6 +/- 6.3 ng/10(6) cells/h, respectively. Degradation is blocked by receptor-associated protein (RAP), indicating a member of the low density lipoprotein (LDL) receptor family is involved in the uptake/degradation of t-PA/PAI-1 complexes by monocytes. Degradation of t-PA/PAI-1 complexes is also inhibited by chloroquine and by pepstatin A, suggesting that a lysosomal aspartyl protease is likely involved. SDS-PAGE and Western blotting demonstrated that the purified lysosomal aspartyl protease, cathepsin D, is capable of digesting t-PA (t1/2 15 min), active PAI-1 (t1/2 2 h), and t-PA/PAI-1 complex (t1/2 30 min). Cathepsin D sequentially cleaves PAI-1 after hydrophobic amino acids, yielding lower molecular weight fragments. PAI-1 conformation influences the degradative efficiency of cathepsin D, with vitronectin-bound PAI-1 and latent PAI-1 exhibiting resistance to proteolysis and > 10-fold prolongation in t1/2. These data provide evidence that t-PA/PAI-1 complexes are internalized by human monocytes via a member of the low density lipoprotein (LDL) receptor family, and identifies cathepsin D-like aspartyl protease activity as largely responsible for the degradation of these complexes. Furthermore, vitronectin-bound PAI-1 and latent PAI-1 are relatively resistant to degradation by cathepsin D, which may be of importance in complex physiological environments.
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PMID:Cathepsin D-like aspartyl protease activity mediates the degradation of tissue-type plasminogen activator/plasminogen activator inhibitor-1 complexes in human monocytes. 766 1

An inhibitor of tissue-type plasminogen activator (tPA)-mediated and plasminogen-dependent fibrinolysis was isolated from human neutrophils. On a G-50 gel filtration column, the antifibrinolytic activity present in neutrophil homogenates comigrated with proteins of < 13 kDa. The inhibitory fraction had only a slight effect on urokinase with plasminogen- or plasmin-mediated fibrinolysis and no effect on urokinase- or plasmin-mediated cleavage of H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251). The neutrophil-derived fraction inhibited tPA with plasminogen activity on S-2251 but not on H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288). The inhibition of tPA-mediated and plasminogen-dependent fibrinolysis or S-2251 cleavage showed a competitive pattern and could be relieved by increasing the concentration of plasminogen. The same fraction also inhibited binding of plasminogen to fibrin. Consecutive purification steps revealed that the molecular mass of the inhibitor was 1-5-kDa. Polylysine-Sepharose affinity chromatography indicated that the inhibitor is a protein of 4 kDa, migrating as one band on SDS-polyacrylamide gel electrophoresis. Amino acid sequence analysis of this band showed the presence of two sequences, differing by one amino acid, which are identical to defensin I and II. Comparison of the sequences of plasminogen and defensin showed homology of defensin to the plasminogen kringles known to contain the lysine binding sites. The close structural similarity between defensin and plasminogen kringles and the ability of defensin to compete with plasminogen on binding to fibrin explain the ability of defensin to inhibit tPA-mediated, plasminogen-dependent fibrinolysis. These results suggest that the antifibrinolytic activity of defensin may have a biological function in preventing the spread of infection.
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PMID:Identification of an inhibitor of tissue-type plasminogen activator-mediated fibrinolysis in human neutrophils. A role for defensin. 772 74

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator, is an important regulator of the blood fibrinolytic system. Elevated plasma levels of PAI-1 are associated with thrombosis, and high levels of PAI-1 within platelet-rich clots contribute to their resistance to lysis by t-PA. Consequently, strategies aimed at inhibition of PAI-1 may prove clinically useful. This study was designed to test the hypothesis that a 14-amino acid peptide, corresponding to the PAI-1 reactive center loop (residues 333-346), can rapidly inhibit PAI-1 function. PAI-1 (0.7 microM) was incubated with peptide (55 microM) at 37 degrees C. At timed intervals, residual PAI-1 activity was determined by addition of reaction mixture samples to t-PA and chromogenic substrate. The T1/2 of PAI-1 activity in the presence of peptide was 4 +/- 3 min compared to a control T1/2 of 98 +/- 18 min. The peptide also inhibited complex formation between PAI-1 and t-PA as demonstrated by SDS-PAGE analysis. However, the capacity of the peptide to inhibit PAI-1 bound to vitronectin, a plasma protein that stabilizes PAI-1 activity, was markedly attenuated. Finally, the peptide significantly enhanced in vitro lysis of platelet-rich clots and platelet-poor clots containing recombinant PAI-1. These results indicate that a 14-amino acid peptide can rapidly inactivate PAI-1 and accelerate fibrinolysis in vitro. These studies also demonstrate that PAI-1 function can be directly attenuated in a physiologic setting and suggest a novel approach for augmenting fibrinolysis in vivo.
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PMID:Peptide-mediated inactivation of recombinant and platelet plasminogen activator inhibitor-1 in vitro. 773 6

BM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated. Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 microgram/ml to 38 min at 10 micrograms/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, alpha 2-antiplasmin and alpha 1-antitrypsin. During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of alpha 2-antiplasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i.e. within 45 min) converted into its two-chain form at concentrations of 5 micrograms/ml BM 06.022 and higher. In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, alpha 2-antiplasmin and alpha 1-antitrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:In vitro stability of a tissue-type plasminogen activator mutant, BM 06.022, in human plasma. 774 Apr 62

Two different techniques have been used to study the complex formation of recombinant human plasminogen activator inhibitor type-1, PAI-1, with either recombinant human two-chain tissue plasminogen activator, tc tPA (EC 3.4.21.68), or the tPA deletion variants tc K2P, containing the kringle 2 domain and the proteinase domain, and P, containing only the proteinase domain. The same value for Kon, 2.10(7) M-1s-1 for binding of PAI-1 was found for the three tPA forms by direct detection of the complex formation in real time by surface plasmon resonance, BIAcore, or indirectly by monitoring the time course of the inhibition of tPA using the chromogenic substrate N-methylsulfonyl-D-Phe-Gly-Arg-4-pNA-acetate. Apparently, no conformational change is involved in the rate-limiting step, since the kon value was found to be independent of the temperature from 20 to 35 degrees C. By the BIAcore technique, it was found that the complex between PAI-1 and tPA covalently coupled to the surface, was stable at 25 degrees C, since no dissociation was seen in buffer. However, extended treatment with 1 M NH4OH destroyed the complex with t 1/2 = 5 h. The same kon values and complex composition were found by measuring either the binding of tPA to PAI-1 captured on the monoclonal antibody MAI-11 or the binding of PAI-1 to tPA captured on the monoclonal antibody 2:2 B10. Quantification of the complex composition between PAI-1 captured on the monoclonal antibody MAI-11 with either tPA, K2P or P gave a one-to-one ratio with the fraction of active PAI-1, consistent with the results from SDS-PAGE and the specific activity of PAI-1. The complexes of the three tPA forms with PAI-1 captured on a large surface of MAI-11 dissociated more rapidly from MAI-11, with the same apparent koff, kdis, = 2.10(-3) s-1, compared with 0.7-10(-3) s-1 for the dissociation of PAI-1 alone. In consistance, the Kd, calculated from the direct determination of the kon and koff for the association of different form of PAI-1 to a small surface of MAI-11, was found to be higher for PAI-1 in complex with tPA than for free active PAI-1. Apparently, upon complex formation, a change is induced in PAI-1 at the binding epitope for MAI-11.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Plasminogen activator inhibitor type-1 interacts exclusively with the proteinase domain of tissue plasminogen activator. 781 90

One feature that distinguishes all of the inhibitory members of the serpin gene family is the presence of a small uncharged residue at the P14 position of the reactive center loop. In this report we examine the effects of mutations at this position, in the serpin, plasminogen activator inhibitor type 1 (PAI-1). Replacement of the native P14 Thr-333 residue by an Arg (Thr-333-->Arg) resulted in complete loss of inhibitory activity toward tissue-type plasminogen activator and urokinase-type plasminogen activator. Comparison of the binding of the mutant inhibitor and wild type PAI-1 (WTPAI-1) to anhydrotrypsin indicated that the initial interaction of the two inhibitors with proteases was identical. However, whereas WTPAI-1 forms SDS-stable complexes with both plasminogen activators, the mutant PAI-1 was efficiently cleaved as a substrate. Amino-terminal sequence analysis indicated that cleavage of the mutant PAI-1 occurred at its reactive center P1-P1' Arg-Met bond. Thermal denaturation studies of native and cleaved PAIs indicated that native Thr-333-->Arg mutant had a thermal stability identical to active WTPAI-1 and that both proteins became significantly more stable following cleavage by elastase (cleaved at the P4-P3 bond). Finally, the function of recombinant PAI-1 variants containing 15 of the possible 19 amino acid substitutions at P14 were analyzed. While residue size appeared to have little effect on inhibitory activity, the presence of either a positive or a negative charge at P14, converted PAI-1 to a substrate. Taken together, these results suggest that while insertion of the reactive center loop is not essential for protease binding, it is a necessary second step required for inhibitor function. The presence of a charged residue at P14 can retard this insertion, resulting in conversion of the serpin to a substrate.
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PMID:Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition. 796 84

Urokinase-type plasminogen activator (uPA) from artificially induced decidual tissue of rat has been purified to homogeneity employing chromatographic techniques and the final preparation has a specific activity of 12,084 I.U./mg. The purified preparation resolves into a single band following SDS-PAGE and has an apparent molecular weight of 45 kDa. HPLC of the purified fraction also yields a single peak at 45 kDa. Decidual uPA is immunogenic in rabbit and a monospecific antiserum raised against it does not cross react with human melanoma tPA or rat Yoshida sarcoma tPA but elicits a precipitin reaction with human uPA and extracts of rat placenta and kidney. The enzyme has a pH optimum of 7.5, a kM of 1.0 microM, is heat stable upto ten minutes at 42 degrees C and inhibited by anti-uPA IgG.
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PMID:Characterization of urokinase-type plasminogen activator of rat decidual tissue. 798 65

Effects of heparan sulfate analogue (HHS-5) and other sulfated polysaccharides on the fibrinolytic system were investigated. Activation rate of native plasminogen (Glu-plg) by tissue plasminogen activator (t-PA), urinary plasminogen activator (u-PA) and single-chain u-PA (scu-PA) was enhanced in the presence of HHS-5 dose dependently up to 10 micrograms/ml. Activation rate of Glu-plg by sct-PA was more enhanced than that by tct-PA in its presence. HHS-5 enhanced the activation of Glu-plg by t-PA or u-PA compared to the activation of Lys-plg. SDS-PAGE confirmed that the enhancement of the hydrolysis of S-2251 by the mixture of Glu-plg, sct-PA and HHS-5 was due to the facilitation of the conversion of Glu-plg to plasmin. HHS-5 only slightly enhanced the amidolytic activity of sct-PA or tct-PA. The enhancement of the activation of plasminogen by t-PA or u-PA was more significant in the presence of HHS-5 than in the presence of chondroitin sulfate C, dextran sulfate or heparin. It is possible that the enhancement of the activation of Glu-plg by activators in the presence of HHS-5 was due to the conformational change of Glu-plg upon interaction with HHS-5.
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PMID:Effects of heparan sulfate analogue or other sulfated polysaccharides on the activation of plasminogen by t-PA or u-PA. 801 15


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