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)

Senile plaques, often surrounded by abnormally grown neurites, are characteristic of Alzheimer's diseased brain. The core of the plaque is mainly composed of amyloid beta protein (beta-AP), two of whose three precursors (APP) have serine proteinase inhibitor regions (APPI). APPI derivatives containing 60, 72 or 88 amino-acid fragments (APPI-60, APPI-72 and APPI-88, respectively) of the longest APP were produced in COS-1 cell culture medium, with the APPI cDNA ligated to the signal sequence of tissue plasminogen activator. The secreted APPIs were purified by sequential acetone precipitation followed by affinity chromatography using immobilized trypsin. These three APPIs and O-glycosylation-site-mutated APPI showed similar inhibitory activity against trypsin, chymotrypsin and plasmin. The purified APPI-72 was found to inhibit trypsin (Ki = 1.1 x 10(-10) M) and chymotrypsin (Ki = 5.8 x 10(-9) M) most strongly, and to inhibit leukocyte elastase (Ki = 7.9 x 10(-7) M) and several blood coagulation proteinases (Ki = 0.46-12 x 10(-7) M), but not urokinase or thrombin. The observed inhibition pattern was quite different from that of protease nexin I, one of serine proteinase inhibitors possessing neurite outgrowth activity. This suggests that the physiological roles of APPI are different from those of protease nexin I, and that APPI could not cause aberrant growth of neurite into the plaque. The presence of APPI having strong inhibitory activity in the brain might lead to the formation of amyloid deposits by preventing complete degradation of APPs.
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PMID:Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of Alzheimer's disease: different properties compared with protease nexin I. 218 Apr 85

The binding of type 1 plasminogen activator inhibitor (PAI-1) to the extracellular matrix (ECM) of cultured bovine aortic endothelial cells was investigated using purified 125I-labeled or L-[35S]methionine-labeled PAI-1 as probes. Little specific binding of latent PAI-1 to ECM previously depleted of endogenous PAI-1 could be demonstrated. In contrast, the guanidine-activated form of PAI-1 bound to ECM in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 60 nM by Scatchard analysis, and approximately 6 pmol of activated PAI-1 was bound per cm2 of ECM. Binding was relatively specific since unlabeled, activated PAI-1 competed with 35S-labeled PAI-1 for binding to ECM, but latent PAI-1 did not. Moreover, PAI-2, protein C inhibitor (i.e. PAI-3), protease nexin-1, and alpha 2-antiplasmin were not able to compete. Tissue-type plasminogen activator (tPA) also inhibited binding, but diisopropyl fluorophosphate-inactivated tPA did not. Pretreatment of ECM with tPA, urokinase-type PA, or thrombin had no effect on its ability to subsequently bind PAI-1, whereas trypsin, plasmin, and elastase pretreatment greatly reduced its ability to bind PAI-1. Guanidine-activated, radiolabeled PAI-1 resembled active endogenous PAI-1 since it was unstable in solution but stable when bound to ECM. In addition, it formed complexes with tPA that had a relatively low affinity for ECM. These data suggest that ECM of bovine aortic endothelial cells contains a protease-sensitive structure that binds active PAI-1 tightly and relatively selectively and that this association stabilizes PAI-1 against the spontaneous loss of activity that occurs in solution.
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PMID:Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured bovine endothelial cells. 249 80

Clonal, fusing, mouse skeletal muscle cells (C2) were grown to the myotube stage (90% confluence) before they were subjected to isotope-containing serum-free media (3H-proline or 35S-methionine). C2 myotubes secrete and organize a biosynthetically labeled matrix which adheres to the plastic after removal of myotubes with detergent and ammonium hydroxide. When these homotypic-labeled myotube matrices were incubated with myoblast-conditioned media containing high specific activity urokinase-type plasminogen activator, slow, but clearly detectable, release of label occurred. However, degradation of matrix, with solubilization of label, was accelerated sixfold by addition of human plasminogen to diluted myoblast-conditioned media. If protease nexin I, a cellular serine protease inhibitor purified from human fibroblast-conditioned media, was added (0.2 microgram/ml) with plasminogen, inhibition of matrix hydrolysis by 52% occurred. Higher concentrations (0.8 microgram/ml or above) of protease nexin 1 completely inhibited the degradation of extracellular matrix components. A similar protease inhibitor was purified from C2 myotube-conditioned media, and this molecule also inhibited the plasminogen-dependent release of extracellular matrix. We propose that protease nexin 1 inhibits the destruction of myotube matrix by inactivating the plasmin/plasminogen activation system and may be the physiologic regulator of this system during muscle development in vivo.
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PMID:Protease nexin I, a serpin, inhibits plasminogen-dependent degradation of muscle extracellular matrix. 250 47

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

A protein that has several similarities to protease nexin I, a fibroblast thrombin and urokinase inhibitor, has been detected on platelets (Gronke RS, Bergman BL, and Baker JB: J Biol Chem 262:3030, 1987). On incubation of platelets with 125I-thrombin, this platelet protein forms complexes with 125I-thrombin that are found both in the incubation medium and, as demonstrated here, associated with purified platelet plasma membranes. The present results indicate that interaction with the platelet surface may modulate the conformation and function of this platelet form of protease nexin I (PNIp) because: (a) an antibody against protease nexin I inhibited released PNIp, but not platelet-bound PNIp from complexing 125I-thrombin, and (b) whereas PNIp extracted from platelets bound both thrombin and urokinase, platelet-bound PNIp bound only thrombin. In experiments using several different platelet isolation methods, PNIp accounted for a large fraction of the rapid high affinity binding of 125I-thrombin to platelets. However, platelets isolated and maintained in the presence of metabolic inhibitors failed to take added thrombin into 125I-thrombin-PNIp complexes. This finding suggests that PNIp is released from inside platelets during activation, and thus does not function to transmit the primary activating signal that is generated by thrombin binding to platelets.
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PMID:A form of protease nexin I is expressed on the platelet surface during platelet activation. 291 87

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

We have prepared a conjugate of the plasminogen activator urokinase (UK) and ferritin, which maintains fibrinolytic activity. Monolayers of BALB/c-3T3 cells and of Rous sarcoma virus-transformed highly malignant line AA12-3T3, subcultured in plasminogen-free serum, were incubated with UK-ferritin at 0 degree and processed for transmission electron microscopy. Under these conditions, both of the lines showed specific receptors on the cell surface that were distributed in singlets, in small or large clusters. In the presence of excess native UK, the binding of ferritin was reduced by 99%, indicating the interaction of UK:ferritin with a specific receptor. The ligand-receptor interaction involves the catalytic site of UK, since the binding was completely impaired by preincubation of UK:ferritin with p-aminobenzamidine, a competitive inhibitor of the catalytic site of UK. The number and density of receptors decreased about one order of magnitude on the membrane of AA12 cells when compared with normal 3T3 cells. Saturation kinetics, using 125I-labeled UK, indicate the presence of 4 X 10(4) and 2.5 X 10(3) receptors on the membrane of 3T3 and AA12 cells, respectively. At 37 degrees, UK:ferritin redistributed on the plane of the membrane, in a process which was faster in malignant than in normal cells. Ferritin particles clustered in large groups on coated areas of the surface and were internalized by adsorptive pinocytosis. After 10 min at 37 degrees, the vesicles showed a progressively deeper internalization and a fusion with lysosomes, and some were observed in the Golgi complex area. Since the experiments were planned in order to exclude the presence of protease-nexin in the incubation medium, these data suggest the existence of a plasminogen-independent novel receptor for the catalytic site of plasminogen activators, the number on the cell surface of which decreases in Rous sarcoma virus-transformed mouse fibroblasts.
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PMID:Receptors for plasminogen activator, urokinase, in normal and Rous sarcoma virus-transformed mouse fibroblasts. 298 11

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

In the present studies we have compared the structural and biochemical properties of human protease nexin-I (PN-I) and a protease inhibitor present in the serum-free culture fluid of normal rat brain astrocytes. The inhibitor binds to and forms covalent complexes with human urokinase and thrombin. The inhibitor has an approximate Mr = 43,000 based on the size of the complexes (deduced from SDS-PAGE) and mediates the cellular binding and uptake of the proteases to which it links. Binding is heparin sensitive and occurs on a cell surface receptor that also binds complexes formed between proteases and a well-characterized cell-secreted protease inhibitor, human PN-I. In addition, the inhibitor co-migrates with PN-I on SDS-PAGE and cross-reacts with anti-PN-I antibody on immunoblots. A similar molecule, designated NPF, is produced by C6 glioma cells in culture and has neurite promoting activity on a neuroblastoma cell line.
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PMID:Identification of a protease inhibitor produced by astrocytes that is structurally and functionally homologous to human protease nexin-I. 304 Jan 75


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