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 levels of fibrinogen and of profibrinolysin (plasminogen) in urokinase-treated plasma as a function of time of incubation were measured. The profibrinolysin concentration was estimated through its complete conversion to fibrinolysin and the inhibition of the enzyme by crystalline soybean trypsin inhibitor. The dissociation constant of the FL-STI complex was determined to be 7 times 10-9 M. The average concentration of profibrinolysin in normal human citrated plasma was found to be 8 times 10-7 M. From the decrease of fibrinogen with time in the urokinase-treated plasma, the free fibrinolysin was calculated. Free fibrinolysin in normal human blood in vivo was estimated from the half-life of fibrinogen and other data obtained in this study to be present at a concentration of 1.7 times 10-10 M. The plasmakinase activity in vivo, expressed as urokinase molarity, is also about 2 times 10-10 M.
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PMID:Molar concentrations of fibrinolytic components, especially free fibrinolysin, in vivo. 12 74

125I-fibrinogen, adsorbed to polystyrene tubes at low ionic strength and treated with thrombin, serves as a substrate for a rapid, convenient, and sensitive test tube assay for plasmin and activators and inhibitors of this enzyme. 125I-labeled digestion products released from the 125I-fibrin-polystyrene matrix are readily separated and quantitated and behave, on gel permeation, in the same manner as plasmin-generated degradation products from an unlabeled conventional fibrin clot. The 125I-fibrin, in probable non-cross-linked form, is firmly bound to the polystyrene and is resistant to nonspecific release, with control (no enzyme) values equivalent to 15.2 ng +/- 1.2 (SD) fibrin (1% of the total bound 125I-fibrin). This fact permits consistent detection of lysis of 30-50 ng 125I-fibrin, which exceeds published sensitivities (1000-5000 ng) using 125I- or fluorochrome-labeled fibrin clots as substrate. The sensitivity for plasmin (0.2 mug/ml) is tenfold greater than that of the fibrin-plate method (2.0-2.5 mug/ml), while sensitivities for streptokinase and urokinase activation of plasmin are 0.02 U/ml and 0.04 CTA U/ml, respectively (sensitivity of fibrin-plate method, 0.5 U/ml for both). The method provides a reasonable analogue of the solid-phase nature of fibrin under physiologic conditions, and the ease of preparation of large batches of tubes makes the method suitable for large-scale screening of factors modulating the plasminogen-plasmin system.
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PMID:A rapid and sensitive 125I-fibrin solid-phase fibrinolytic assay for plasmin. 12 94

The effect of a cadaver-derived vascular plasminogen activator (VA) on the degradation of fibrinogen, soluble fibrin monomer, and fibrin was studied and compared with the effect of equivalent fibrinolytic potencies of streptokinase (SK), urokinase (UK), and plasmin. The proteolytic activity of the three activators and plasmin was determined by a standard fibrin plate assay and was expressed in CTA units from a UK reference curve. Fibrinogen degradation was measured by clottable protein determinations and by an electrophoretic technique sensitive to small changes in the molecular weight of fibrinogen. When VA was incubated in plasma, no degradation of fibrinogen occurred, whereas rapid fibrinolysis took place after the plasma was clotted. By contrast, equivalent potencies of SK, UK, and plasmin caused extensive fibrinogenolysis. Since the plasmin added and that formed by the three activators had equivalent fibrinolytic activity, the failure of VA to induce fibrinogen degradation was attributed to antiactivators rather than antiplasmins. VA activity in plasma was consumed by clotting, whereas the antiactivator activity remained in the serum, suggesting dissociation of the VA-antiactivator complex on the fibrin clot. Fibrinogen and its soluble derivatives resisted degradation by VA in plasma because a solid phase appeared necessary for the complex to dissociate. The findings indicated that the degradation of fibrinogen or soluble fibrin in blood as a result of plasminogen activation by VA was unlikely to occur due to a large excess of antiactivator activity. Alternative pathways for their catabolism are discussed.
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PMID:The resistance of fibrinogen and soluble fibrin monomer in blood to degradation by a potent plasminogen activator derived from cadaver limbs. 12 95

A method is described by which the heavy chain of human plasmin, obtained by partial reduction of urokinase-activated plasminogen with 2-mercaptoethanol, is adsorbed on lysine coupled to polyacrylamide. The heavy chain is recovered from the adsorbent by elution with 6-aminohexanoic acid (yield 60-65%). Sulfhydryl titrations of the heavy chain showed that the partial reduction involved primarily the cleavage of the sole interchain disulfide bridge of plasmin. Dodecylsulfate-polyacrylamide electrophoresis gave essentially a single band corresponding to a component of about 60000 molecular weight. The NH2-terminal amino acid was predominantly threonine. 6-Aminohexanoic acid at different concentrations caused significant variations of the sedimentation and diffusion constants of the heavy chain indicating inhibitor-induced conformational alterations of the protein. The present results suggest that in plasmin only the heavy chain is capable of interacting with 6-aminohexanoic acid, and it appears that it is primarily this chain which plays an important role in the inhibition of the enzyme by 6-aminohexanoic acid.
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PMID:A new method of isolation and some properties of the heavy chain of human plasmin. 12 54

Radionuclides have been used in the evaluation of intravascular thrombosis for several years. The most widely accepted nonimaging technique is I-125 labeled fibrinogen. It is commonly used for patients who have a high probability of developing a thrombus (e.g., those undergoing surgery). Other techniques are emerging as having value in thrombosis detection. These techniques include radionuclide venography and clot localization scanning techniques utilizing the affinity of clot for small radiolabeled particles, as well as imaging techniques using I-131 labeled fibrinogen. More recently, a number of the above-mentioned techniques have been applied to patient problems. The FDA ban on commercial radiolabeled fibrinogen in the United States has promoted the development of alternative techniques and the use of autologous fibrinogen. A number of other techniques for thrombosis detection have also been explored on a more experimental basis. They include the use of thrombolytic agents such as streptokinase, urokinase, and white blood cells, as well as platelets. The common lung or pulmonary perfusion scan using macroaggregates of albumin or microspheres radiolabeled also gives information as to the presence of thrombosis of embolus within the pulmonary arteries, by showing the effect upon the perfusion pattern. This review will explore in detail the principles, as well as the present and prospective usefulness of the techniques currently available.
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PMID:Thrombosis detection using radionuclide techniques. 13 Feb 28

Affinity chromatography forms, 1 and 2, were each isolated from human Glu- and Lys-plasminogens by gradient elution from a L-lysine-substituted Sepharose column with a linear gradient of epsilon-aminocaproic acid. Although each of the two zymogen forms contains two affinity chromatography forms, the relative concentrattions of these forms in each of the zymogen preparations depended upon the plasma sample or enriched plasma fraction used for the preparation of the zymogen. Specific analytical acrylamide gel electrophoretic systems were used for the characterization of the zymogen and enzyme forms, and their component affinity chromatography forms, 1 and 2. The four zymogen affinity chromatography forms, Glu-1-plasminogen, Glu-2-plasminogen, Lys-1-plasminogen, and Lys-2-plasmingoen, show distinct stepwise differences in their molecular size and charge. The Glu-1-form is the largest in molecular size and the most acidic, and the Lys-2-form is the smallest in molecular size and the most basic. The proteolytically altered Lys-1- and Lys-2- forms appear to be specifically df the zymogen affinity chromatography forms showed a different distribution of isoelectric forms. The major isoelectric forms isolated from Glu-plasminogen with pI values of 6.2, 6.3, 6.4, and 6.6, and the major isoelectric forms isolated from Lys-plasminogen with pI values of 6.7, 7.2, 7.5, 7.8, and 8.1, (Summaria, L., Arzadon, L., Bernabe, P., Robbins, K. C., and Barlow, G. H. (1973) J. Biol. Chem. 248, 2984-2991) were shown to be mixtures of the Glu-1- and Glu-2- forms, or the Lys-1- and Lys-2- forms, respectively. Although the sialic acid contents of the Glu- and Lys- forms appear to be similar, the isolated affinity chromatography forms show distinct differences. The sialic acid contents of the Glu-1- and Lys-1- forms are identical, and are substantially higher than the sialic acid contents of the Glu-2- and Lys-2- forms which are also identical to each other. It is possible that the charge difference between the zymogen-1- and -2- forms may be related to the differences in their sialic acid content. Each of the four zymogen affinity chromatography forms, when activated by urokinase in the presence of the plasmin inhibitor, Trasylol, was converted to an apparently unique and different enzyme form. The four enzyme forms show distinct stepwise differences in molecular size; Glu-1-plasmin is the largest in size whereas Lys-2-plasmin is the smallest in size. Each plasmin-derived carboxymethyl heavy(A) chain was found to be different in molecular size, but the two carboxymethyl light(B) chains found in each of the four enzyme forms appeared to be identical and of the same molecular sizes. The four heavy(A) chains show a stepwise difference in molecular size; the Glu-1-heavy(A) chain is the largest in size whereas the Lys-2-heavy(A) chain is the smallest in size...
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PMID:Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. 13 40

When human plasminogen (Glu-Pga) is activated by urokinase in the presence of pancreatic trypsin inhibitor, the plasmin produced (Glu-Pma) exclusively contains a heavy chain (Glu-Ha) derived intact from the original NH2 terminus of Glu-Pga. Similar activations, utilizing a low molecular weight synthetic plasmin acylating agent, p-nitrophenyl-p-(pyridiniummethyl) benzoate, still result in a plasmin molecule with approximately 50% of the plasmin heavy chain containing the intact NH2 terminus of the original Glu-Pga. Activations performed at high levels of urokinase in the absence of any inhibitors initially produce Glu-Pma. However, the final stable plasmin, Lys-Pmb, which is obtained contains a heavy chain (Lys-Hb) which arises by plasminolysis of a small peptide from the NH2 terminus of Glu-Ha. Alternatively, Lys-Pmb can be formed in a separate series of reactions initially involving plasminolysis of Glu-Pga to yield Lys-Pgb. The peptide removed in this step is identical to the peptide removed in the Glu-Ha to Lys-Hb reaction. Next, urokinase catalyzes the conversion of Lys-Pgb to Lys-Pmb without further loss of peptide material. This latter pathway involving Lys-Pgb is probably the major pathway for human Lys-Pmb generation. These studies support a mechanism of activation of human plasminogen which involves at least two bond cleavages in Glu-Pga. However, these same studies strongly indicate that the Nh2-terminal peptide need not be released from Glu-Pga prior to plasmin formation. Further, we feel that plasmin and not urokinase catalyzes cleavage of the NH2-terminal peptide bond from Glu-Pga and the Glu-Ha heavy chain of Glu-Pma.
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PMID:Mechanism of the urokinase-catalyzed activation of human plasminogen. 13 42

Fibrinolytic activity of normal plasma and blood has been measured by 125l-fibrin solid phase assay. Activity of plasma is not affected by removal of plasminogenplasmin by affinity chromatography. Activities of euglobulin and pseudoglobulin fractions are approximately equal. epsilon-aminocaproic acid (EACA) (10 mM), tranexamic acid (10 mM), diisopropylfluorophosphate (DFP, 50 mM), and soybean and lima bean trypsin inhibitors (100 mug/ml) do not inhibit plasma activity at concentrations that inhibit pure plasmin and urokinase-activated plasma. Activity is not affected by glass contact and is not inhibited by inhibitors of contact or enzymatic activation of Hageman factor (hexadimethrine bromide, 100 mug/ml; cytochrome C, 250 mug/ml; spermidine, 2 mM; phenylmethylsulfonylfluoride, 1 mM). It is inhibited partially (30%-40%) by heating (56 degrees C, 30 min) and by zymosan (2.5 mg/ml; 40%-90% inhibition), and is increased by hydrazine (20 mM), salicylaldoxime (20 mM), DFP (50 mM), and tosyl-L-arginine methyl ester (TAMe, 10 mM)-the latter two at concentrations known to inhibit Cls of the classic, and factor D of the alternate complement pathways. Increase fibrinolytic activity with TAMe is associated with reciprocal decrease in classic and alternate complement pathway activity. It is concluded that normal plasma fibrinolytic activity is relatively independent of plasmin as the ultimate fibrinolytic enzyme, that Hageman factor-dependent pathways are of minor importance, and that significant heat-stable and heat-labile nonplasmin fibrinolytic activities are operative. These may include proteinases involved in complement activation, and in common control of classic and alternate complement pathways, as well as other nonplasmin proteinases.
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PMID:Fibrinolysis in normal plasma and blood: evidence for significant mechanisms independent of the plasminogen-plasmin system. 13 51

A procedure is presented for purifying a novel proteinase inhibitor in human plasma whose apparent unique biological property is to inhibit efficiently the lysis of fibrin clots induced by plasminogen activator. The final product is homogeneous as judged by disc gel electrophoresis, and immunoelectrophoresis. Its molecular weight estimated by sodium dodecyl sulfate gel electrophoresis or sedimentation equilibrium is 67,000 and 63,000, respectively. The inhibitor is a glycoprotein consisting polypeptide chain containing 11.7% carbohyrate. It migrates in the alpha2-globulin region in immunoelectrophoresis. The inhibitor is chemically and immunologically different from all the other known inhibitors in plasma. Inhibition of plasmin by the inhibitor is almost instantaneous even at 0 degrees, in contrast to the slow inhibition of urokinase (plasminogen activator in urine). Plasminogen activation by urokinase-induced clot lysis is inhibited by the inhibitor mainly through a mechanism of instantaneous inhibition of plasmin formed and not through the inhibition of urokinase. The inhibitor also inhibits trypsin. Consequently, it is suggested that this newly identified inhibitor is named alpha2-plasmin inhibitor or alpha2-proteinase inhibitor. A specific antibody directed against the inhibitor neutralizes virtually all inhibitory activity of plasma to activator-induced clot lysis. Immunochemical quantitation of the inhibitor was specific antiserum to the inhibitor and the purified inhibitor as a standard indicates that the concentration of the inhibitory in the serum of a healthy man is in or near the range of 5 to 7 mg/100 ml, which is the lowest concentration among the concentration of the proteinase inhibitors in plasma. The inhibitor and plasmin, trypsin, or urokinase form a complex which cannot be dissociated with denaturing and reducing agents. The formation of the enzyme-inhibitor complex occurs on a 1:1 molar basis and is associated with the cleavage of a unique peptide bone, which is most clearly demonstrated in the interaction of the inhibitor and beta-trypsin. In the complex formation between the inhibitor and plasmin, the inhibitor is cross-linked with the light chain which contains the active site of plasmin. It is suggested that, in a fashion analogous to complex formation between alpha1-antitrypsin and trypsin, the cross-links are formed between the active site serine of the enzyme and the newly formed COOH-terminal residue of the inhibitor, with cleavage of a peptide bond.
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PMID:Isolation and characterization of alpha2-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis. 13 98

Fresh plasma was seeded with trace amounts of highly purified biologically intact iodine-labelled plasminogen and the plasmin-inhibitor complexes formed after activation with streptokinase or urokinase separated by gel filtration. Two radioactive peaks were observed, the first one eluted in the void volume and the second one just before the 7-S globulin peak. In incompletely activated samples, the second peak was always predominant over the first one. Both components were purified with high yield by a combination of affinity chromatography on lysine-agarose and gel filtration, and investigated by dodecylsulphate-polyacrylamide gel electrophoresis and immunoelectrophoresis. Neither component reacted with antisera against alpha1-antitrypsin, antithrombin III, C1-esterase inhibitor, inter-alpha-trypsin inhibitor or alpha1-antichymotrypsin. The component of the first peak appeared to be a complex between plasmin and alpha2-macroglobulin which reacted with antisera against human plasminogen and against alpha2-macroglobulin. The component of the second peak had a molecular weight (Mr) of 120000-140000 by dodecyl-sulphate-polyacrylamide gel electrophoresis and lpon reduction displayed a doublet band with an Mr of 65000-70000 and a band with Mr 11000. It reacted with antisera against plasminogen and with antisera raised against this complex and absorbed with purified plasminogen. The latter antisera reacted with a single component in plasma which is different from the above-mentioned plasma protease inhibitors. Specific removal of this component from plasma by immuno-absorption resulted in disappearance of the fast-reacting antiplasmin activity whereas alpha2-macroglobulin was found to represent the slower-reacting plasmin-neutralizing activity. In the presence of normal plasma levels of these proteins, the specific removal or absence of alpha1-antitrypsin, antithrombin III or C1-esterase inhibitor did not alter the inactivation rate of plasmin when added to plasma in quimolar amounts to that of plasminogen. It is concluded that only two plasma proteins are important in the binding of plasmin generated by activation of the plasma plasminogen, namely a fast-reacting inhibitor which is different from the known plasma protease inhibitors and which we have provisionally named antiplasmin, and alpha2-macroglobulin, which reacts more slowly.
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PMID:Identification and some properties of a new fast-reacting plasmin inhibitor in human plasma. 13 45


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