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

Optical density measurements of plasma clot formation and lysis were recorded using a platelet aggregometer and strip chart recorder. It was discovered that, by adding standard solutions of ellagic acid-activated partial thromboplastin, urokinase, and CaCl2, and monitoring the reaction via the recorder, characteristic curves would be generated by normal human plasma. The curve segments were labeled Tc (clotting time), which correlated with the activated partial thromboplastin time, Fc (maximum optical density change), which paralleled fibrinogen concentration, and Tl (lysis time), which corresponded generally to plasminogen levels. Deviations from normal curve segments, observed in disseminated intravascular coagulation, hypo- and hyperfibrinogenemia, factor VIII deficiency, severe hepatocellular disease, juvenile rheumatoid arthritis, and neonates (normally low in plasminogen), indicated abnormalities which were substantiated by standard procedures. This new test, given the acronym "CLUE" for clotting and lysis, urokinase enzyme activated, appears to be sensitive, inexpensive and easily performed on a sample of 0.2 ml. of plasma in only 15 minutes.
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PMID:The CLUE test. A multiparameter coagulation and fibrinolysis screening test using the platelet aggregometer. 111 Dec 77

Fibronectin immobilized onto polystyrene surface was found to bind plasminogen and tissue-type plasminogen activator (t-PA) but only slightly the urokinase type as determined using mono- and polyclonal antibodies against the activators. Of the defined fibronectin fragments tested, the Mr 120,000-140,000 fragment was found to bind both plasminogen and t-PA. Proteolytically modified plasminogen (Lys-plasminogen) bound considerably better than the native form (Glu-plasminogen). Experiments with 125I-plasminogen yielded Kd = 9.1 X 10(-8) M for the binding to immobilized fibronectin. The partially or completely inactive single-chain form of t-PA (pro-t-PA) bound considerably better than the activated two-chain form. Lysine at greater than 3 mM inhibited the binding of plasminogen. The interaction was independent of calcium ions. CaCl2 (greater than 0.5 mM) and NaCl (greater than 0.2 M) inhibited the binding of pro-t-PA and of t-PA. Fibronectin-bound t-PA retained its ability to activate plasminogen. The observed interactions may operate in directional proteolysis localizing plasminogen and plasminogen activator to degrade fibronectin-containing extracellular matrix including fibrin clots.
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PMID:Plasminogen and tissue-type plasminogen activator bind to immobilized fibronectin. 241 22

The formation of thrombi in vivo includes the activation of both platelets and the coagulation cascade. Conventional thrombolytic therapy is primarily directed toward the dissolution of fibrin. To evaluate the possibility that platelet activity impairs the lysis of thrombi, we studied the effects of aspirin and platelet-deaggregating prostaglandin E1 on thrombolysis with urokinase. Combined platelet and fibrin thrombi were produced in vitro by adding CaCl2 and collagen (1 microgram/ml) to citrated platelet-rich plasma (250,000 platelets per microliters). Urokinase (500-10,000 units/ml) caused a dose-dependent weight loss of the thrombi that was maximal at 2,000 units/ml. The addition of aspirin (10-200 micrograms/ml) to platelet-rich plasma before thrombus formation markedly enhanced thrombolysis with urokinase. This effect was most pronounced at 20 micrograms/ml aspirin. However, when aspirin was added after completion of thrombus formation, no significant effect on thrombolysis was noted. Prostaglandin E1 (1-100 mumol/l) improved the lysis with urokinase of the combined platelet and fibrin thrombi. This effect was maximal at 20 mumol/l prostaglandin E1. When pure fibrin thrombi were produced in platelet-free plasma, prostaglandin E1 was without effect on lysis. Thus, in vitro lysis with urokinase of combined platelet and fibrin thrombi was enhanced by the addition of platelet-deaggregating prostaglandin E1 and by pretreatment with aspirin.
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PMID:Effects of aspirin and prostaglandin E1 on in vitro thrombolysis with urokinase. Evidence for a possible role of inhibiting platelet activity in thrombolysis. 272 Sep 29

The release of plasminogen activators (PA) from human isolated glomeruli has been studied by a sensitive radioenzymatic assay using 125I-fibrin coated tubes and plasminogen. The glomerular fibrinolytic activity (GFA) was detectable after 15 minutes of incubation. Then it increased with time, the glomerular protein concentration, and with the plasminogen concentration (P less than 0.001 for all). CaCl2 (1 mM) increased the GFA (9.7 +/- 0.9 versus 4.9 +/- 0.4 micrograms fibrin/mg/30 min, P less than 0.05). The GFA was also enhanced when pH increased. Arachidonic acid (AA, 1 to 20 micrograms/ml) increased the GFA in a saturable manner. Inhibitors of cyclooxygenase (aspirin) or of lipoxygenase (nordihydroguaiaretic acid) did not modify the basal and AA-stimulated GFA. Other polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), eicosatrienoic acid (ETA), eicosatetraynoic acid (ETYA), or dihomo-gamma-linoleic acid (DHL), also stimulated the GFA whereas linoleic acid and oleic acid did not. Polyunsaturated fatty acids also stimulated the fibrinolytic activity of glomerular supernatants. Specific antibodies to t-PA, and to a lesser extent to u-PA, decreased this fibrinolytic activity whether or not AA was added. Furthermore, AA and EPA were found to increase the activity of purified u-PA and t-PA. We conclude that human glomeruli release both t-PA and u-PA, and that this release is increased by calcium and alkaline pH. The polyunsaturated fatty acids enhanced the GFA, mainly by a stimulatory effect of PA activity rather than an increased release of PA from glomerular cells.
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PMID:Polyunsaturated fatty acids increase fibrinolytic activity of human isolated glomeruli. 309 75

Several monoclonal antibodies (MABs) against specific parts of the plasminogen molecule were developed. One of them, MPW1PG, recognizes only the native form Glu-plasminogen, while the other one, MPW2PG, reacts equally well with Glu-plasminogen and its proteolytically degraded derivative Lys-plasminogen as confirmed by immunoblotting experiments. Both MABs do not alter the cleavage of the low molecular weight substrate S-2251 by plasmin in a purified system but rather increase the rate of plasmin formation by urokinase and t-PA in a system without fibrin. In the presence of fibrin MPW1PG has no effect on plasmin formation by t-PA, while MPW2PG exhibits mixed type inhibition. To investigate whether this effect can also be seen in a whole blood clot lysis system, the Chandler loop was used (PVC tubes, 4 mm inner diameter, 28 mm length), 125I-fibrinogen was added to citrated whole blood (0.36% sodium citrate) to about 5,000 cpm/10 microliter sample. 1.5 ml of the mixture were pipetted into each tube, the tubes were closed, put on a rotary plate, tilted to an angle of 23 degrees, and rotated at 16 rpm. 10 microliter samples were taken for radioactive counting before clotting by addition of 10 microliter 3.02M CaCl2 (100%-value), 45 min after clotting (0%-value), and 30, 60, 120, 180, 360 minutes after addition of activators. Lysis was started one hour after recalcification. Different amounts of MABs were added either 30 min before recalcification (MAB endogen) or together with the activator (MAB exogen) Percent lysis was calculated from the radioactivity released into the fluid phase. Double determinations were done in all experiments.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of anti plasminogen monoclonal antibodies on whole blood clot lysis. 312 10

125I-labeled heparin cofactor II (HCII) was mixed with plasma and coagulation was initiated by addition of CaCl2, phospholipids, and kaolin or tissue factor. In the presence of 67 micrograms/ml of dermatan sulfate, radioactivity was detected in a band which corresponded to the thrombin-HCII complex (Mr = 96,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No other complexes were observed. The thrombin-HCII complex was undetectable when 5 units/ml of heparin was present or when prothrombin-deficient plasma was used. In experiments with purified proteases, HCII did not significantly inhibit coagulation factors VIIa, IXa, Xa, XIa, XIIa, kallikrein, activated protein C, plasmin, urokinase, tissue plasminogen activator, leukocyte elastase, the gamma-subunit of nerve growth factor, and the epidermal growth factor-binding protein. HCII inhibited leukocyte cathepsin G slowly, with a rate constant of 8 X 10(4) M-1 min-1 in the presence of dermatan sulfate. These results indicate that the protease specificity of HCII is more restricted than that of other plasma protease inhibitors and suggest that the anticoagulant effect of dermatan sulfate is due solely to inhibition of thrombin by HCII.
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PMID:The protease specificity of heparin cofactor II. Inhibition of thrombin generated during coagulation. 383 15

The plasminogen activators (PA) and cell types responsible for the fibrinolytic activity of isolated human glomeruli were studied by an indirect spectrophotometric method for plasminogen activation and immunohistological techniques with specific antibodies. Our results indicate that human glomeruli possess the ability to release both tissue-type PA (t-PA) and urokinase (UK). This has been shown for t-PA by quenching its fibrin-dependent activity with rabbit anti-t-PA antibodies and for UK by quenching its fibrin-independent activity with goat anti-UK antibodies. On the other hand, immunohistochemical analysis performed with a murine monoclonal antibody to plasma-t-PA and goat polyclonal antibodies to UK allowed the exclusive localization of t-PA in the endothelial cell lining of the glomerular flocculus and UK in the cytoplasma of glomerular epithelial cells. In addition, arachidonic acid and CaCl2 were shown to enhance glomerular fibrinolytic activity by stimulating the release of either UK or t-PA, respectively. This particular distribution and regulation of glomerular PA's may be important in the physiopathology of the intra and extracapillary fibrin deposits observed in several glomerulopathies.
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PMID:Identification and cellular localization of plasminogen activators from human glomeruli. 393 66

We have investigated the factors governing the plasminogen-dependent fibrinolysis catalyzed by the serine proteinase, plasminogen activator (EC 3.4.21.-), under physiologic conditions. We found that live rabbit fibroblasts digested much less fibrin than predicted by cell-free assay of the secreted plasminogen activator. The reduced catalytic activity of plasminogen activator expressed by cells growing on fibrin was regulated by the salt concentration of culture medium. The plasminogen activators of cells from several mammalian species were inhibited by physiologic salt concentrations (0.15 M NaCl) in cell-free assays. CaCl2 and KCl, but not D-glucose, were also effective inhibitors. The catalytic activity of purified human urokinase and of plasmin was unaffected by increased ionic strength. Plasminogen activators secreted both spontaneously and in response to stimulation by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate, were inhibited by 0.15 M NaCl. Physiologic salt concentration appeared to function by interacting with plasminogen activator, or plasminogen, and a third component, possibly a reversible inhibitor. One consequence of this regulation of plasminogen activator under physiologic conditions is the limitation of plasminogen-dependent fibrin degradation by living cells.
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PMID:Expression of the catalytic activity of plasminogen activator under physiologic conditions. 645 62

Keratinocytes propagated in low calcium (30 microM CaCl2) serum-free media grow in a monolayer and exhibit morphologic and biosynthetic phenotypes most similar to those of keratinocytes in the basal layer of the normal epidermis. When the calcium in the media is elevated to 1 mM, the cells stratify and differentiate. The effects of calcium on human foreskin keratinocyte expression of urokinase type (uPA) and tissue type (tPA) plasminogen activator enzymes and plasminogen activator inhibitor 1 and 2 (PAI-1, PAI-2) were assessed by Northern analyses. Our data show that keratinocytes, cultured in the presence of low and high CaCl2 concentrations, express transcripts for uPA and PAI-2. Message levels for uPA were dramatically reduced in cultures stimulated with calcium, whereas those for PAI-2 were only slightly decreased. Little PAI-1 mRNA and no tPA mRNA were detected, independent of calcium levels. Actin mRNA levels were not modulated consequent to calcium stimulation. Hybridizations to 28S ribosomal RNA confirmed that equal amounts of RNA were analyzed from cells grown under low and high calcium conditions. These data demonstrate that keratinocytes, propagated in serum-free media under low and high calcium conditions, are similar to normal human epidermis with respect to their expression of regulators of plasminogen activation. Additionally, they suggest that the ratio of PAI-2 to uPA increases with keratinocyte differentiation.
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PMID:Calcium modulates the expression of urokinase plasminogen activator and plasminogen activator inhibitor 2 by human keratinocytes. 792 56

Heat-stable proteases produced by the psychrotroph Pseudomonas fluorescens M3/6 have been shown to affect the plasmin system in milk, which in turn will affect the quality of processed milk. The M3/6 proteases cause dissociation of plasmin from casein in minimally processed milk. The objective of this work was to study the effect of M3/6 protease on the plasmin system, as well as its role in plasminogen activation, under commonly applied cheese-making conditions. Isolated M3/6 protease was added to raw milk, which then was pasteurized, and subjected to pH adjustments and CaCl2 addition. Casein and whey fractions were separated by chymosin treatment then analyzed for plasmin activity. Individual and interaction effects of M3/6 protease addition, pH treatment, and CaCl2 addition on plasmin activity were studied. Enzyme activity assays were carried out to study individually the effect of M3/6 protease on plasmin system components. Kinetic parameters were calculated to characterize the effect of M3/6 protease on plasminogen activation. Plasmin activity increased in the curd fractions of the protease-treated milk that was subjected to conditions most resembling cheese-making conditions, indicating that M3/6 protease triggered plasminogen activation rather than dissociation of plasmin from casein micelles. Results from the studies on plasminogen activation confirmed that the observed activation of plasminogen in protease-treated samples subjected to cheese making conditions was attributed to the stimulatory effect M3/6 protease had on plasminogen activators (PA). The M3/6 protease stimulated human and bovine PA by increasing their activity 4.5- and 2.5-fold, respectively. Similarly, the catalytic efficiencies of human urokinase-type PA and bovine PA were increased in the presence of M3/6 protease by 12- and 4-fold, respectively. Our research presented a basic step toward fully understanding the effect of bacterial proteases under different processing conditions, where the gathered information can aid in better control of processing conditions based on the desired outcome.
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PMID:Effects of Pseudomonas fluorescens M3/6 bacterial protease on plasmin system and plasminogen activation. 1616 12


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