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)

Various inducers endow human leucocytes with a procoagulant activity of tissue factor type. We have observed a novel plasma factor which in combination with streptokinase induces powerful leucocyte procoagulant activity. This streptokinase dependent factor (SKDF) is present in normal plasma or serum albeit quantitatively different in individual donors. The generation of tissue factor activity as a function of streptokinase-plasma complex shows a specific and saturable sigmoidal dose-response curve. The Hill plot shows a straight line with Hill coefficient, H = 2.2, suggesting a strong positive cooperativity for the binding of this streptokinase-plasma complex to the leucocyte surface receptor for the signal transduction leading to the biosynthesis of tissue factor apoprotein. It also suggests that the leucocyte surface receptor for streptokinase-plasma complex differs from that for endotoxin lipopolysaccharides. SKDF is of apparent high molecular weight. It does not appear to be an antibody to streptokinase since its level does not correlate with the level of antibodies to streptokinase, and it does not correlate with the antistreptolysin titre. Furthermore, SKDF does not bind to protein A. It has a narrow pH range of stability, and is destroyed at 56 degrees C, or at freeze-drying, Urokinase, another plasminogen activator, or plasmin were unable to activate SKDF to induce the leucocyte procoagulant activity. SKDF may play a role in thrombolytic therapy.
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PMID:A streptokinase dependent plasma factor (SKDF) induces leucocyte tissue factor activity. 297 1

The mammalian serine protease zymogen, plasminogen, can be converted into the active enzyme plasmin by vertebrate plasminogen activators urokinase (uPA), tissue plasminogen activator (tPA), factor XII-dependent components, or by bacterial streptokinase. The biochemical properties of the major components of the system, plasminogen/plasmin, plasminogen activators, and inhibitors of the plasminogen activators, are reviewed. The plasmin system has been implicated in a variety of physiological and pathological processes such as fibrinolysis, tissue remodeling, cell migration, inflammation, and tumor invasion and metastasis. A defective plasminogen activator/inhibitor system also has been linked to some thromboembolic complications. Recent studies of the mechanism of fibrinolysis in human plasma suggest that tPA may be the primary initiator and that overall fibrinolytic activity is strongly regulated at the tPA level. A simple model for the initiation and regulation of plasma fibrinolysis based on these studies has been formulated. The plasminogen activators have been used for thrombolytic therapy. Three new thrombolytic agents--tPA, pro-uPA, and acylated streptokinase-plasminogen complex--have been found to possess better properties over their predecessors, urokinase and streptokinase. Further improvements of these molecules using genetic and protein engineering tactics are being pursued.
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PMID:Plasminogen activation: biochemistry, physiology, and therapeutics. 297 9

Urokinase is one of the two plasminogen activators that catalyze the conversion of inactive plasminogen to plasmin. By combining somatic cell genetics, in situ hybridization, and Southern hybridization, we localized the human urokinase gene on the distal third of the long arm (q24-qter) of chromosome 10.
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PMID:Human urokinase gene is located on the long arm of chromosome 10. 298 21

The capacity of cells to interact with the plasminogen activator, urokinase, and the zymogen, plasminogen, was assessed using the promyeloid leukemic U937 cell line and the diploid fetal lung GM1380 fibroblast cell line. Urokinase bound to both cell lines in a time-dependent, specific, and saturable manner (Kd = 0.8-2.0 nM). An active catalytic site was not required for urokinase binding to the cells, and 55,000-mol-wt urokinase was selectively recognized. Plasminogen also bound to the two cell lines in a specific and saturable manner. This interaction occurred with a Kd of 0.8-0.9 microM and was of very high capacity (1.6-3.1 X 10(7) molecules bound/cell). The interaction of plasminogen with both cell types was partially sensitive to trypsinization of the cells and required an unoccupied high affinity lysine-binding site in the ligand. When plasminogen was added to the GM1380 cells, a line with high intrinsic plasminogen activator activity, the bound ligand was comprised of both plasminogen and plasmin. Urokinase, in catalytically active or inactive form, enhanced plasminogen binding to the two cell lines by 1.4-3.3-fold. Plasmin was the predominant form of the bound ligand when active urokinase was added, and preformed plasmin can also bind directly to the cells. Plasmin on the cell surface was also protected from its primary inhibitor, alpha 2-antiplasmin. These results indicate that the two cell lines possess specific binding sites for plasminogen and urokinase, and a family of widely distributed cellular receptors for these components may be considered. Endogenous or exogenous plasminogen activators can generate plasmin on cell surfaces, and such activation may provide a mechanism for arming cell surfaces with the broad proteolytic activity of this enzyme.
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PMID:The plasminogen system and cell surfaces: evidence for plasminogen and urokinase receptors on the same cell type. 302

Urokinase-related proteins were purified from 60-liter batches of human urine collected into the protease inhibitor aprotinin to prevent proteolytic degradation. Three homogeneous species were obtained by chromatography on zinc chelate-Sepharose, SP-Sephadex C-50, Sephadex G-100, benzamidine-Sepharose, and immunoadsorption on a murine anti-human urokinase monoclonal antibody. One urokinase-related protein with Mr 95,000 representing a complex of two-chain urokinase with an inhibitor accounts for about 70% of the total urokinase-related antigen in urine. Nucleophilic agents dissociate the complex into active two-chain urokinase and a protein with Mr 45,000-50,000 which is immunologically distinct from urokinase. Approximately 25% of the urinary urokinase-related antigen represents a single-chain molecule with Mr 54,000. This highly purified single-chain molecule was obtained with a yield of 5 micrograms/liter of urine. Only trace amounts (less than 5%) of the urokinase-related antigen were recovered as free two-chain urokinase. The urinary single-chain urokinase-related protein has no specific affinity for fibrin. It has a very low activity on Pyroglu-Gly-Arg-p-nitroanilide, a urokinase-specific synthetic substrate, but directly activates plasminogen following Michaelis-Menten kinetics with Km = 0.7 microM and kcat = 0.0011 S-1. The single-chain molecule is rapidly converted to active two-chain urokinase by plasmin. Active two-chain urinary urokinase has a very high amidolytic activity and activates plasminogen with Km = 60 microM and kcat = 1.4 S-1. It is concluded that the urokinase-related proteins in human urine consist of about 25% of single-chain urokinase (10-20 micrograms/liter) and of about 75% two-chain urokinase (40-50 micrograms/liter), the bulk of which is complexed to an inhibitor. Because even in freshly voided urine most of the urokinase-related antigen is already converted to two-chain urokinase, urine does not seem to be a suitable source for the large-scale purification of single-chain urokinase. In view of the very significant intrinsic plasminogen-activating properties of single-chain urokinase, it should not be considered to be a proenzyme form of urokinase. The dramatic differences of its kinetic constants from those of urokinase render the designation single-chain urokinase equally inadequate. Consequently, the designation "single-chain urokinase-type plasminogen activator" was recently adopted by the International Committee on Thrombosis and Haemostasis (Annual Meeting, San Diego, CA, July 13-14, 1985).
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PMID:Urokinase-related proteins in human urine. Isolation and characterization of single-chain urokinase (pro-urokinase) and urokinase-inhibitor complex. 308 Apr 22

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

Plasminogen activators convert plasminogen into plasmin, a serine protease that initiates extracellular proteolysis. Two types of plasminogen activator activities have recently been demonstrated in granulosa cells, and the proteolysis-inducing enzymes are believed to be involved in ovulation. However, little attention has been paid to the presence of these enzymes in oocytes. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique, we studied plasminogen activator activity in oocytes. Denuded oocytes collected from ovaries of hypophysectomized, estrogen-treated immature rats contained a tissue-type plasminogen activator (tPA), but not urokinase (uPA). In contrast, oocyte-free granulosa cells in these preantral follicles contained uPA, but not tPA. The tPA activity found in oocytes was plasminogen-dependent; incubation with increasing numbers (25-200) of denuded oocytes resulted in a dose-dependent increase in fibrinolysis only in the presence of plasminogen. Cellular localization of tPA was studied in the preantral follicles using an immuno-cytochemical method. Positive tPA staining was detected in the cytoplasm, but not in the germinal vesicle or zona pellucida of the oocytes. Furthermore, analysis using a reverse fibrin-overlay method did not reveal the presence of a plasminogen activator inhibitor. Culturing of denuded oocytes for 24 h increased the cellular content of tPA, but the enzyme activity was not further enhanced by treatment with FSH or forskolin. Also, no tPA activity was detected in the medium. We further studied plasminogen activator activities in the cumulus-oocyte complexes. Although only tPA activity was detected in freshly obtained cumulus-oocyte complexes, incubation for 24 h increased both tPA and uPA activity. Furthermore, tPA, but not uPA, activity was stimulated by treatment with FSH or forskolin. This was accompanied by the secretion of tPA into the medium. The identity of tPA and uPA in the cumulus-oocyte complexes was further confirmed by immunoprecipitation with specific antibodies. Isolation of denuded oocytes and cumulus cells after hormonal stimulation of the cumulus-oocyte complexes suggested that tPA activity was stimulated in both cell types and that the cumulus cells may mediate the action of FSH and forskolin on oocytes. In conclusion, the detection and regulation of tPA activity in cumulus-oocyte complexes suggest possible involvement of this enzyme in ovulation or the process of cumulus cell expansion and dispersion. Changes in oocyte tPA content may also serve as an indicator of oocyte development.
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PMID:Identification and regulation of tissue plasminogen activator activity in rat cumulus-oocyte complexes. 309 95

Early reperfusion of occluded coronary arteries offers great promise as a method for minimizing myocardial damage after acute myocardial infarction. Such reperfusion is usually attempted via administration of fibrinolytic agents. Urokinase may hold marginal advantages over streptokinase, especially in patients with high preexisting titers of antistreptokinase antibodies. These minor differences, however, pale in comparison to important advantages demonstrated by the newly developed agent, tissue plasminogen activator (t-PA). The advantages of t-PA derive primarily from its property of binding to, and being activated by, fibrin. Consequently the generated plasmin is also fibrin-bound, the bound plasmin is protected from circulating antiplasmin and therefore more efficiently utilized, and circulating fibrinogen is spared. Preliminary clinical experience indicates that the frequency of favorable response after intravenous administration of t-PA is considerably greater than after SK. A major determinant of clinical benefit after reperfusion is the brevity of ischemia. Selective intracoronary infusion of fibrinolytic agent produces faster lysis than does intravenous infusion, and rate of lysis may be further accelerated by transcatheter disruption of clot and intrathrombic injections of highly concentrated urokinase or t-PA. Even maximally accelerated lysis, however, cannot fully compensate for the inherent delay imposed by catheterization. For that reason, prompt intravenous infusion of fibrinolytic agents, presumably t-PA, seems preferable to the intracoronary route. In the effort to initiate fibrinolytic therapy at the earliest feasible time after infarction, administration by paramedics, or even home administration after training, is a program worthy of exploration.
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PMID:Streptokinase, urokinase, and tissue plasminogen activator: pharmacokinetics, relative advantages, and methods for maximizing rates and consistency of lysis. 310 38

Urokinase- and tissue-type plasminogen activators (u-PA and t-PA) were identified immunohistochemically during reepithelialization of mouse and human skin wounds, by means of polyclonal and monoclonal antibodies. In incised mouse skin wounds u-PA immunoreactivity was found in keratinocytes at the edge of the wound after 12 h, and at days 2 to 10 after wounding it was found in virtually all keratinocytes of the epithelial outgrowth that gradually covered the wound. At day 14, the epidermis appeared normal and no u-PA immunoreactivity was detected. t-PA immunoreactivity was found from day 5 to day 10 in some keratinocytes located superficially in the epidermal outgrowths near the edge of the mouse wounds. In 3- and 5-day old human skin wounds, u-PA immunoreactivity was found in keratinocytes in the epithelial outgrowths, whereas no t-PA immunoreactivity was detected. No u-PA and no t-PA immunoreactivity was found in normal mouse and human epidermis. The specificity of the staining was supported by a variety of controls, including absorption of the polyclonal antibodies with highly purified u-PA and t-PA preparations and zymographic analysis of extracts of wound tissue. The function of the plasminogen activators during reepithelialization is discussed and it is suggested that the keratinocytes use plasmin activated by u-PA for dissecting their way through the provisional matrix in the upper part of the granulation tissue.
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PMID:Urokinase- and tissue-type plasminogen activators in keratinocytes during wound reepithelialization in vivo. 313 40

The effect of tissue plasminogen activator (TPA) or urokinase on the specific binding of human Glu-plasminogen to fibrin I formed in plasma by clotting with Reptilase was studied using 125I-plasminogen and 131I-fibrinogen. In the absence of TPA, small amounts of plasminogen were bound to fibrin I. TPA induced binding of plasminogen to plasma fibrin I that was dependent upon the concentrations of TPA and plasminogen as well as upon the time of incubation. Plasminogen binding occurred in association with fibrin clot lysis and the formation in the clot supernatant of alpha 2-plasmin inhibitor-plasmin complexes. Urokinase also induced binding of plasminogen to plasma fibrin I that was concentration- and time-dependent. The molecular form of plasminogen bound to the fibrin I plasma clot was identified as Glu-plasminogen by dodecyl sulfate-polyacrylamide gel electrophoresis and by fast performance liquid chromatography. Further studies demonstrated that fibrin I formed from fibrinogen that had been progressively degraded by plasmin-bound Glu-plasminogen. The mole ratio of plasminogen bound increased with the time of plasmin digestion. Glu-plasminogen did not bind to fibrin I formed from fibrinogen progressively digested by human leukocyte elastase, thereby demonstrating the specificity of plasmin. These studies demonstrate that plasminogen activators regulate the binding of Glu-plasminogen to fibrin I by catalyzing plasmin-mediated modifications in the fibrin substrate.
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PMID:Tissue plasminogen activator and urokinase mediate the binding of Glu-plasminogen to plasma fibrin I. Evidence for new binding sites in plasmin-degraded fibrin I. 315 57


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