Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q9UIJ5 (Rec)
 58,342 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism of the activation of plasminogen by recombinant pro-urokinase (Rec-pro-UK), obtained by expression of the human pro-urokinase gene in Escherichia coli, was investigated in purified systems. In mixtures of Rec-pro-UK and plasminogen, both active urokinase and plasmin are quickly generated. Addition of plasmin inhibitors (aprotinin or alpha 2-antiplasmin) abolishes the conversion of Rec-pro-UK to urokinase but not the activation of plasminogen to plasmin, suggesting that Rec-pro-UK activates plasminogen directly. Human plasma competitively inhibits the activation of plasminogen by pro-urokinase with a Ki of 0.2% (v/v). This explains the relative stability of Rec-pro-UK in plasma and the lack of activation of the plasma fibrinolytic system in the absence of fibrin. The competitive inhibition by plasma is abolished by the addition of CNBr-digested fibrinogen although Rec-pro-UK has no specific affinity for fibrin. These findings suggest that the fibrin specificity of the activation of plasminogen by pro-urokinase is due to neutralization by fibrin of the competitive inhibition exerted by plasma and not to fibrin-enhanced activation of plasminogen.
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PMID:Activation of plasminogen by pro-urokinase. I. Mechanism. 293 28

The fibrinolytic and fibrinogenolytic properties of recombinant pro-urokinase (Rec-pro-UK) and recombinant urokinase (Rec-UK) obtained by expression of the human urokinase cDNA in E. coli, were compared with those of natural urokinase (Nat-UK) of urinary origin and of tissue-type plasminogen activator (t-PA) in a system, composed of a radioactive human plasma clot immersed in citrated human plasma. The specific fibrinolytic effects of Nat-UK, Rec-pro-UK and Rec-UK were very similar, causing significant clot lysis at concentrations of 100 IU/ml plasma or more. t-PA caused equivalent degrees of clot lysis at 10-fold lower concentrations. Activation of the fibrinolytic system in the plasma (fibrinogenolysis), was not observed with t-PA in concentrations which induced complete clot lysis within 5 hr (20-30 IU/ml plasma). With Nat-UK and Rec-UK, all concentrations which caused significant clot lysis (100-200 IU/ml plasma) also caused extensive activation of the plasma fibrinolytic system. With Rec-pro-UK an intermediate response was obtained. The highest amounts required for complete clot lysis in 5 hr (200 IU/ml plasma) also caused significant fibrinogenolysis. At intermediate concentrations (50-100 IU/ml), however, significant clot lysis (40-80%) was observed without systemic fibrinolytic activation.
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PMID:Biological and thrombolytic properties of proenzyme and active forms of human urokinase--I. Fibrinolytic and fibrinogenolytic properties in human plasma in vitro of urokinases obtained from human urine or by recombinant DNA technology. 649 59

The turnover of recombinant pro-urokinase (Rec-pro-UK), recombinant urokinase (Rec-UK) and natural urinary urokinase (Nat-UK) was studied in rabbits and in squirrel monkeys (Saimiri sciureus). Following intravenous injection, urokinase activity disappeared rapidly from the blood. The initial disappearance rate could be described by a single exponential term with a t 1/2 of 3 to 6 min for each molecular form of urokinase in both species. Urokinase related antigen, measured with a radioimmunoassay in the plasma of the squirrel monkeys disappeared with a t 1/2 of 3.5 min for Rec-pro-UK, 6.0 min for Rec-UK and 8.0 min for Nat-UK. The clearance and organ distribution of Rec-pro-UK, Rec-UK and Nat-UK was studied with the use of 125I-labeled preparations. In each case the radioactivity initially disappeared rapidly from the plasma, also with a t 1/2 of a few min, but then the disappearance rate slowed down. Labeled Rec-UK in which the active site histidine was irreversibly blocked by alkylation, disappeared equally rapidly from the plasma. Measurement of the organ distribution of 125I at different time intervals revealed that all three types of urokinase were rapidly accumulated in the liver, which was followed by release of degradation products in the blood. Experimental hepatectomy prolonged the t 1/2 of each type of urokinase very markedly (t 1/2 greater than 30 min). These findings indicate that urokinase is rapidly removed from the blood by clearance and degradation in the liver. Recognition by the liver does not require a functional active site and is not mediated via carbohydrate side chains. Inactivation by plasma protease inhibitors does not represent a significant pathway of urokinase inhibition in vivo.
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PMID:Biological and thrombolytic properties of proenzyme and active forms of human urokinase--II. Turnover of natural and recombinant urokinase in rabbits and squirrel monkeys. 649 60

The fibrinolytic and fibrinogenolytic properties of recombinant pro-urokinase (Rec-pro-UK) and recombinant urokinase (Rec-UK) were compared with those of natural urokinase (Nat-UK) and of tissue-type plasminogen activator (t-PA) in an in vitro system consisting of 125I-labeled autologous plasma clots immersed in plasma of humans, five primate species, dogs, rabbits and pigs. With each of the four plasminogen activators, a dose-dependent clot lysis was observed, the degree of which differed, however, very markedly from one species to the other. At a concentration of 100 IU/ml of urokinase extensive plasma clot lysis was obtained in plasma of man, Macaca mulatta, Macaca fascicularis and Macaca radiata, while the plasma clots of Papio cynocephalus, Papio anubis and rabbit, dog and pig were much more resistant to lysis. No significant differences in the extent of lysis were observed between Rec-pro-UK and Rec-UK nor between Rec-UK and Nat-UK. Comparable degrees of lysis were obtained with t-PA at 3- to 5-fold lower concentrations. Lysis with Rec-UK or Nat-UK was always associated with extensive activation of the fibrinolytic system in plasma, evidenced by fibrinogen breakdown and plasminogen activation and alpha 2-antiplasmin consumption. With t-PA, extensive clot lysis was obtained in the absence of fibrinolytic activation in the plasma. With Rec-pro-UK the response was intermediate; at high concentrations (200 IU/ml) extensive lysis in the reactive species was associated with fibrinogen consumption, while at intermediate concentrations (50-100 IU/ml) significant clot lysis was obtained in the reactive species in the absence of marked activation of the fibrinolytic system in the plasma.
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PMID:Biological and thrombolytic properties of proenzyme and active forms of human urokinase--IV. Variability in fibrinolytic response of plasma of several mammalian species. 649 61

The thrombolytic properties of recombinant pro-urokinase (Rec-pro-UK), recombinant active urokinase (Rec-UK) and natural urinary urokinase (Nat-UK) were compared with those of tissue-type plasminogen activator (t-PA) in rabbits with a radiolabeled thrombus in the jugular vein. The thrombolytic agents were infused intravenously over a time period of 4 hr and the extent of thrombolysis measured two hours later. In control animals the extent of thrombolysis was 11 +/- 2% (n = 8) after 6 hr. Nat-UK and Rec-UK had very similar thrombolytic properties. Significant thrombolysis was only obtained with infusion of 240,000 IU per kg (41 +/- 2%, n = 4 for Nat-UK and 37 +/- 4%, n = 4 for Rec-UK) and this was associated with a marked systemic activation of the fibrinolytic system, as evidenced by consumption of plasminogen and alpha 2-antiplasmin and fibrinogen breakdown. Infusion of Rec-pro-UK induced thrombolysis at a dose of 60,000 IU per kg (44 +/- 8%, n = 3) but without associated systemic activation of the fibrinolytic system. In this respect the properties of Rec-pro-UK were similar to those of t-PA, which, however, had a 2- to 4-fold higher specific thrombolytic activity (30,000 IU/kg yielding 48 +/- 1% lysis, n = 4). It is concluded that Rec-UK has very similar thrombolytic properties as Nat-UK and that Rec-pro-UK has a better thrombus-selectivity and less systemic side effects than the active enzymes.
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PMID:Biological and thrombolytic properties of proenzyme and active forms of human urokinase--III. Thrombolytic properties of natural and recombinant urokinase in rabbits with experimental jugular vein thrombosis. 654 17

Urokinase-type plasminogen activator (uPA) binds with high affinity to a specific cell surface glycosyl phosphatidyl-inositol (GPI)-anchored receptor, the urokinase receptor (uPAR). Pro-uPA, the enzymatically inactive single-chain form of uPA after having been activated by certain proteases, converts plasminogen into plasmin. This activation of pro-uPA to enzymatically active uPA can be prevented by the action of thrombin on pro-uPA. This inactivation process is accelerated in the presence of thrombomodulin (TM). The present study investigated whether pro-uPA bound to uPAR is still susceptible to inactivation by thrombin in the presence or absence of TM. A truncated soluble form of the uPAR lacking the GPI-anchor was cloned and expressed in CHO-cells (rec-uPAR277). Rec-uPAR277 efficiently inhibited the thrombin-mediated inactivation of pro-uPA up to 90% in a concentration dependent manner. The protective effect of rec-uPAR277 was far less pronounced when thrombin was complexed with TM. Enzyme kinetic experiments with varying concentrations of pro-uPA showed that in the presence of TM the catalytic efficiency (kcat/Km) of thrombin-mediated inactivation raised from 0.010 microM-1 s-1 to 0.50 microM-1 s-1 corresponding to a fifty-fold increase. In the presence of rec-uPAR277, however, the catalytic efficiency dropped by 4.1-fold (0.5 microM-1 s-1 to 0.122 microM-1 s-1). The inactivation kinetics of pro-uPA by thrombin (no TM added) in the presence of an excess of rec-uPAR277 could not be determined since virtually no inactivation occurred. Our data suggest that pro-uPA once bound to uPAR, is significantly protected from inactivation by thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inactivation of receptor-bound pro-urokinase-type plasminogen activator (pro-uPA) by thrombin and thrombin/thrombomodulin complex. 784 Sep 2

A recombinant soluble human urokinase receptor comprising amino acids 1-277 was cloned and transfected into CHO cells. The mutant protein (rec-uPAR277), purified from the CHO cell supernatant by affinity chromatography on immobilized urokinase (uPA), in a four-fold excess, completely abolished the binding of FITC-labeled pro-uPA to the human ovarian cancer cell line, OV-MZ-6. This invasive and tumorigenic cancer cell line expresses uPA, its inhibitor PAI-1, and the high-affinity receptor for uPA, uPAR. Rec-uPAR277 significantly reduced the proliferation of OV-MZ-6 cells in a concentration-dependent manner without altering the viability of the cells. Invasion of OV-MZ-6 cells tested in an in vitro Matrigel invasion assay was inhibited by rec-uPAR277 up to 75%. In conclusion, these results demonstrate that rec-uPAR277 can function as a scavenger for uPA in vitro by inhibiting proliferation and invasion of human cancer cells.
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PMID:Recombinant soluble urokinase receptor as a scavenger for urokinase-type plasminogen activator (uPA). Inhibition of proliferation and invasion of human ovarian cancer cells. 828 66

Thrombomodulin (TM), a membrane proteoglycan on endothelial cells, binds thrombin in a 1:1 complex, accelerates the protein C activation by thrombin, promotes the thrombin inactivation by antithrombin III and inhibits the procoagulant properties of thrombin. The inactivation of single-chain urokinase-type plasminogen activator (scu-PA) by thrombin is accelerated about 70-fold by TM [De Munk, Groeneveld and Rijken (1991) J. Clin. Invest. 88, 1680-1684]. The present study investigates the role of the O-linked glycosaminoglycan moiety of TM in the latter reaction. In the presence of an excess of a fully-glycosylated soluble recombinant human TM mutant (high-Mr rec-TM), 0.11 nM thrombin inactivated 50% of 4.4 nM scu-PA in 45 min at 37 degrees C. In the presence of a soluble recombinant TM mutant lacking the glycosaminoglycans (low-Mr rec-TM), 1.9 nM thrombin was needed to inactivate 50% scu-PA, as compared with 4.7 nM thrombin in the absence of TM. Using the scu-PA inactivation assay the dissociation constant for the thrombin-TM interaction was found to be 0.4 nM for high-Mr rec-TM and 14 nM for low-Mr rec-TM. Treatment of high-Mr rec-TM with chondroitinase ABC to digest the glycosaminoglycans decreased the accelerating effect to the level of low-Mr rec-TM. A similar decrease was observed after treatment of solubilized rabbit TM with chondroitinase ABC. As expected, chondroitinase ABC had no influence on the accelerating effect of low-Mr rec-TM. The free glycosaminoglycans obtained by alkaline treatment of TM or chondroitin sulphate A also accelerated the inactivation of scu-PA by thrombin, but about 1000-fold higher concentrations than with TM were needed to obtain the same acceleration. It is concluded that the major glycosaminoglycan of TM plays a pivotal role in the inactivation of scu-PA by the TM-thrombin complex, both in the formation and in the activity of the complex.
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PMID:Role of the glycosaminoglycan component of thrombomodulin in its acceleration of the inactivation of single-chain urokinase-type plasminogen activator by thrombin. 838 42