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)

Newborn rat cerebellum microexplants cultured in Minimal Essential Medium with glucose and insulin released plasminogen activator (PA), which was detected in living cultures by a substrate overlay assay. Gel electrophoresis of cerebellum-conditioned medium followed by zymography resolved PA activity in two separate bands of 48,000 and 75,000 daltons apparent mol. wt. Using specific antisera, these bands were shown to be respectively urokinase and tissue-type PA. Cerebellum conditioned medium as well as purified human urokinase induced the proliferation and outgrowth of glial fibrillary acid protein-positive cells from newborn cerebellar microexplants. The effect was suppressed by the serine protease inhibitor phenyl methanesulfonylfluoride. Since PAs are most likely of neuronal origin, we suggest that at least one of these proteases acts as a neuronoglial mitogenic signal during development.
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PMID:Plasminogen activator is a mitogen for astrocytes in developing cerebellum. 403 64

The serine protease inhibitor protein C inhibitor is present in semen at a relatively high concentration and forms in vivo complexes with two plasminogen activators also present in semen, urokinase-type and tissue-type plasminogen activators. Therefore, the fact that prostate-specific antigen (PSA), a major prostate enzyme, complexes and inactivates protein C inhibitor (PCI) in semen could have implications in human reproduction. The present study was undertaken to develop an enzyme-linked immunosorbent assay (ELISA) for complexes of PSA with PCI (PSA:PCI) with purified PSA:PCI complexes as a standard. Seminal plasma was utilized as the starting material for purification of complexes by affinity chromatography on heparin-Sepharose and gel filtration. The final preparation contained equimolar concentrations of PSA and PCI and was used for calibration of an ELISA for PSA:PCI complexes involving polyclonal anti-PSA and horseradish peroxidase-labeled anti-PCI antibodies. The ELISA had a detection limit of about 0.2 ng/ml of complex and was specific for PSA:PCI complexes because no color was developed at PSA or PCI concentrations up to 100 microgram/ml. Normal plasma or plasma from patients with prostate carcinoma who had high PSA levels had no detectable PSA:PCI complexes. Seminal plasma from voluntary donors collected in the absence of inhibitors and incubated at room temperature for at least 3 hours had PSA:PCI complex levels ranging from 30 to 46 micrograms/ml, accounting for up to 34% of the total PCI in seminal plasma.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A quantitative ELISA for the measurement of complexes of prostate-specific antigen with protein C inhibitor when using a purified standard. 750 42

HGF is a powerful mitogen for both rat and human hepatocytes, epithelial cells and endothelial cells in vitro, and is angiogenic in vivo. It has considerable homology with plasminogen and has been shown to upregulate urokinase-type plasminogen activator (u-PA) in endothelial cells as well as u-PA and its receptor in kidney epithelial cells. In this study, we report that human recombinant HGF stimulates expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue factor (TF) in the human hepatoma cell line HepG2. PAI-1 antigen as determined by a specific enzyme-linked immunosorbent assay increased up to threefold in conditioned media of HepG2. This increase was dose dependent with maximum stimulation achieved with a concentration of 50 ng/mL of hepatocyte growth factor (HGF). PAI-1 antigen also increased up to fourfold in the extracellular matrix in HGF treated HepG2. The production of the PAI-1 binding protein vitronectin (Vn) was not affected by HGF. In contrast, TF activity in HepG2 treated with HGF increased up to twofold. As determined by Northern blotting, PAI-1 and TF-specific mRNA were increased significantly in the presence of HGF, whereas Vn mRNA was not affected. The increase in PAI-1 and TF mRNA was also seen when HepG2 were incubated with HGF in the presence of cycloheximide, thereby indicating that de novo protein synthesis is not required to mediate the effect. u-PA could be detected neither in unstimulated or HGF-stimulated HepG2 cells on the antigen level nor on the mRNA level. In conclusion, our data give evidence that HGF, in addition to its proliferative effect for different cell types, is also involved in the local regulation of fibrinolysis and coagulation. One could speculate that HGF might modulate processes requiring matrix degradation by increasing the expression of the protease u-PA in one cell type and by upregulating the expression of the serine protease inhibitor PAI-1 in a different cell type. Because u-PA has been shown to activate latent HGF to the active form, it could furthermore be speculated that by upregulating PAI-1, which in turn could inhibit u-PA, HGF might regulate its own activation.
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PMID:Hepatocyte growth factor stimulates expression of plasminogen activator inhibitor type 1 and tissue factor in HepG2 cells. 751 5

PC-3 cells, whose growth is androgen-independent, were shown to be capable of slow proliferation in serum-free medium and in the absence of added growth factor for 7 days. They secreted insulin-like growth factor (IGF)-II but no detectable IGF-I. This IGF-II, although produced in small amounts, plays a role in their proliferation because growth could be inhibited dose dependently by up to 80% in the presence of monoclonal antibodies directed against IGFs or the type 1 IGF receptor. PC-3 cells also secreted IGF binding proteins (IGFBPs) -2, -3, -4, and -6. Immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of the same molecular size as those generated from IGFBP-3 in vivo. With the addition to the culture medium of a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc-SC), at concentrations < 0.2 mM that were nontoxic to the cells, cell proliferation was dose dependently inhibited up to 80% and, at the same time, proteolysis of the IGFBP-3 secreted by the cells was depressed. Urokinase activity detected in the conditioned media was depressed by Pefabloc, suggesting that the urokinase-type plasminogen activator was involved in the proteolysis of IGFBP-3. In addition, 0.01-5 micrograms/ml plasminogen induced a dose-dependent increase in both proliferation and the proportions of proteolysed IGFBP-3 in the media. The stimulation of proliferation was totally blocked in the presence of anti-type 1 IGF receptor antibody. Recombinant human IGF-II (5-200 ng/ml) added to cell-free medium conditioned by 48 h of culture dose dependently stimulated PC-3 cell proliferation. At concentrations < or = 100 ng/ml, its mitogenic action was potentiated when medium had been conditioned by cells cultured in the presence of plasminogen but inhibited when medium had been conditioned by cells cultured in the presence of Pefabloc. We conclude from these results 1) that IGF-II is involved in the autocrine control of PC-3 cell proliferation via the type 1 IGF receptor; and 2) that this proliferation is directly dependent on IGF-II bioavailability that itself is modulated by the limited IGFBP-3 proteolysis induced, at least in part, by urokinase-type plasminogen activator and plasmin.
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PMID:Autocrine regulation of cell proliferation by the insulin-like growth factor (IGF) and IGF binding protein-3 protease system in a human prostate carcinoma cell line (PC-3). 758 99

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unique in its ability and mechanism of inhibiting serine proteases of a broad range of substrate specificity. However, although the catalytic domain of human urokinase-type plasminogen activator (uPA) has 40% identity to bovine trypsin and the substrate specificities of these two proteases are virtually identical, ecotin inhibits uPA almost 10,000-fold less efficiently than trypsin. Ecotin was expressed on the surface of filamentous bacteriophage (ecotin phage) to allow the isolation of more potent inhibitors of uPA from a library of ecotin variants. The 142-amino acid inhibitor was fused to the C-terminal domain of the M13 minor coat protein, pIII, through a Gly-Gly-Gly linker and assembled into phage particles. The ecotin phage were shown to react with anti-ecotin antibodies, revealing a stoichiometry of approximately one ecotin per bacteriophage. The ecotin displayed on the surface of phage inhibited trypsin with an equilibrium dissociation constant of 6.7 nM, in close approximation to that of free ecotin, indicating that phage-associated ecotin is correctly folded and functionally active. Reactive-site amino acids 84 and 85 of ecotin were then randomized and a library of 400 unique ecotin phage was created. Three hundred thousand members of the library were screened with immobilized uPA and subjected to three rounds of binding and in vitro selection. DNA sequence analysis of the selected ecotin phage showed that ecotin M84R/M85R predominated while ecotin M84R, M84K, and M84R/M85K were present at a lower frequency. The four ecotin variants were overexpressed and purified and their affinities toward uPA were determined. Each of the selected ecotin variants exhibited increased affinity for uPA when compared to wild-type ecotin with ecotin M84R/M85R showing a 2800-fold increase in binding affinity.
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PMID:Isolation of a high affinity inhibitor of urokinase-type plasminogen activator by phage display of ecotin. 774 76

Tegumental extracts from adult worms of Schistosoma mansoni contain an inhibitory activity to the S. mansoni 28-kDa serine protease and to pancreatic elastase. By using biotinylated elastase and streptavidin-agarose, the postulated protease inhibitor has been isolated from the crude worm extract in a single step. Monospecific rabbit antibodies raised against the protease inhibitor have immunoprecipitated a 56-kDa [35S]Met-labeled serine protease inhibitor which was designated Smpi56 (S. mansoni protease inhibitor, 56 kDa). Smpi56 binds tightly to and inhibits the 28-kDa protease of S. mansoni and pancreatic and neutrophil elastase but not papain, pepsin, thrombin, trypsin, chymotrypsin, proteinase K, urokinase and acetylcholinesterase. The biological function of Smpi56 is still not known, but in view of its elastase inhibitory activity it may be speculated that the parasite is employing Smpi56 to protect itself from activated neutrophils. Smpi56 may also potentially protect the parasite from its endogenous 28-kDa protease.
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PMID:Schistosoma mansoni: isolation and characterization of Smpi56, a novel serine protease inhibitor. 811 69

Plasminogen activator inhibitor 1 (PAI-1), a member of the serine protease inhibitor (Serpin) superfamily, is the primary inhibitor of the plasminogen activators tPA and uPA. PAI-1 is produced in an active form but converts to an inactive or latent form with a half-life of approximately 1 h at pH 7.5, 37 degrees C. This study describes the construction, expression, and characterization of PAI-1 mutants with increased functional stability. Three mutations that disrupt an ion pair, present in latent PAI-1, between Arg-30 and Glu-350 (P4'), were introduced into recombinant PAI-1. All three mutant proteins maintained normal functional activity against both uPA and tPA. However, the half-life of each purified PAI-1 mutant was extended compared to the 1.1 h observed for wild-type PAI-1 (wtPAI-1) (1.2 h for Glu-350-->Arg, 2.0 h for Glu-350-->Pro, and 2.1 h for the Arg-30-->Glu mutation). An additional PAI-1 variant containing a second mutation designed to potentially reconstitute the ion pair (Arg-30-->Glu, Glu-350-->Arg) failed to restore the wild-type half-life. Circular dichroism spectra analysis indicated that the active and latent forms of wtPAI-1 and all four mutants contained similar secondary structural elements. Thermal stability determinations showed that latent wtPAI-1 was much more structurally stable than the active conformation. However, the latent form for all four mutants was significantly less stable than the corresponding wtPAI-1 conformer. This is the first report of PAI-1 mutants which have been specifically engineered to produce enhanced functional stability.
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PMID:Engineering plasminogen activator inhibitor 1 mutants with increased functional stability. 814 62

Since the serine protease inhibitor, protein C inhibitor (PCI), is present in seminal plasma at approximately 3 microM, complexes of PCI with urokinase (uPA) and tissue type (tPA) plasminogen activator were quantitated using sandwich enzyme-linked immunosorbent assays (ELISA's). Seminal plasma (N = 10) collected in the absence of extrinsic inhibitors had a mean of 25 +/- 5 ng/ml uPA:PCI, 76 +/- 23 ng/ml tPA:PCI, and 4 +/- 2 ng/ml of tPA complexes with plasminogen activator inhibitor-1 (tPA:PAI-1). 93% of the uPA and 17% of the tPA antigen in seminal plasma was in complex with PCI and, when complexation was inhibited by collecting semen into an 1,10-phenanthrolinium solution, 33% of the uPA and 7% of the tPA was complexed to PCI. Urine (N = 10) contained 4 +/- 1 ng/ml uPA:PCI. In purified system, complexation of uPA and tPA to PCI paralleled the inhibition of the enzymes. In vitro studies in blood and seminal plasma showed that heparin stimulated complexation of uPA and tPA with PCI, suggesting that negatively charged glycosaminoglycans in blood vessels and in the reproductive system may regulate PCI reactions with uPA and tPA. These results suggest that PCI is a physiologic regulator of uPA and tPA in male reproductive tissues and raises questions about a potential role of PCI in human fertility and in uPA-dependent cell invasiveness.
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PMID:Evidence for the regulation of urokinase and tissue type plasminogen activators by the serpin, protein C inhibitor, in semen and blood plasma. 816 23

Protease Nexin-1 (PN-1) also known as Glia-Derived Nexin (GDN) inhibits the activity of several serine proteases including thrombin, tissue (tPA)- and urokinase (uPA)-type plasminogen activators. These and other serine proteases seem to play roles in development and tissue homeostasis. To gain insight into where and when PN-1 might counteract serine protease activities in vivo, we examined its mRNA and protein expression in the mouse embryo, postnatal developing nervous system and adult tissues. These analyses revealed distinct temporal and spatial PN-1 expression patterns in developing cartilage, lung, skin, urogenital tract, and central and peripheral nervous system. In the embryonic spinal cord, PN-1 expression occurs in cells lining the neural canal that are different from the cells previously shown to express tPA. In the developing postnatal brain, PN-1 expression appears transiently in many neuronal cell populations. These findings suggest a role for PN-1 in the maturation of the central nervous system, a phase that is accompanied by the appearance of different forms of PN-1. In adults, few distinct neuronal cell populations like pyramidal cells of the layer V in the neocortex retained detectable levels of PN-1 expression. Also, mRNA and protein levels did not correspond in adult spleen and muscle tissues. The widespread and complex regulation of PN-1 expression during embryonic development and, in particular, in the early postnatal nervous system as well as in adult tissues suggests multiple roles for this serine protease inhibitor in organogenesis and tissue homeostasis.
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PMID:Variable and multiple expression of Protease Nexin-1 during mouse organogenesis and nervous system development. 830 78

Hereditary angioedema is caused by a genetic deficiency of C1-inhibitor, a serine protease inhibitor that regulates activation of complement, contact, and fibrinolytic systems. Symptoms (bouts of subcutaneous and mucous swelling) depend on the release of a vasoactive mediator, probably through activation of these three systems. We studied the interrelationship among complement, contact, and fibrinolytic activation in 23 patients with hereditary angiodema, 18 during remission and five during an attack, by measuring plasma levels of C1-C1 inhibitor, factor XIIa-C1 inhibitor, kallikrein-C1 inhibitor, and plasmin-alpha 2-antiplasmin complexes, tissue plasminogen activator, and urokinase plasminogen activator. In addition, cleavage of high-molecular weight kininogen was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and quantified by densitometry. During remission, plasma levels of C1-C1 inhibitor complexes were elevated (p = 0.0002), whereas the other parameters were within the normal range. During acute attacks, not only plasma levels of C1-C1 inhibitor complexes but also those of plasmin-alpha 2-antiplasmin complexes (P = 0.0009) and cleaved high-molecular weight kininogen were elevated. A positive correlation between plasmin-alpha 2-antiplasmin complexes and cleaved high-molecular weight kininogen was observed (r = 0.75, p < 0.001). This article presents the first in vivo evidence that supports the concept that release of vasoactive mediators in hereditary angiodema attacks is associated with the activation of the fibrinolytic system.
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PMID:Generation of plasmin during acute attacks of hereditary angioedema. 842 80


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