Magnetic urokinase: targeting of urokinase to fibrin clot

A plasminogen activator of human origin, urokinase, was endowed with magnetic property. The magnetic urokinase was composed of magnetite, polyethylene glycol derivative and urokinase, and dispersed in saline. Its particle size of magnetite was approximately 30-60 nm. It was selectively delivered to fibrin clot by magnetic force in continuously circulating plasma and exerted fibrinolytic activity without degrading fibrinogen.     

Chemical modification of enzymes with activated magnetic modifier

An activated magnetic modifier, which could render biological materials magnetic property, was synthesized in following two steps: oxidation of ferrous ions (Fe2+) with hydrogen peroxide in the presence of alpha, omega-dicarboxymethylpoly(oxyethylene) (DCPEG) to obtain DCPEG-magnetite (Fe3O4); free carboxyl groups in the DCPEG-magnetite were activated with N-hydroxysuccinimide. By coupling the activated magnetic modifier to amino groups of lipase or L-asparaginase, magnetic enzymes were prepared. They dispersed stably not only in aqueous solution but also in organic solvents with high enzymic activities. Magnetic enzymes were readily recovered from reaction mixture in a magnetic field of 6000 Oe without loss of enzymic activity.     

Regulation of extracellular plasminogen activator by human fibroblasts. The role of protease nexin

When the plasminogen activator urokinase was radioiodinated and incubated at 40 ng/ml in medium conditioned by human foreskin (HF) cells, within 30 min over 80% of the added plasminogen activator was complexed to cell-released protease nexin (PN). The urokinase complexed to PN had little if any activity. Incubation of purified PN with urokinase confirmed that PN is an inhibitor of this plasminogen activator. However, a widely used plasminogen-dependent fibrinolysis assay for plasminogen activator indicated that abundant endogenous plasminogen activator activity co-existed with PN in HF cell-conditioned medium. The source of this activity was electrophoretically and immunologically indistinguishable from urokinase. Furthermore, gel exclusion chromatography showed that about 90% of the urokinase antigen detected in conditioned medium had a molecular weight similar to that of free active urokinase. These paradoxical findings are resolved by evidence that this "PN-resistant urokinase-like" plasminogen activator is actually urokinase proenzyme that is activated by plasmin or conditions in the fibrinolysis assay for plasminogen activator. It is shown that the activated form of HF cell plasminogen activator is sensitive to inhibition by PN. PN may thus be an important component in the cellular regulation of endogenous plasminogen activator activity.     

IFN-gamma, tumor necrosis factor-alpha, and urokinase regulate the expression of urokinase receptors on human monocytes

The ability of macrophages to reach inflammatory loci is crucial in the function of cellular immunity. Invasive properties of macrophages may be due to the proteinase urokinase which binds to cell surface receptors, and thereby confers on macrophages the capacity for localized proteolysis of the interstitium. Here, we investigated the role of the macrophage-activating factors IFN-gamma, TNF-alpha, and granulocyte-macrophage-CSF and of urokinase on the expression of urokinase receptors by human cultured monocytes. IFN-gamma and TNF-alpha induced increased urokinase binding to human cultured monocytes in a time- and dose-dependent fashion. At optimal concentrations, IFN-gamma (200 U/ml) increased the number of receptors/cell from 14,000 to 64,000, TNF-alpha (50 U/ml) to 30,000, and combinations of IFN-gamma and TNF-alpha to 90,000. Granulocyte-macrophage-CSF had no effect. The enhanced urokinase binding is due to increased numbers of urokinase receptors and not an increased affinity of the receptor for urokinase. In the presence of urokinase during monocyte activation, IFN-gamma induced only 25,000 receptors/cell. However, urokinase does not inhibit increased receptor expression when the cells are activated with TNF-alpha. The effect of urokinase on induction of urokinase receptors by combinations of IFN-gamma and TNF-alpha varied with the dosage of TNF-alpha: A combination of IFN-gamma (200 U/ml) and TNF-alpha (15 U/ml) induced 38,000 receptors/cell in the presence and 90,000 receptors/cells in the absence of urokinase, whereas IFN-gamma (200 U/ml) and TNF-alpha (20 U/ml) induced 90,000 receptors/cell in the absence and presence of urokinase. These studies demonstrate that IFN-gamma, TNF-alpha, and urokinase collectively regulate the number of urokinase receptors on human monocytes. The induction of urokinase receptors may be responsible for increased invasiveness of the activated macrophage.     

Magnetization of activated sludge by an external magnetic field

Magnetization of acclimated activated sludge with the application of an external magnetic field was observed using a magnetic force microscope and can be expressed as the visual magnetic flux density graph. FeCl₃ addition up to 0.1% (w/v) into the sludge, which was acclimated with synthetic sewage wastewater, enhanced the magnetization. Such magnetization was also observed in the activated sludge obtained from the practical wastewater treatment plants of a food processing plant and sewage wastewater. FeCl₃ addition also enhanced the sludge magnetization. The possibility is suggested that this magnetization causes the flock to enlarge and enhance sedimentation of the activated sludge on application of an external magnetic field.     

双专－性抗体介导的纤溶作用

用双功能团试剂将抗尿激酶单克隆抗体Ｎ３４的ＩｇＧ和抗人活化血小板α－颗粒膜蛋白ＧＭＰ－１４０单克隆抗体ＳＺ－５１的Ｆａｂ片段通过二硫键共价偶联,偶联的抗体保留了对各自抗原的亲和性。这种对尿激酶和血栓同时具有亲和活性的双专一性抗体（Ｎ３４－ＳＺ－５１）提高低分子量尿激酶的溶栓效率３８倍,且对血浆中纤维蛋白原的含量基本上不影响。     

Effects of an external magnetic field on the flock size and sedimentation of activated sludge

Effects of an external magnetic field on sedimentation enhancement were investigated using three kinds of practical activated sludge. An indoor experiment was setup (3.5l aeration vessel) equipped with a broth circulation system and 800 G of external magnetic field. The application of an external magnetic field to the activated sludge enhanced the sedimentation irrespective of the nature of the activated sludge. At the same time, the flock size of the sludge was enlarged by the external magnetic field. However, the surface zeta potentials of the sludge were not changed by the external magnetic field. Addition of FeCl₃ to the sludge enhanced the effects of the external magnetic field. Based on these results, the following mechanism for the enhancement of sedimentation by the external magnetic field is suggested: activated sludge containing iron was magnetized during entry to the magnetic field and coagulated with the magnetic force. As a consequence, the flock size was enlarged. Almost the same sedimentation enhancement by external magnetization was confirmed by a pilot scale magnetization system (2.0 m³ of aeration vessel) when the airlift type of mild broth circulation system was equipped with a 5000 G magnetic field.     

Competition of activated protein C and urokinase for a heparin-dependent inhibitor

Human urine contains a hitherto unrecognized heparin-dependent inhibitor of activated protein C (APC) (Mr approximately 50,000) that coelutes from heparin-Sepharose together with the only observed peak of urokinase inhibitory activity at a position (0.35 M NaCl) similar to that of plasma protein C (PC) inhibitor. Based on functional assays and immunoblot studies, urokinase and APC compete for this crude inhibitor in the absence or presence of heparin. These results suggest that the same heparin-dependent urinary inhibitor that is immunologically different from several known protease inhibitors is responsible for the observed inhibition of APC and urokinase. In the absence of heparin this inhibitor inhibits APC and urokinase with similar rates, and heparin enhances its inhibitory activity toward both enzymes with more pronounced stimulation of its PC inhibitory activity than its urokinase inhibitory activity. Half-maximal stimulation of inhibition of APC occurs at about 2 mU/ml and maximal stimulation (approximately 10-fold increase of the pseudo-first-order rate constant) at greater than or equal to 50 mU/ml of heparin. This is the first demonstration of competition between APC and urokinase for a heparin-dependent inhibitor. These results may therefore represent a new link between the two major antithrombotic pathways, the PC pathway and the fibrinolytic system.     

Activation of pro-urokinase by the human T cell-associated serine proteinase HuTSP-1

The human T cell-associated serine proteinase-1 (HuTSP-1) is expressed by activated T lymphocytes and is exocytosed upon their interaction with target cells. Here, we report that HuTSP-1 is able to convert single-chain human pro-urokinase into the active two-chain enzyme. Time-dependent activation by HuTSP-1 of recombinant human pro-urokinase as well as natural pro-urokinase derived from human melanoma cells was demonstrated in a chromogenic assay specific for active urokinase type plasminogen activator and in immunoblotting experiments revealing the conversion of single-chain into two-chain urokinase. Control experiments excluded plasmin as the activating agent. These data suggest a novel pathway for plasmin generation during T cell-mediated processes such as immune responses and extravasation of immune cells.     

Identification and characterization of urokinase receptors in natural killer cells and T-cell-derived lymphokine activated killer cells.

Fluorescence-activated cell scanning analysis of human blood cells revealed novel urokinase receptors in large granular lymphocytes and a small subset of T-cells (CD3+). Culturing of T-cells with interleukin-2 to generate CD3+ lymphokine-activated killer cells caused a large increase in urokinase binding, suggesting that the urokinase receptor is an activation antigen. The receptor in lymphocytes was similar to that in monocytes with regard to size, affinity and ligand specificity, but did not mediate degradation of urokinase-inhibitor complexes. It is suggested that lymphocyte-bound pro-urokinase is activated, e.g. by the human T-cell-specific serine proteinase, HuTSP-1, and thereby starts a cascade of plasminogen activation important for extravasation of the cells.     

Sensitive fluorescence assays for urokinase using synthetic peptide 4-methoxy-beta-naphthylamide substrates

Two assays for the plasminogen activator urokinase using peptide fluorogenic substrates are described. N-carbobenzoxy-glycyl-glycyl-l-arginine-4-methoxy-β-naphthylamide (CBZ-Gly-Gly-Arg-4MβNA) can be used in a direct assay that is simple, rapid, and sensitive to as little as 0.5 IU/ml urokinase. Additional sensitivity, to 0.01 IU/ml urokinase, is obtained in a second method that uses plamsinogen as the primary substrate followed by a fluorogenic substrate assay employing N-carbobenzoxy-l-alanyl-l-alanyl-l-lysine-4-methoxy-β-naphthylamide (CBZ-Ala-Ala-Lys-4MβNA) as a specific substrate for the activated plasmin. These assays are as sensitive as the best assays presently in use and are simpler to perform. In addition, these assays can readily be used for kinetic analysis of the hydrolytic activity of urokinase or other plasminogen activators.     

Lattice spacing changes accompanying isometric tension development in intact single muscle fibers.

The myosin lattice spacing of single intact muscle fibers of the frog, Rana temporaria, was studied in Ringer's solution (standard osmolarity 230 mOsm) and hyper- and hypotonic salines (1.4 and 0.8 times standard osmolarity respectively) in the relaxed state, during "fixed end" tetani, and during shortening, using synchrotron radiation. At standard tonicity, a tetanus was associated with an initial brief lattice expansion (and a small amount of sarcomere shortening), followed by a slow compression (unaccompanied by sarcomere length changes). In hypertonic saline (myosin lattice compressed by 8.1%), these spacing changes were suppressed, in hypotonic saline (lattice spacing increased by 7.5%), they were enhanced. During unloaded shortening of activated fibers, a rapid lattice expansion occurred at all tonicities, but became larger as tonicity was reduced. This expansion was caused in part by the change in length of the preparation, but also by a recoil of a stressed radial compliance associated with axial force. The lattice spacing during unloaded shortening was equal to or occasionally greater than predicted for a relaxed fiber at that sarcomere length, indicating that the lattice compression associated with activation is rapidly reversed upon loss of axial force. Lattice recompression occurred upon termination of shortening under standard and hypotonic conditions, but was almost absent under hypertonic conditions. These observations indicate that axial cross-bridge tension is associated with a compressive radial force in intact muscle fibers at full overlap; however, this radial force exhibits a much greater sensitivity to lattice spacing than does the axial force.     

Enhanced expression of an antibody-targeted plasminogen activator in Escherichia coli by fusion to decorsin

To improve the thrombolytic specificity of plasminogen activators, an antibody-targeted plasminogen activator was constructed consisting of a single-chain variable fragment of a monoclonal antibody SZ-51 raised specifically against human P-selectins on activated platelets and a low molecular weight single-chain urokinase. After fusion to the 3' end of the gene coding for decorsin, originally isolated from the leech Macrobdella decora, expression of the antibody-targeted plasminogen activator gene in E. coli strain Rosetta (DE3) pLysS was greatly enhanced.     

Mechanism of the urokinase-catalyzed activation of human plasminogen.

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.     

Enhanced biological treatment of saline wastewater by using halophilic bacteria

Biological treatment of saline wastewater by conventional activated sludge culture usually results in low removal of chemical oxygen demand (COD) because of plasmolysis of the organisms at high salt concentrations. Since salt removal operations by physicochemical processes before biological treatment are costly, a salt-tolerant organism (Halobacter halobium) was used for effective biological treatment of saline wastewater in this study. Halobacter halobium was used in activated sludge culture for COD removal from saline wastewater (1–5% salt) by fed-batch operation of an aeration tank. Inclusion of Halobacter halobium into activated sludge culture improved the rate and extent of COD removals especially with salt above 2% (w/v).     

Plasminogen activation by single-chain urokinase in functional isolation. A kinetic study

The kinetics of the activation of Glu- and Lys-plasminogen by single-chain urokinase (sc urokinase) derived from the transformed human kidney cell line TCL-598 have been studied and compared with two-chain urokinase (tc urokinase). Plasminogen activation was determined by the increase in fluorescence polarization of fluorescein-labeled aprotinin, a high affinity inhibitor of plasmin. This methodology allows plasmin generation by sc urokinase to be measured in functional isolation, with no interfering generation of tc urokinase, sc urokinase was found to activate plasminogen to plasmin with apparent Michaelis-Menten-type kinetics. The Km for Glu-plasminogen activation was 47.7 microM, with a catalytic constant of 2.91 min-1. Lys-plasminogen activation by sc urokinase was characterized by a Km of 11.7 microM and a kcat of 5.60 min-1. The Km values for the activation of Glu- and Lys-plasminogen by tc urokinase were found to be similar to those for activation by sc urokinase (36.8 and 9.0 microM, respectively), but the catalytic constants were higher at 36.0 and 118 min-1, respectively. Therefore, on the basis of the catalytic efficiency kcat/Km, sc urokinase seems to have 16-27-fold lower activity than tc urokinase. This activity of sc urokinase is in contrast to its lack of activity against a low molecular weight peptide substrate (less than 0.2% of the activity of sc urokinase). The activation of sc urokinase to tc urokinase by plasmin was also characterized (Km = 3.0 microM, kcat = 105 min-1). Using these data, it was possible to calculate the theoretical rate of plasminogen activation by sc urokinase in the absence of aprotinin, when tc urokinase is generated by the action of plasmin. The calculated rate was in good agreement with that determined experimentally using the chromogenic substrate D-Val-Leu-Lys-p-nitroanilide. These data demonstrate that sc urokinase has properties which distinguish it from conventional serine protease zymogens. The lack of activity against low molecular weight peptide substrates demonstrates the inaccessibility of the substrate-binding pocket. However, there is a moderate activity against plasminogen, suggesting that plasminogen may be acting as both an effector and a substrate for sc urokinase.     

Heparin binding to the urokinase kringle domain.

The binding of urokinase to immobilized heparin and dextran sulfate was studied using activity assays of the bound urokinase. The markedly higher binding observed with high M(r) urokinase compared to low M(r) urokinase indicated a role for the amino-terminal fragment (ATF). This was confirmed by the use of inactive truncated urokinase and monoclonal antibodies specific for the ATF in competition assays of urokinase binding. Antibody competition assays suggested a site in the kringle domain, and a synthetic decapeptide Arg-52-Trp-62 from the kringle sequence (kringle numbering convention) was competitive in assays of urokinase binding to dextran sulfate and heparin. Heparin binding to the urokinase kringle was unambiguously demonstrated via 1H NMR spectroscopy at 500 MHz. Effective equilibrium association constants (K(a)*) were determined for the interaction of isolated kringle fragment and low M(r) heparin at pH 7.2. The binding was strong in salt-free 2H2O (K(a)* approximately 57 mM-1) and remained significant in 0.15 M NaCl (K(a)* approximately 12 mM-1), supporting a potential physiological role for the interaction. This is the first demonstration of a function for the kringle domain of urokinase, and it suggests that while the classical kringle structure has specificity for lysine binding, there may also exist a class of kringles with affinity for polyanion binding.     

Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma

Urokinase-related proteins in human urine occur mainly as a 1:1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogenous glycoprotein of apparent Mr 50,000 was obtained with a yield of 40 micrograms/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 degrees C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with beta-mobility. Inhibition of urokinase occurred with a second order rate constant (k) of 8 X 10(3) M-1 s-1 in the absence and of 9 X 10(4) M-1 s-1 in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinase-type plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below 10(3) M-1 s-1 and thrombin with a k of 4 X 10(4) M-1 s-1 in the absence and 2 X 10(5) M-1 s-1 in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 +/- 0.7 micrograms/ml (mean +/- S.D., n = 25). The protein purified from plasma by immunoabsorption had the same Mr, amino acid composition, and immunoreactivity as the urinary protein. Furthermore, when urokinase was added to plasma, time-dependent urokinase-urokinase inhibitor complex formation was observed at a rate similar to that observed for the inhibition of urokinase by the purified inhibitor from urine. This urokinase inhibitor, purified from human urine, most probably represents a new plasma protease inhibitor.     

The matrix metalloproteinase pump-1 catalyzes formation of low molecular weight (pro)urokinase in cultures of normal human kidney cells.

The enzyme responsible for the metalloproteinase activity which cleaves the Glu143-Leu144 bond of (pro)urokinase has been isolated from the conditioned medium of cultured normal human kidney cells. Using S-Sepharose and Cibacron Blue-agarose chromatography, then C-4 reversed phase high pressure liquid chromatography, a protein of about 20,000 Da was isolated. Through an identical amino-terminal sequence, the protein was shown to be the matrix metalloproteinase previously referred to in the literature as "pump-1" (putative metalloproteinase). When aprotinin was added during the course of the purification, the major species isolated was the zymogen form (28,000 Da) of pump-1. Pump-1 has been shown to efficiently cleave the susceptible bond of both pro-urokinase (single-chain) and active (two-chain) urokinase and thereby produce the corresponding low molecular weight forms. The amino-terminal sequences of the A and B chains of low molecular weight urokinase prepared by action of pump-1 on recombinant high molecular weight urokinase are identical to those of the low molecular weight urokinase isolated from human kidney cell culture. Since the reaction of urokinase with this metalloproteinase results in separation of its serine proteinase region from the domain which mediates binding to the urokinase receptor, it may be of importance in the regulation of the functional activity of the plasminogen activator in cellular processes.     

Characteristics of a thrombin inhibitor secreted by activated platelets

A 77-kDa complex of thrombin and a protein secreted by activated platelets had little if any thrombin amidolytic activity, indicating that the secreted protein is an inhibitor. The molecular weight of the inhibitor before reaction with thrombin was approximately 50,000. The apparent second-order rate constant for complex formation was estimated to be 1.3 x 10(6) M-1 s-1 (mean of four measurements); it was not affected by heparin or heparinase. These properties distinguish this inhibitor from other protease inhibitors secreted by platelets. The inhibitor reacted with trypsin and possibly with urokinase but not with factor Xa.