Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.7 (plasmin)
9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protease nexin-1 is a protein proteinase inhibitor that is secreted by a variety of cultured cells and rapidly forms complexes with thrombin, urokinase, and plasmin; the complexes then bind back to the cells and are internalized and degraded. In fibroblast cultures, protease nexin-1 is localized to the extracellular matrix. Here we report that protease nexin-1, which is bound to the surface of fibroblasts, forms complexes with thrombin, but not urokinase or plasmin. Experiments were conducted to determine directly if protease nexin-1 binding to the fibroblast surface alters its proteinase specificity. To do this, cell surface protease nexin-1 was inhibited using anti-protease nexin-1 monoclonal antibodies that stoichiometrically block its ability to form complexes with target proteinases. Then, purified protease nexin-1 was added to these cells; the cell-bound molecule formed complexes with thrombin, but not urokinase or plasmin. Similar experiments showed that protease nexin-1 bound to preparations of fibroblast extracellular matrix also formed complexes with thrombin, but not urokinase or plasmin. Components of the extracellular matrix other than heparin-like glycosaminoglycans are required for this regulation since heparin did not block the formation of complexes between protease nexin-1 and urokinase or plasmin. These results suggest that protease nexin-1 is primarily a thrombin inhibitor in interstitial fluids where much of it would be bound to cell surfaces.
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PMID:Binding of protease nexin-1 to the fibroblast surface alters its target proteinase specificity. 264 83

Previous studies have shown that neuroblastoma cells and several types of primary neuronal cells in culture rapidly extend neurites when switched from serum-containing to serum-free medium. The present studies on cloned neuroblastoma cells show that thrombin blocked this spontaneous differentiation at 2 nM with a half-maximal potency of 50 pM. This required the catalytic activity of thrombin and was reversed upon thrombin removal. Thrombin also caused cells in serum-free medium to retract their neurites at equally low concentrations. Two other serine proteases, urokinase and plasmin, did not block or reverse neurite extension even at 100-fold higher concentrations. A specific assay for thrombin indicated that thrombin detected in serum-containing medium from neuroblastoma cultures was derived from serum and that it was likely responsible for much of the known capacity of serum to maintain neuroblastoma cells in a nondifferentiated state. This was supported by the finding that heparin addition reduced the thrombin concentration in serum-containing medium and stimulated neurite outgrowth from neuroblastoma cells in serum-containing medium. Studies on the ability of thrombin to modulate neurite outgrowth by other agents showed that it blocked and reversed the neurite outgrowth activity of two thrombin inhibitors: protease nexin-1 (which is identical to glial-derived neurite-promoting factor) and hirudin. Thrombin, however, did not block the neurite-promoting activity of dibutyryl cAMP or prostaglandin E1. These results suggest a specific role for thrombin in control of neurite outgrowth.
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PMID:Thrombin modulates and reverses neuroblastoma neurite outgrowth. 283 73

Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits plasminogen activator (PA) activity secondary to the induction of a specific acid-stable inhibitor of plasminogen activation (Cwikel, B. J., Barouski-Miller, P.A., Coleman, P.L., and Gelehrter, T.D. (1984) J. Biol. Chem. 259, 6847-6851). We have further characterized this inhibitor with respect to its interaction with both urokinase and tissue plasminogen activator, and its protease specificity. The HTC PA inhibitor rapidly inhibits urokinase and tissue plasminogen activator with an apparent second-order rate constant of 3-5 x 10(7) M-1 X s-1. The inhibitor forms stable covalent complexes with both urokinase and tissue plasminogen activator, with which plasmin, trypsin, and factor Xa apparently do not compete. Complex formation is saturable and requires the active site of the PA. The mass of the inhibitor-PA complex is 50,000 daltons greater than that of PA alone, consistent with an Mr for the PA inhibitor of 50,000 as demonstrated directly by reverse fibrin autography. The HTC PA inhibitor does not inhibit thrombin and differs in its kinetic and biochemical properties from protease nexin.
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PMID:Characterization of the dexamethasone-induced inhibitor of plasminogen activator in HTC hepatoma cells. 293 42

Fibroblasts as well as several other cell types, secrete a number of protease inhibitors into their culture media. Among these inhibitors are the protease nexins, a class of proteins which covalently bind serine proteases, thereby inactivating their specific targets. Protease nexin-I, first discovered in human foreskin fibroblasts, binds thrombin, plasmin, and urokinase with high affinity, forming covalently linked complexes. Human fibroblasts bind complexes of protease nexin-I and its target protease via a cell-surface, high-affinity receptor. We have analyzed a number of characteristics of this receptor, and found them to be typical of class II receptors in general. At 4 degrees C binding of PN-I:protease complexes was competed by heparin. In addition, binding was independent of the particular protease bound to the PN-I; purified complexes of PN-I with thrombin or urokinase competed equipotently for [125]I-thrombin:PN-I binding. As the pH of the binding buffer was lowered, binding to cells increased. A twofold increase in binding was attained by lowering the pH from 7.5 to 4.5. This phenomenon was not due to irreversible, pH-induced changes to either the cell surface or the labeled complexes. At 37 degrees C, the removal of labeled complexes from culture medium was rapid; approximately 80% was removed by 4 hours under given conditions. The internalization of complexes was also very rapid, with an estimated ke (endocytic rate constant) of 1.0 min-1. At neutral pH, fibroblasts bind complexes in a saturable manner. Scatchard analysis yields a receptor number of 250,000 per cell and a Kd of 1 nM.
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PMID:Characterization of the receptor for protease nexin-I:protease complexes on human fibroblasts. 303 24

Protease nexin-I (PN-I, Mr approximately 43,000) is representative of a newly described class of cell-secreted protease inhibitors. PN-I has been purified to apparent homogeneity, partially sequenced, and monospecific antibodies have been raised against it. PN-I is a potent inhibitor of urokinase, thrombin, plasmin, and trypsin. In addition, cells have specific receptors that mediate the uptake of covalently linked complexes formed between PN-I and its protease substrates. In the present studies, we have investigated the relationship between human PN-I and a protease inhibitor derived from C6 glioma cells in culture that has neurite-promoting activity. On the basis of co-purification on heparin-Sepharose, identical molecular weight, antibody cross-reactivity, and receptor cross-reactivity, we conclude that PN-I and the glioma-cell-derived inhibitor are equivalent molecules.
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PMID:The glioma cell-derived neurite promoting activity protein is functionally and immunologically related to human protease nexin-I. 304 Jul 80

Urokinase-related proteins in human urine occur mainly as a 1:1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogenous glycoprotein of apparent Mr 50,000 was obtained with a yield of 40 micrograms/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 degrees C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with beta-mobility. Inhibition of urokinase occurred with a second order rate constant (k) of 8 X 10(3) M-1 s-1 in the absence and of 9 X 10(4) M-1 s-1 in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinase-type plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below 10(3) M-1 s-1 and thrombin with a k of 4 X 10(4) M-1 s-1 in the absence and 2 X 10(5) M-1 s-1 in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 +/- 0.7 micrograms/ml (mean +/- S.D., n = 25). The protein purified from plasma by immunoabsorption had the same Mr, amino acid composition, and immunoreactivity as the urinary protein. Furthermore, when urokinase was added to plasma, time-dependent urokinase-urokinase inhibitor complex formation was observed at a rate similar to that observed for the inhibition of urokinase by the purified inhibitor from urine. This urokinase inhibitor, purified from human urine, most probably represents a new plasma protease inhibitor.
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PMID:Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma. 309 4

Serum-free culture medium collected from primary monolayer cultures of human articular chondrocytes was found to inhibit human urokinase [EC 3.4.21.31] activity. Although chondrocyte culture medium contained a small amount of endothelial-type plasminogen activator inhibitor which could be demonstrated by reverse fibrin autography, most of the urokinase inhibitory activity of chondrocyte culture medium was shown to be due to a different molecule from endothelial-type inhibitor, since it did not react with a specific antibody to this type of inhibitor. The dominant urokinase inhibitor in chondrocyte culture medium was partially purified by concanavalin A-Sepharose affinity chromatography. The partially purified inhibitor inhibited high-Mr urokinase more effectively than low-Mr urokinase, but no obvious inhibition was detected against tissue-type plasminogen activator, plasmin, trypsin, and thrombin. The inhibitor had an apparent Mr of 43,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and it was unstable to sodium dodecyl sulfate, acid, and heat treatments. Inhibition of urokinase by the inhibitor was accompanied with the formation of a sodium dodecyl sulfate-stable high-Mr complex between them. Inhibition and complex formation required the active site of urokinase. The partially purified inhibitor was thought to be immunologically different from the known classes of plasminogen activator inhibitors, including endothelial-type inhibitor, macrophage/monocyte inhibitor, and protease nexin, since it did not react with specific antibodies to these inhibitors.
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PMID:Detection and partial characterization of a specific plasminogen activator inhibitor in human chondrocyte cultures. 314 40

A plasminogen activator inhibitor was purified to apparent homogeneity from conditioned media of U138 cells. The inhibitor is a glycoprotein with a pI of 5.4 and an apparent molecular weight of 45,000. The inhibitor forms sodium dodecyl sulfate-stable complexes with plasminogen activators and trypsin but not with plasmin, thrombin, or pancreatic kallikrein. Some biochemical and immunochemical characteristics of the U138 inhibitor distinguish it from other known plasminogen activator inhibitors. The expression of this inhibitor by U138 cells could be modulated by incubation in phorbol myristate acetate, interleukin-1, tumor necrosis factor, and gamma interferon, but not in beta interferon. Thus, the expression of the plasminogen activator inhibitor can be influenced by biological response modifiers known to be active in the brain and in the neural response to inflammatory stimuli. Therefore, this inhibitor, along with protease nexin, may be involved in brain development and regulation.
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PMID:Purification and partial characterization of a plasminogen activator inhibitor from the human glioblastoma, U138. 314 98

Protease nexin-1 (PN-1) is a protease inhibitor that is secreted by fibroblasts and several other cultured cells. PN-1 forms complexes with certain serine proteases in the extracellular environment including thrombin, urokinase, and plasmin. The complexes then bind to the cells and are rapidly internalized and degraded. This report demonstrates that PN-1 is present on the surface of fibroblasts, bound to the extracellular matrix. Immunofluorescent studies showed that PN-1 colocalized with fibronectin on both intact cells and in preparations of extracellular matrix made from these cells. In contrast, PN-1 did not colocalize with the epidermal growth factor receptor, a plasma membrane marker. An enzyme-lined immunosorbent assay was developed which showed that the extracellular matrix contained at least 60-80% of the cellular immunoreactive PN-1. Extraction of the matrix with 2 M NaCl removed PN-1 in a form which reacted with 125I-thrombin to form complexes which were immunoprecipitated by anti-PN-1 IgG and were of identical size as complexes made from soluble PN-1 and 125I-thrombin. These data indicate that in addition to its role as a soluble protease inhibitor, PN-1 is also a component of the extracellular matrix and might control its proteolysis.
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PMID:Localization of protease nexin-1 on the fibroblast extracellular matrix. 327 57

Human fibrosarcoma (HT-1080) cells, in contrast to normal fibroblasts, rapidly hydrolyze the glycoprotein, collagen, and elastin extracellular matrix (ECM) synthesized by cultured rat aortic smooth muscle cells. This degradation occurs at a rapid rate in the presence of serum, indicating that the cellular proteases responsible are relatively insensitive to serum proteinase inhibitors. Here it is shown that protease nexin I (PNI), a fibroblast-secreted inhibitor of urokinase, plasmin, and certain other serine proteinases, effectively inhibited the HT-1080 cell-mediated degradation of this ECM. PNI at 2.0 nM significantly inhibited matrix destruction for 1-2 days and at 0.2 microM caused a virtually complete inhibition that persisted for the entire 10-day period of observation. Inhibition of ECM destruction was accompanied by a transient arrest of HT-1080 cell proliferation that took place during the first 3 days after PNI addition. PNI did not inhibit the growth of normal fibroblasts and also did not inhibit the growth of HT-1080 cells that were seeded onto plastic dishes rather than onto ECM. Like many types of malignant cells, HT-1080 cells release large amounts of urokinase. Antibody against this plasminogen activator partially protected ECM from HT-1080 cell-mediated hydrolysis, indicating that it may have been a target of PNI. One potential physiological function of PNI could be to help maintain the integrity of connective tissue matrices, protection that malignant cells could overcome by secreting proteinases in excessive amounts.
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PMID:Inhibition of tumor-cell-mediated extracellular matrix destruction by a fibroblast proteinase inhibitor, protease nexin I. 351 69


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