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)

Human RC2A myelomonocytic leukemia cells are able to activate the prourokinase (pro-u-PA) they secrete so that active u-PA is present both in serum-free conditioned medium from these cells, as well as on the cell surface. When the cells are grown in serum-containing medium, no u-PA activity can be found in the medium but active u-PA is found bound to the cell surface where it can generate bound plasmin. This distribution of u-PA activity was shown to be, first, the net result of slow inactivation of free active u-PA by serum inhibitor(s) and simultaneous rapid uptake of u-PA onto the cell surface. Binding to cells was at least six times faster than inactivation by 10% serum. The principal serum inhibitor of u-PA was identified as alpha 2-macroglobulin (alpha 2M), and prior inactivation of u-PA by purified human alpha 2M was also shown to prevent uptake of u-PA activity onto cells. Second, although endogenous u-PA could form covalent complexes with purified alpha 2M in the culture medium of RC2A cells, covalent alpha 2M complexes were not formed by u-PA on the cell surface; the u-PA taken up in this compartment was protected against alpha 2M inhibition. u-PA anchored to plastic surfaces via monoclonal antibodies to the amino-terminal region of u-PA was also protected against alpha 2M, suggesting that the protection of cell surface u-PA results from a steric effect. These results provide evidence as to how the active u-PA produced by leukemia cells can contribute to proteolytic activity on their cell surface in the presence of serum inhibitors.
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PMID:Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells. 172 17

Extracellular proteolysis is believed to be an essential component of the angiogenic process. The effects of VEGF, a recently described angiogenic factor, were assessed on PA activity and PA and PAI-1 mRNA levels in microvascular endothelial cells. u-PA and t-PA activity were increased by VEGF in a dose-dependent manner, with maximal induction at 30 ng/ml. u-PA and t-PA mRNAs were increased 7.5- and 8-fold respectively after 15 hours, and PAI-1 mRNA 4.5-fold after 4 hours exposure to VEGF. At equimolar concentrations (0.5 nM), VEGF was a more potent inducer of t-PA mRNA than bFGF, while bFGF was a more potent inducer of u-PA and PAI-1 mRNAs. In addition, VEGF induced u-PA and PAI-1 mRNAs with kinetics similar to those previously demonstrated for bFGF. These results demonstrate the regulation of PA and PAI-1 production by VEGF in microvascular endothelial cells and are in accord with the hypothesis that extracellular proteolysis, appropriately balanced by protease inhibitors, is required for normal capillary morphogenesis.
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PMID:Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells. 175 66

The signal transduction pathways of urokinase (u-PA) in A431 cells, a human epidermoid carcinoma cell line, were studied using the inducers EGF and TPA. Based on the following observations, we conclude that the two pathways are different: (1) The effects are additive; (2) EGF induction of u-PA was not compromised either by down-regulation of PKC or selective inhibition of the same by H7; (3) 8-Br-cAMP has no effect by itself; however, it inhibits the EGF effect and doubles the TPA induction of u-PA; (4) after EGF-induced tyrosine phosphorylations are completed, addition of TPA can still induce u-PA, an effect that can be blocked by the addition of the tyrosine phosphorylation inhibitor, genistein, indicating that the two agents induce different tyrosine phosphorylation; (5) in 2-dimensional electrophoresis of 32P-labeled cell lysates, inducer-specific differences in the intensity of spots can be observed. Some of the spots weakened by genistein (tyrosine phosphorylations) are different, depending on the inducer used.
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PMID:Epidermal growth factor and 12-tetradecanoyl phorbol 13-acetate induction of urokinase in A431 cells. 179 95

The content of PAI was measured in carcinoma tissues from the stomach and colorectum divided macroscopically into three portions: the central part of the carcinoma, the marginal part of the carcinoma containing some normal mucosa, and the normal mucosa. The PAI-1 antigen was highest in the central part of the carcinoma. On the other hand, no PAI-1 antigen or activity was observed in the normal mucosa. The PAI-1 in the carcinoma tissues showed a nonlytic zone with a molecular weight of 54 kd by reverse fibrin autography, and this 54 kd band reacted with anti-PAI-1 immunoglobulin G (IgG) on an immunoblotted nitrocellulose membrane. The contents of PAI-2 in the carcinoma tissues were not significantly different from those in the normal mucosa of the stomach and colorectum, respectively. In both the stomach and colorectal carcinomas, the highest value of u-PA/total PA (sum of u-PA and t-PA) was observed in the central part of the carcinoma, followed by the marginal part of the carcinoma, and was lowest in the normal mucosa. We conclude that increased levels of PAI-1 in malignant tissue of the stomach and colorectal tract may serve to modulate extracellular proteolysis by u-PA.
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PMID:Plasminogen activator inhibitor in stomach and colorectal carcinomas. 179 98

In the normal stomach of male rats marked differences in plasminogen activator activity (PAA) and plasmin inhibition (PI), but not in plasminogen activator inhibition (PAI), were noted among cardiac area, body and pyloric region. Chronic ethanol consumption (for 15 or 30 days) at the concentration of 6% or 12% in the drinking water induced an increase in PAA in the pyloric region and the body of the stomach (the higher concentration after 15 days and both concentrations after 30 days). The response was time- and dose-dependent. At the cardiac area no change of PAA was noted. Ethanol at both concentrations induced after 30 days a decreased PAI in the pyloric region and the body of the stomach, which was expressed against u-PA, but not against t-PA. A decreased PI was noted at both concentrations of ethanol after 30 days only in the pyloric region. Therefore, changes in PAA, PAI and PI after chronic ethanol consumption were dependent on the concentration, the period of the consumption and the area along the gastric wall.
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PMID:Enhancement of plasminogen activator activity in the gastric wall after chronic ethanol consumption. 182 83

Using DNA synthesis technology we constructed two synthetic DNAs, designated syn-uPA-DNA and mut-uPA-DNA. Syn-uPA-DNA contains the complete coding sequence of human presecretion form of single-chain u-PA. Mut-uPA-DNA was derived from syn-uPA-DNA by deleting 18 base pairs coding for amino acids Arg179-Ser184. Each synthetic DNA was inserted into a bovine papilloma viral genome-based expression vector to obtain expression in mouse cells. The results indicate that both syn-uPA and mut-uPA proteins are secreted predominantly in single-chain form. The single-chain form of both enzymes can be completely converted to two-chain form by treatment with plasmin. Both enzymes are as active as natural urokinase (std-uPA) isolated from urine. Analysis of enzymatic activity indicates that under conditions where syn-uPA and std-uPA are completely inhibited by endothelial-type plasminogen activator inhibitor (PAI-1), mut-uPA retains 90% activity. In identical experiments with placental-type PAI (PAI-2), mut-uPA retains 80% activity. Syn-uPA is capable of forming a approximately 100-kDa complex with PAI, whereas mut-uPA can not. PAI-treated mut-uPA has kinetic properties similar to untreated syn-uPA or std-uPA. Overall, the data indicate that amino acids Arg179-Ser184 function at least in part as a binding site for PAI. Resistance to PAI inhibition may increase the potency of mut-uPA as a thrombolytic agent.
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PMID:A synthetic DNA encoding a modified human urokinase resistant to inhibition by serum plasminogen activator inhibitor. 182 21

Evidence has recently been presented that activated macrophages (M phi) express both urinary (u-PA) and tissue type (t-PA) plasminogen activator. Major cell products of M phi and polymorphonuclear neutrophils (PMN) are reactive oxidants of the HOCl/chloramine type. Since PMN and M phi are involved in inflammatory and fibrinolytic processes, we were interested in the interaction of u-PA, t-PA, and plasmin with oxidants of the leukocyte type. The enzymes were treated with chloramine-T, which at pH 8.5 is a selective oxidant for methionine residues. Oxidation by chloramine-T of t-PA abolishes about 40% of both stimulation susceptibility of t-PA by fibrinogen degradation products (FDP) and affinity of t-PA to FDP. However, the plasminogenolytic and amidolytic activity of unstimulated t-PA as well as the plasminogenolytic activity of u-PA and the amidolytic activity of plasmin are not impaired. Identification of the amino acid residues in the t-PA responsible for the interaction with fibrin might be of great importance in order to understand the mechanism of the clot- selectivity of t-PA. The present study gives evidence that fibrin specificity of t-PA partly depends on chloramine oxidizable amino acids, presumably methionine residues. Hence, experimental data on the interaction between t-PA and fibrin, using oxidized and labelled t-PA should be interpreted with caution. It may be suggested that oxidants of the leukocyte type might regulate t-PA activity and selectivity for fibrin.
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PMID:Effect of oxidants on proteases of the fibrinolytic system: possible role for methionine residues in the interaction between tissue type plasminogen activator and fibrin. 182 45

c[Arg-aB-(CH2+SCH3 phi)-Gly4] was designed and studied as a mechanism-based inactivator (suicide substrate) for plasminogen activators (u-PA and t-PA) and plasmin. This compound inhibited u-PA and fulfills criteria expected for the involvement of an enzyme-activated inhibitor: first-order and irreversible process, saturation kinetics, protection by substrate. The limiting first-order rate constant kinact and the apparent enzyme-inhibitor dissociation constant KI were 0.021 s-1 and 9 microM, respectively at pH 7.5 and 25 degrees C. The activation of plasminogen by u-PA is compromised after this enzyme has been treated by the reagent. Plasmin and t-PA were inactivated 40- and 2330-fold less efficiently than u-PA, respectively.
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PMID:A cyclopeptidic suicide substrate preferentially inactivates urokinase-type plasminogen activator. 182 86

The murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against ligand-induced binding sites [LIBS] on human platelet glycoprotein IIb/IIIa) were conjugated with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) using the cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The conjugates (rscu-PA/MA-TSPI-1, rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, and rscu-PA/MA-LIBS-1), purified by immunoadsorption and gel filtration, were obtained with recoveries of 34% to 45%, with an average stoichiometry of 1.6 to 1.8 IgG molecules per rscu-PA molecule, and with unaltered specific activities and affinities. Preincubation of human platelet-rich plasma with rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, or unconjugated rscu-PA resulted in partial inhibition of ADP-induced aggregation; 25% inhibition was obtained with 63 micrograms/mL rscu-PA and with 6 micrograms u-PA/mL rscu-PA/MA-PMI-2 or 1.2 micrograms u-PA/mL rscu-PA/MA-PMI-1. In an in vitro system composed of a 125I-fibrin-labeled platelet-rich human plasma clot immersed in normal human plasma, the conjugates had threefold to greater than 15-fold less fibrinolytic potency than unconjugated rscu-PA. The thrombolytic potency of rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 was compared with that of rscu-PA and that of a control conjugate rscu-PA/MA-1C8 in a pulmonary embolism model in the hamster, using clots prepared from platelet-poor or platelet-rich human plasma. Lysis was measured 30 minutes after the end of a 60-minute intravenous infusion of the thrombolytic agents. rscu-PA, rscu-PA/MA-PMI-1, rscu-PA/MA-LIBS-1, as well as rscu-PA/MA-1C8 had comparable thrombolytic potencies (percent lysis per dose administered) towards platelet-poor human plasma clots. In contrast, the thrombolytic potency of rscu-PA/MA-PMI-1 and of rscu-PA/MA-LIBS-1 towards platelet-rich clots was 2.3- to 3-fold higher than that of rscu-PA (P less than .005) and fivefold to sevenfold higher than that of the control conjugate (P less than .01).
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PMID:Effect of chemical conjugation of recombinant single-chain urokinase-type plasminogen activator with monoclonal antiplatelet antibodies on platelet aggregation and on plasma clot lysis in vitro and in vivo. 183 Oct 57

The functional operation of the cell surface pro-u-PA and plasminogen activating system has previously been shown to depend on the assembly of u-PA receptors, plasminogen binding sites, and their respective ligands at the focal adhesions of cell extensions. We now show that additional factors operate that affect the persistence of functional activity and that evidently involve charge interactions mediated by polyanions, such as those found in the cell surface proteoglycans. Heparin-like compounds and protamine were identified as fast-acting stimulators of cell surface plasminogen activation. Heparin stabilized surface u-PA activity during plasminogen activation, and we propose that a heparin binding site exists in the kringle structure of u-PA. Heparin at 40 micrograms/ml could reduce u-PA loss to only 20% compared with 60% on control cells activating plasminogen. Protamine (25 micrograms/ml) exerted a strong stimulatory effect on the level of generated bound plasmin and notably prolonged the persistence of this activity, so that 100 minutes after addition of plasminogen the level of plasmin on protamine-treated cells was five times higher than on control-treated cells. The effect of protamine on plasmin clearance suggests that an unknown plasmin inhibitor may be produced by rhabdomyosarcoma cells, whose action is accelerated by endogenous polyanions, in an analogous manner to thrombin inactivation by antithrombin III and protease nexin on endothelial cells and fibroblasts, respectively. The stimulatory effects of heparin and protamine do not affect the inactivation of cell surface u-PA by recombinant PAI-2.
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PMID:Stimulation of cell surface plasminogen activation by heparin and related polyionic substances. 183 80


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