Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

A fraction of the 125I-thrombin that binds to human platelets is taken into a sodium dodecyl sulfate-resistant 77 kDa complex with a platelet factor (Bennett, W. F., and Glenn, K. C. (1980) Cell 22, 621-627). Here we show that this platelet factor is in several respects similar to protease nexin I (PNI), a fibroblast thrombin inhibitor. The complexes are of the appropriate size, bind to Sepharose that has been derivatized with anti-PNI antibody, do not form when the thrombin active site has been blocked with diisopropylphosphofluoridate, and do not appear on platelets when heparin is present. However, the platelet factor does not bind urokinase, indicating that this "platelet PN" may be distinct from PNI. Following brief incubation with 125I-thrombin, platelet PN X 125I X thrombin complexes are found both associated with the platelets and free in the binding medium. 125I-Thrombin has a higher affinity for platelet PN than for platelet receptors. In 30-s binding incubations carried out with thrombin at concentrations below 0.3 nM, formation of the 77-kDa complex accounts for most of the platelet specific binding of 125I-thrombin. Subtracting this large contribution to 125I-thrombin-specific binding reveals that the reversible binding of 125I-thrombin to platelet receptors exhibits sigmoidal thrombin dose-dependence. Thrombin stimulation of platelet [14C]serotonin release exhibits similar thrombin dose dependence. These results indicate that platelets may possess a mechanism for suppressing their interaction with active thrombin at thrombin doses below 0.3 nM. It is possible that platelet PN carries out this function by capturing thrombin before thrombin binds to its signal-transmitting receptors.
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PMID:Thrombin interaction with platelets. Influence of a platelet protease nexin. 310 83

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

Plasminogen activation has been shown to be inhibited by the cell-specific production of a number of protease inhibitors belonging to the serine protease inhibitor family. In the bovine testis this inhibitor production is induced by glucocorticoids. Monospecific antibodies raised against the three known classes of plasminogen activator inhibitor were used to identify which type of inhibitor was secreted by bovine Sertoli cell-enriched cultures. Immunoblot analysis and [35S]methionine labelling of newly synthesized proteins revealed that a novel protein with an apparent molecular weight of 49 kDa, which shares antigenic determinants with placental and macrophage PAI and fibroblast protease nexin, is secreted in response to dexamethasone stimulation. This protein was shown by immunoadsorption to be a functionally active inhibitor of both tissue-type and urokinase-type plasminogen activators.
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PMID:Characterization of a plasminogen activator inhibitor induced by glucocorticoids in immature bovine Sertoli cell-enriched cultures. 325 23

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

Protease nexin 1 (PN-1) is a protease inhibitor secreted by cultured fibroblasts that forms complexes with certain serine proteases; the complexes bind back to the cells and are internalized and degraded. In the present studies, a panel of PN-1 monoclonal antibodies (mAbs) was isolated; none showed detectable cross-reactivity with four related plasma protease inhibitors. Four purified mAbs (mAbp1, mAbp6, mAbp9, and mAbp18) were tested for their ability to block the formation of complexes between PN-1 and target proteases. mAbp1, as well as a rabbit polyclonal anti-PN-1 IgG preparation, did not block formation of 125I-thrombin-PN-1 complexes. mAbp6, mAbp9, and mAbp18 blocked the formation of 125I-thrombin-PN-1 and 125I-urokinase-PN-1 complexes at stoichiometric concentrations of mAb and PN-1. Studies on their ability to block formation of 125I-trypsin-PN-1 complexes showed that mAbp18 also blocked this reaction at stoichiometric concentrations with PN-1 whereas mAbp6 and mAbp9 blocked less effectively. Thus, mAbp18 appears to bind at or close to the reactive center of PN-1. The blocking mAbs should be useful in studies to probe physiological functions of PN-1.
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PMID:Monoclonal antibodies to protease nexin 1 that differentially block its inhibition of target proteases. 337 52

UK-I, a 60-kDa urokinase-inhibitor derived from human fibroblasts, inhibited 54-kDa urokinase (EC 3.4.21.31) activity dose-dependently on ordinary fibrin-agar autograms. This UK-I formed an SDS-stable approximately 75-kDa complex with radioiodinated urokinase (33 kDa) on an autoradiogram following SDS/polyacrylamide gel electrophoresis. Benzamidine hydrochloride inhibited its formation, indicating UK-I to bind at the active site of urokinase and form an inactive complex. UK-I did not form a complex with [125I]thrombin (EC 3.4.21.5). It is thus evident that UK-I is one type of urokinase-inhibitor derived from human fibroblasts with properties differing from protease nexin, another urokinase-inhibitor derived from the same source.
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PMID:A fibroblast-derived urokinase-inhibitor differing from protease nexin. 342 13

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

This review summarizes the mechanisms by which several serine proteases, particularly urokinase, thrombin, and elastase, interact with cultured fibroblasts. Many of these studies were prompted by findings that interactions of these proteases with cells and the extracellular matrix are important in a number of physiologic and pathologic processes. Two main pathways have been identified for specific interactions of these proteases with fibroblasts. One involves surface binding sites for the free protease that appear to bind only one particular protease. An unusual feature collectively shared by the binding sites for urokinase, thrombin, and elastase is that the bound protease is not detectably internalized by the fibroblasts. The other pathway by which serine proteases interact with fibroblasts involves proteins named protease nexins (PNs). Three PNs have been identified. They are secreted by fibroblasts and inhibit certain serine proteases by forming a covalent complex with the protease catalytic site serine. The complexes then bind back to the fibroblasts via the PN portion of the complex and are internalized and degraded. Recent studies showing that the fibroblast surface and extracellular matrix accelerate the inactivation of thrombin by PN-1 support the hypothesis that the PNs control protease activity at and near the cell surface. The PNs differ from plasma protease inhibitors in their molecular properties, absence in plasma, site of synthesis, and site of clearance of the inhibitor:protease complexes.
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PMID:Interactions of serine proteases with cultured fibroblasts. 354 29


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