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)

The binding of plasminogen to preformed human plasma clots immersed in citrated human plasma was measured and correlated with the sensitivity of these clots to lysis with recombinant tissue-type plasminogen activator (rt-PA), recombinant single-chain urokinase-type plasminogen activator (rscu-PA) or two chain urokinase-type plasminogen activator (tcu-PA, urokinase). When 0.15 ml plasma clots were compressed mechanically to about 1% of their original weight, and immersed in 0.15 ml plasma, 131I-labeled native plasminogen (Glu-plasminogen) adsorbed progressively from the plasma milieu onto the clot; binding was 3 +/- 1% (n = 10) after 1 h, 7 +/- 1% after 12 h and 12 +/- 1% after 48 h. This was associated with an increased sensitivity of the clot to lysis; 50% clot lysis in 4 h was obtained with 65 +/- 5 ng/ml (n = 3) rt-PA before and 30 +/- 5 ng/ml (n = 3) after 48 h preincubation in plasma (p less than 0.01), with corresponding values of 660 +/- 55 ng/ml (n = 3) and 280 +/- 25 ng/ml (n = 3) for rscu-PA, (p less than 0.01), and 800 +/- 85 ng/ml (n = 3) and 270 +/- 35 ng/ml (n = 3) for urokinase (p less than 0.01). Additional binding of plasminogen and increased sensitivity to lysis were reduced or abolished when the clot was preincubated in plasminogen-depleted or in t-PA-depleted plasma, or when 20 mM 6-aminohexanoic acid or 2,000 KIU/ml aprotinin were added.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Correlation between progressive adsorption of plasminogen to blood clots and their sensitivity to lysis. 209 91

The rate of activation by urokinase of porcine plasminogen is accelerated by 6-aminohexanoate, although the maximally enhanced rate is 10-fold less than that of human plasminogen without the amino acid. 6-Aminohexanoate facilitates only activation of native porcine plasminogen (asp-plasminogen), but has no effect on activation of des-kringle1-4-plasminogen. Sodium chloride, on the other hand, inhibits activation by urokinase of both porcine asp-plasminogen and des-kringle1-4-plasminogen. It is concluded that 6-aminohexanoate exerts its effect via kringle1-4 domains of plasminogen, whereas Cl- acts, at least in part, through effects on the kringle5 or proteinase domains.
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PMID:6-aminohexanoate and chloride ion in the activation by urokinase of porcine plasminogens. 237 95

1. Possible interactions between fibrin(ogen) and heparin in the control of plasminogen activation were studied in model systems using the thrombolytic agents tissue-type plasminogen activator (t-PA), urokinase and streptokinase.plasminogen activator complex and the substrates Glu- and Lys-plasminogen. 2. Both t-PA and urokinase activities were promoted by heparin and by pentosan polysulphate, but not by chondroitin sulphate or hyaluronic acid. The effect was on Km. 3. In the presence of soluble fibrin (and its mimic, CNBr-digested fibrinogen) the effect of heparin on t-PA was attenuated, although not abolished. In studies using a monoclonal antibody and 6-aminohexanoic acid, it was found that heparin and fibrin did not seem to share a binding site on t-PA. 4. The activity of t-PA B-chain was unaffected by heparin, so the binding site is located on the A-chain of t-PA (and urokinase). 5. Fibrin potentiated the activity of heparin on urokinase. The activity of streptokinase.plasminogen was unaffected by heparin whether or not fibrin was present. 6. If these influences of heparin and fibrin also occur in vivo, then, in the presence of heparin, the relative fibrin enhancement of t-PA will be diminished and the likelihood of systemic activation by t-PA is increased.
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PMID:Kinetic studies on the effect of heparin and fibrin on plasminogen activators. 244 77

Enzyme kinetic plots relating the initial rate of activation of pro-urokinase to urokinase by plasmin, according to the concentration of substrate, were smooth downward curves and indicated that an apparent decrease in binding affinity occurred with increase in the concentration of pro-urokinase. Such nonlinear plots were obtained with plasmin 1 and also plasmin 2. Over sections of each curve it was possible to estimate apparent kinetic constants. At the uppermost concentrations of substrate tested, these were Km 2.9 microM and kcat 35.5 min-1 for plasmin 1, and at the lowermost concentrations, Km 9.5 nM and kcat 2.0 min-1. Linear plots were obtained when the single proteolytic cleavage was made by K5-plasmin or undegraded plasmin in the presence of 1.0 mM 6-aminohexanoic acid (6-AHa). Constants were estimated for catalysis of this reaction by K5 plasmin to be Km 6.0 microM and kcat 38 min-1 (r = 0.987). The catalytic efficiency of plasmin, at the lowermost concentrations of pro-urokinase tested, was therefore 33-fold higher than that of K5-plasmin. Plotting of data for the cleavage of pro-urokinase by plasmin 1 (in the absence of 6-AHa) according to the model of Hill, gave a slope of 0.5 at the lowermost concentrations of pro-urokinase increasing to 1.0 at higher concentrations (greater than 0.3 microM); such a profile is characteristic of negative cooperativity. The rates of formation of plasmin and urokinase in a mixture containing a low concentration of plasminogen and pro-urokinase were measured and compared to those predicted by a computer program designed to calculate theoretical rates using available kinetic data. The observed rates of generation of both plasmin and urokinase coincided to those predicted from the negative cooperativity model. The mechanism of the negative cooperativity may reside in a conformational change induced by binding of pro-urokinase to the kringle structure of plasmin. This property may be of significance in controlling the fibrinolytic properties of the urokinase-type plasminogen activator system.
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PMID:Activation of pro-urokinase by plasmin: non-Michaelian kinetics indicates a mechanism of negative cooperativity. 252 53

The binding of urokinase-type plasminogen activators (u-PA) to receptors on various cell types has been proposed to be an important feature of many cellular processes requiring extracellular proteolysis. We have investigated the effect of single-chain u-PA binding to the monocyte-like cell line U937 on plasminogen activation. A 16-fold acceleration of the activation of plasminogen was observed at optimal concentrations of single-chain u-PA. This potentiation was abolished by the addition of either 6-aminohexanoic acid or the amino-terminal fragment of u-PA, thus demonstrating the requirement for specific binding of both single-chain u-PA and plasminogen to the cells. The mechanism of the enhancement of plasmin generation appears to be due primarily to an increase in the rate of feedback activation of single-chain u-PA to the more active two-chain u-PA by cell-bound plasmin, initially generated by single-chain u-PA. This increased activity of the plasminogen activation system in the presence of U937 cells provides a mechanism whereby u-PAs may exert their influence in a variety of cell-associated proteolytic events.
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PMID:Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. Potentiation by U937 monocytes. 252 25

The gene transfer technique was used to examine the role of plasminogen activator (PA) in the invasive and metastatic behavior of tumorigenic cells. H-ras-transformed NIH 3T3 clonal cells producing a very low level of PA were generated and further transfected with an expression plasmid containing a cDNA sequence encoding either the urokinase-type or the tissue-type human PA. Compared with the parental transformed cells, clonal cells expressing high levels of both types of recombinant PA invaded more rapidly through a basement membrane reconstituted in vitro. Furthermore, cells expressing high levels of recombinant urokinase-type PA also caused a higher incidence of pulmonary metastatic lesions after intravenous injection into nude mice. Both activities were reduced by the serine proteinase inhibitor EACA; invasion was also suppressed by antibodies blocking the activity of human PAs and by the synthetic collagenase inhibitor SC-44463. These findings provide direct genetic evidence for a causal role of PA in invasive and metastatic activities.
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PMID:Expression of human recombinant plasminogen activators enhances invasion and experimental metastasis of H-ras-transformed NIH 3T3 cells. 274 45

Photoaffinity labeling of human plasmin using 4-azidobenzoylglycyl-L-lysine inhibits clot lysis activity, while the activity toward the active-site titrant, p-nitrophenyl-p'-guanidinobenzoate, or alpha-casein are maintained. Photoaffinity labeling of native Glu-plasminogen with the same reagent causes incorporation of approximately 1.5 mol label per mol plasminogen. This labeled plasminogen can be activated to plasmin by either urokinase or streptokinase. The resulting plasmin has full clot lysis activity and can be subsequently photoaffinity labeled with a loss of clot lysis activity. The rate of activation of labeled plasminogen by urokinase is increased relative to that of native plasminogen. epsilon-Aminocaproic acid blocks incorporation of photoaffinity label into both plasminogen and plasmin, indicating that the labeling is specific to the lysine-binding sites. The labels are located in the kringle 1+2+3 fragment in either photoaffinity-labeled plasminogen or plasmin. These results indicate that the specific lysine-binding site blocked in plasmin acts in concert with the active-site in binding and using fibrin as a substrate. This clot lysis regulating site is not available for labeling in plasminogen, but is exposed or changed upon activation to plasmin. The different lysine-binding sites labeled in plasminogen may regulate the conformation of the molecule as evidence by an enhanced rate of activation to plasmin.
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PMID:Photoaffinity labeling of functionally different lysine-binding sites in human plasminogen and plasmin. 316 Mar 89

The kinetics of activation of Lys-plasminogen (Lys-77-Asn-790) and miniplasminogen (Val-442-Asn-790) catalysed by low-molecular-weight urokinase (LMW-urokinase) was investigated in the presence and absence of ligands that bind to the AH-site of the plasminogens. 6-Aminohexanoic acid and alpha-N-acetyl-L-lysine methyl ester (AcLysMe) were used. Saturation of the AH-sites of the plasminogens result in similar, but rather small positive effects on the kinetics of activation of the two plasminogens. Michaelis constants decrease approx. 2-fold and second-order rate constants (kc/Km)Pg increase approx. 1.2-fold. Michaelis constants (KPg values) were obtained using a new approach; the values were determined from the competing effects of the plasminogens on urokinase-catalysed hydrolysis of a synthetic substrate. In the pH range 7.4-8.0, only minor alterations of the values of the kinetic parameters are observed. At 25 degrees C, values of (kc/Km)Pg are approx. 3-fold less than the value at 37 degrees C, whereas KPg is not changed. We conclude that kc/Km values are approx. 10(5) M-1.s-1 and that KPg values are approx. 40 microM of urokinase-catalysed conversions of Lys- and miniplasminogen to their respective plasmins.
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PMID:Urokinase-catalysed plasminogen activation. Effects of ligands binding to the AH-site of plasminogen. 319 Nov 43

Activation of human Glu-plasminogen, Lys-plasminogen and low-Mr plasminogen (lacking lysine-binding sites) by pro-urokinase (pro-UK), obtained from a human lung adenocarcinoma cell line (Calu-3, ATCC), obeys Michaelis-Menten kinetics. Activation occurs with a comparable affinity (Km 0.40-0.77 microM), while the catalytic rate constant (kcat) is comparable for Glu-plasminogen (0.0022s-1) and low-Mr plasminogen (0.0034 s-1), but is somewhat higher for Lys-plasminogen (0.0106 s-1). The rate of activation of plasminogen by pro-UK is not significantly influenced by the presence of 6-aminohexanoic acid, purified fragments LBS I or LBS II or histidine-rich glycoprotein, indicating that the high affinity of pro-UK for plasminogen is not mediated via the high-affinity lysine-binding site of plasminogen located in kringles 1-3 (LBS I) nor via the low-affinity lysine-binding site comprised within kringle 4 (LBS II). The site(s) in plasminogen involved in the high-affinity interaction with pro-UK thus appear to be located within the low-Mr plasminogen moiety.
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PMID:Characterization of the high-affinity interaction between human plasminogen and pro-urokinase. 392 92

The catalytic efficiency (kcat/Km) of high-molecular-mass urokinase for the activation of Glu-plasminogen is increased about 10-fold in the presence of CNBr-digested fibrinogen. This stimulation is similar to that observed with 6-aminohexanoic acid, and yields kinetic parameters comparable to those for the activation of Lys-plasminogen by urokinase. The increase of the activation rate of Glu-plasminogen by urokinase in the presence of CNBr-Fg can thus be explained by a conformational change in the plasminogen molecule similar to that observed upon conversion of Glu-plasminogen to Lys-plasminogen and upon binding of 6-aminohexanoic acid to Glu-plasminogen. Stabilization of the Michaelis complex between urokinase and plasminogen by formation of a cyclic ternary complex with CNBr-Fg, which has been invoked to explain the dramatic stimulatory effect of CNBr-Fg on the activation of plasminogen by tissue-type plasminogen activator, does not appear to play a significant role in the increased activation rate.
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PMID:Influence of cyanogen-bromide-digested fibrinogen on the kinetics of plasminogen activation by urokinase. 648 41


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