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)

A rapid and quantitative fibrinolytic assay has been used to measure the overall activity of a recombinant tissue plasminogen activator (rTPA) preparation for dissolution of a fibrin clot by its ability to activate [Glu1]plasminogen (containing glutamic acid at position 1) to plasmin. A standard curve constructed for wild-type two-chain rTPA that contains, from the amino terminus, the finger (F)-growth factor (E)-kringle 1 (K1)-kringle 2 (K2)-serine protease (P) domains was used to assess the overall fibrin-dissolving abilities of variant recombinant molecules. Two-chain deletion mutants lacking the E domain, the F-E domains, the F-E-K1 domains, and the K1-K2 domains yielded activities ranging from 22% to 35% of the overall activity of wild-type two-chain rTPA, suggesting that both the K2 and F domains are individually responsible for a portion of the function of the molecule. Comparison of variant molecules containing F-K1-K2-P and F-K2-K2-P domains showed that the latter variant possessed a 4-fold higher activity (1.4-fold greater than that of wild-type two-chain rTPA), indicating that, for the activity measured, the presence of K2 leads to a greater effectiveness than that of K1. A plasmin cleavage-resistant mutant (Arg-275----Ser) has been used to assess possible differences in one- and two-chain rTPA in this overall activity, the former displaying 86% of the activity of the latter, suggesting that such differences are indeed small. Finally, the proper covalent attachment of the light and heavy chains of two-chain rTPA are very important to its overall fibrinolytic activity, since replacement of Cys-264 with glycine and concomitant disruption of one of the covalent attachment sites of the two chains provides a variant of rTPA with less than 2% of the activity of the wild-type two-chain molecule. The effector molecule, epsilon-amino hexanoic acid (epsilon Ahx; epsilon-aminocaproic acid), inhibits the overall fibrinolytic effect of rTPA in this system, with an effective Ki of approximately 1.5 mM. Its efficacy, as measured by the Ki, is independent of the presence of the epsilon Ahx binding regions of plasminogen and rTPA and is similar to the efficacy obtained when urokinase was the activator in place of wild-type two-chain rTPA or when activation of plasminogen was bypassed as a result of provision of preformed plasmin to the assay. The results suggest that in the overall clot lysis system, an important epsilon Ahx binding site may exist on fibrin that inhibits its dissolution by plasmin.
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PMID:Plasmin-mediated fibrinolysis by variant recombinant tissue plasminogen activators. 252 73

Certain group A streptococci are known to possess a receptor for the human enzyme plasmin. Plasmin is a member of a super gene family that includes other serine proteases and kringle containing proteins. In this study we have examined the interaction of a group A streptococcus with structurally related proteins, including plasmin, glu-plasminogen, tissue plasminogen activator, kallikrein, factor XII, prothrombin, thrombin, trypsin, and urokinase. Our studies indicate that only the key fibrinolytic enzyme, plasmin, demonstrates significant binding activity to the group A streptococcus.
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PMID:Group A streptococci bind human plasmin but not other structurally related proteins. 255 Oct 62

The effects of plasma fibronectin on the fibrinolytic system were studied in vitro. Fibronectin caused a time and concentration-dependent increase (up to 99% with 330 micrograms/ml) in the amidolytic activity of tissue plasminogen activator (TPA) but not of urokinase. In the presence of fibronectin the Km of the amidolytic activity of TPA decreased without a change in Vmax. It also caused a concentration-dependent increase in lys-plasminogen activation by TPA (up to 825% with 375 micrograms/ml) and by urokinase (up to 400% with 250 micrograms/ml), as well as in the amidolytic activity of plasmin (up to 55% with 300 micrograms/ml). Fibronectin did not enhance the activation of glu-plasminogen. In the presence of fibronectin the Km of lys-plasminogen activation decreased without a change in Vmax. In purified systems fibronectin significantly shortened the clot lysis time (CLT) by up to 28% and 30% in TPA- and plasmin-activated lysis, respectively. The presence of Ca2+ did not change fibronectin's effect on CLT. Clots of non-fibronectin-depleted plasma were lysed up to about twice as fast as the clots of fibronectin-depleted plasma. In conclusion, physiologic concentrations of fibronectin enhanced the fibrinolytic system in vitro. Further studies will be required to elucidate the mechanisms involved and to document whether fibronectin has a similar effect in vivo.
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PMID:Plasma fibronectin enhances fibrinolytic system in vitro. 293 66

Lysine-plasminogen (Lys-PLG), the plasmin-modified form of native glutamic acid-plasminogen (Glu-PLG), displays enhanced binding affinity for fibrin and also enhanced activation by urokinase and tissue plasminogen activator. We previously demonstrated high-affinity, specific, and functional binding of Glu-PLG as well as tissue plasminogen activator to cultured human umbilical vein endothelial cells (HUVEC). In the present study, we demonstrate binding of Lys-PLG to HUVEC, as well as conversion of Glu-PLG to Lys-PLG at the cell surface. Binding of Lys-PLG to HUVEC was saturable, reversible, epsilon-aminocaproic acid-sensitive, and involved two saturable sites with Kd's of 142 pM and 120 nM, respectively. Upon incubation with Glu-PLG, HUVEC, as well as endothelium in situ, partially converted the ligand to a Lys-PLG-like species. Conversion by HUVEC was blocked by diisopropyl-fluorophosphate, but not by other serine protease inhibitors, including alpha 2-plasmin inhibitor. Eluates of intact umbilical cord vessels contained Lys-PLG by immunoblot analysis. Lys-PLG was also identified immunohistochemically on the endothelial surface of vessels from a variety of normal and inflamed tissues. Thus, endothelial cells appear to actively modify circulating Glu-PLG, bind Lys-PLG to their surface, and thus enhance the fibrinolytic potential of the blood vessel wall.
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PMID:Endothelial cell-mediated conversion of Glu-plasminogen to Lys-plasminogen. Further evidence for assembly of the fibrinolytic system on the endothelial cell surface. 314 82

Since the introduction of thrombolytic treatment based on the activation of plasminogen (PLG) by streptokinase (SK) and urokinase (UK) the search for new and improved methods has been continuing. The pivotal issue is how to achieve clot-specific fibrinolysis without producing systemic fibrinogenolysis. One out of various approaches to enhance lysis rates has been the use of PLG either alone or in combination with UK or SK in the light of the fact that fibrinolytic treatment, particularly using SK, is associated with a consumption of PLG, and that thrombi contain relatively small amounts of native PLG, however, are capable of incorporating added PLG in vitro. PLG-concentrates from various manufactures have been administered intravenously for treatment of deep venous thrombosis, mainly in combination with SK, and of pulmonary embolism in combination with UK. Local intracoronary and intraarterial administration in combination with UK has been reported in patients with myocardial infarction, and peripheral arterial occlusions, respectively. Lysis rates obtained in these studies were in most cases superior to results obtained with SK or UK alone, without increasing the incidence of bleeding complications. In addition, excellent results in larger group of patients with cerebral thrombosis were obtained with PLG alone. The encouraging results of these studies may be explained by the fact that all of the preparations used contained partially activated forms of PLG (commonly designated lys-PLG) to a greater or lesser extent. Lys-PLG has a higher affinity for fibrin than the native glu-PLG and is activated by UK or SK by a manyfold faster. These properties allow for a rapid formation of plasmin which--bound to fibrin--is also protected from the attack of neutralizing antiplasmin. The design and results of previous studies with lys-PLG concentrates will be reviewed and approaches to further improve fibrinolytic regimens with lys-PLG-concentrates discussed.
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PMID:Review of studies with plasminogen concentrates and proposals for further therapeutic strategies with plasminogen concentrates. 328 Apr 22

Like a number of the components of the fibrinolytic and coagulation systems, plasminogen (plgn) is a multifunctional molecule. As a proenzyme, a number of its activities such as its binding to fibrin, histidine-rich glycoprotein (HRGP) and alpha 2-antiplasmin (AP) are expressed while its major enzymatic activity remains unexpressed. This latter activity has been used as a yardstick of plasminogen potency, despite the fact that no such activity resides in the native plasminogen molecule. Assay procedures usually involve the activation of the plasminogen to plasmin using an activator such as streptokinase (SK) or urokinase (UK) and a major problem involves the establishment of a properly-timed plasminogen-activator ratio to fully express the plasminogen as the active enzyme plasmin (Gaffney, P.J. et al. Activation of plasminogen as a feature of its assay. Haemostasis 1977, 6, 72-78). Substrates such as casein, fibrinogen and fibrin have been used to assess the plasmin activity developed while more recently the tripeptide chromogenic substrate S-2251 has been successfully used. These assays have been standardised using a reference preparation of the active enzyme, plasmin, and both a 1st and 2nd International Reference Preparation (IRP) have been established. These IRP's differed in that the fibrin binding kringle-structures were missing in the 1st IRP yielding differing fibrinolytic and chromogenic activities (Philo, R.D. and Gaffney, P.J. Plasmin potency estimates. Influence of substrate used in assay. Thrombosis and Haemostasis 1981, 45, 107-109). Activation procedures of plasminogen and subsequent assays of plasmin using a variety of substrates have been recently superseded by an assay which involves the formation of a plgn-SK complex which complex has an active site which hydrolyses the chromogenic substrate S-2251. This avoids the problems highlighted above involved in measuring plasminogen activity at the optimum stage during activation. While plasmin standards have been suitable for the standardisation of plasminogen when it is measured by activation-based procedures, a British Standard for glutamic acid-plasminogen has now been established in order to standardise the plgn-SK assay (Gaffney, P.J. and Curtis, A.D. The establishment of a standard for plasminogen (glu-type). Thrombosis and Haemostasis 1984, 51, 376-378). The calibration of this standard using the 2nd IRP for plasmin and the value of this standard in the measurement of plasminogen in plasma is discussed.
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PMID:Standardization of plasminogen assays. 328 Apr 25

An ideal thrombolytic (or fibrinolytic) agent is one which would generate the formation of plasmin only where it is required, i.e. bound to fibrin within the thrombus. However, the capacity of even the newer thrombolytic agents to achieve localised plasmin generation within the thrombus is relative and depends on the concentration of the agent administered. For all available activators, the concentration required for effective clinical thrombolysis is also capable of converting plasminogen to plasmin within the circulation (plasminaemia). Since the action of plasmin is not specific to fibrin, plasminaemia results in dissolution not only of fibrin but also of several other clotting factors. For example, plasmin can degrade fibrinogen and cause impaired haemostasis. The plasminogen activators which are available, or have been developed to date, include streptokinase, urokinase, pro-urokinase, anisoylated plasminogen-streptokinase activator complex (APSAC) and tissue plasminogen activator (t-PA). All of these agents have the same biochemical mechanism of action, cleaving an arginine-valine bond in the plasminogen molecule to form plasmin, but they differ with regard to other important properties. The first property to be considered is clot specificity; the ability to dissolve fibrin as opposed to fibrinogen, and also to dissolve the clot as opposed to a haemostatic plug. Unfortunately, fibrin specificity does not equate entirely with thrombus specificity, and all currently developed plasminogen activators, by dissolving fibrin, will induce the destruction of haemostatic extravascular plugs as well as intravascular thrombi. Thus, no agent is thrombus-specific in this respect. The degree of fibrinogenolysis does vary between plasminogen activators. Those which have the least effect on haemostasis or clotting capability would seem, at first, to be preferable. However, a short term reduction in fibrinogen could also be beneficial, since it may reduce the incidence of early reocclusion and, by reducing blood viscosity, improve microcirculation to the infarct zone. The intrinsic efficiency of the plasminogen activators is a second important property. In vitro, under conditions pertaining to the circulation, urokinase is about 10 times more efficient than t-PA at converting glu-plasminogen to plasmin (on the basis of the Vmax to Km ratio), while streptokinase-plasmin is 20 times more efficient. The efficiency of these activators is increased in the presence of fibrin and lys-plasminogen, 1800-fold for t-PA, 8-fold for urokinase and 180-fold for streptokinase-plasmin.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Preclinical pharmacological evaluation of anisoylated plasminogen streptokinase activator complex. 331 13

When combining angioplasty and local lysis with urokinase (UK) in treatment of peripheral arterial occlusions we have observed marked differences in the individual patient's response irrespective of the age of the thrombus. The extensive arteriosclerotic changes revealed by angiography in some of these patients suggest a reduced fibrinolytic potential depending on the underlying disease. In the standard in vitro test system we measured the UK-dependent thrombolysis in blood samples from 10 normal controls at UK concentrations of 150, 200, and 300 IU/ml of whole blood. In comparison we determined the whole blood thrombolysis time (WBTT) of 10 patients with AOD using UK concentrations of 150, 200, and 300 IU/ml of whole blood. The mean WBTT values for normal controls obtained at UK concentrations of 150 IU/ml, 200 IU/ml, and 300 IU/ml, respectively, amounted to 9.5, 5.5, and 3.5 minutes, respectively, while in patients mean values of 20.7, 8.1, and 5.5 minutes, respectively, were found. Studies on plasma samples had shown that the lysis time could be shortened in a dose-dependent manner by addition of lys-plasminogen (LYS-PLASMINOGEN Steam Treated) and to some extent also glu-plasminogen. Since lys-plasminogen gave clearly superior results we tried to improve the lytic potential in terms of a shortening of the WBTT by adding different doses of lys-plasminogen (0.14-0.56 CU/ml whole blood) to each patient sample. Although the individual response varied, the addition of lys-plasminogen to the patient samples resulted in a clear dose-dependent improvement of pathologically prolonged lysis times.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reduced fibrinolytic potential in patients with arterial occlusive disease (AOD) in comparison with normal subjects. 335 Mar 95

A rapid and highly sensitive solid phase assay was compared with the fibrin plate method for the measurement of urokinase, streptokinase and the plasminogen activators in human euglobulin fractions. The solid phase assay was run using glu - or lys - plasminogen, and significant differences were observed in the activation of the plasminogens by urokinase and streptokinase. Plasminogen activator levels in euglobulin fractions were also measureable. Very good agreement was obtained between the fibrin plate and solid phase methods in all cases.
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PMID:The comparison of solid phase and fibrin plate methods for the measurement of plasminogen activators. 635 44

We have investigated the effects of diffusive and convective transport on fibrinolysis. Using a constant pressure drop (delta P/L) from 0 to 3.7 mmHg/cm-clot to drive fluid permeation, various regimes of lytic agents were delivered into fine and coarse fibrin gels (3 mg/ml) and whole blood clots. Using plasmin (1 microM) delivered into pure fibrin or urokinase (1 microM) delivered into glu-plasminogen (2.2 microM)-laden fibrin, the velocity at which a lysis front moved across fibrin was greatly enhanced by increasing delta P/L. Lysis of fine and coarse fibrin clots by 1 microM plasmin at delta P/L of 3.67 and 1.835 mmHg/cm-clot, respectively, led to a 12-fold and 16-fold enhancement of the lysis front velocity compared to lysis without pressure-driven permeation. For uPA-mediated lysis of coarse fibrin at delta P/L = 3.67 mmHg/cm-clot, the velocity of the lysis front was 25-fold faster than the lysis front velocity measured in the absence of permeation. Similar permeation-enhanced phenomenon was seen for the lysis of whole blood clots. Without permeation, the placement of a lytic agent adjacent to a clot boundary led to a reaction front that moved at a velocity dependent on the concentration of plasmin or uPA used. Overall, these studies suggest that transport phenomena within the clot can play a major role in determining the time needed for reperfusion during fibrinolysis.
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PMID:Transport phenomena and clot dissolving therapy: an experimental investigation of diffusion-controlled and permeation-enhanced fibrinolysis. 797 57


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