Inhibition of urokinase by complex formation with human
Abstract
Human was prepared from fresh human plasma by (NH4)2SO4-precipitation, gel filtration, affinity chromatography on concanavalin A, ion exchange chromatography and isotachophoresis. Human urokinase (EC 3.4.99.26) (plasminogen activator from urine) with 46 000 and 36 000 was further purified from Urokinase Leo reagent preparation by gel filtration on Sephadex G-100 Superfine. The hydrolytic activity of urokinase on acetyl-glycyl-l-lysine methyl ester acetate (Ac-Gly-Lys-OMeAc) was inhibited in a strong timedependent manner by . Complex formation between enzyme and inhibitor could be demonstrated in crossed immunoelectrophoresis against and anti-urokinase serum as well as by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The latter method revealed the formation of 1:1 and 2:1 molar enzyme-inhibitor complexes.
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Cited by (28)
Proteinases from the fibrinolytic and coagulation systems: Analyses of binding to pregnancy zone protein, a pregnancy-associated plasma proteinase inhibitor
1995, Fibrinolysis and ProteolysisThe inhibitory effects of pregnancy zone protein (PZP) on proteinases within the fibrinolytic and coagulation systems have been studied and compared to that of human α2-macroglobulin (α2-M). Plasmin, t-PA, urokinase, thrombin, human plasma kallikrein, human and porcine tissue kallikrein were tested for binding to PZP and α2-M.
PZP was cleaved at the ‘bait’ region as seen in SDS-PAGE, by both human and porcine tissue kallikrein, but not by any of the other proteinases tested and we therefore suggest that PZP may have a role in inhibition of tissue kallikrein.
Plasmin, thrombin, and plasma kallikrein were found to be bound and inhibited by α2-M by cleavage of the ‘bait’ regions. Minor amounts of cleavage products of α2-M were detected with t-PA and urokinase after prolonged incubation at room temperature. Cleavage of α2-M was detected following incubation with porcine tissue kallikrein, but no cleavage was seen following incubation with human tissue kallikrein. The fast inhibition of plasmin, thrombin, and plasma kallikrein suggest that α2-M may be physiological relevant as an inhibitor for these proteinases.
Despite the similarities between α2-M and PZP, significant differences are observed in the inhibition of proteinases. These results suggest distinctive differences in the function of the two human α-macroglobulins. PZP does only seem to inhibit human tissue kallikrein of the proteinases tested from the fibrinolytic and coagulation systems.
The fibrinolytic system in man
1986, Critical Reviews in Oncology and HematologyThe fibrinolytic system comprises a proenzyme, plasminogen, which can be activated to the active enzyme plasmin, that will degrade fibrin by different types of plasminogen activators. Inhibition of fibrinolysis may occur at the level of plasmin or at the level of the activators. Fibrinolysis in human blood seems to be regulated by specific molecular interactions between these components. In plasma, normally no systemic plasminogen activation occurs. When fibrin is formed, small amounts of plasminogen activator and plasminogen adsorb to the fibrin, and plasmin is generated in situ. The formed plasmin, which remains transiently complexed to fibrin, is only slowly inactivated by α2-antiplasmin, while plasmin, which is released from digested fibrin, is rapidly and irreversibly neutralized. The fibrinolytic process, thus, seems to be triggered by and confined to fibrin. Thrombus formation may occur as the result of insufficient activation of the fibrinolytic system and (or) the presence of excess inhibitors, while excessive activation and/or deficiency of inhibitors might cause excessive plasmin formation and a bleeding tendency. Evidence obtained in animal models suggests that tissue-type plasminogen activator, obtained by recombinant DNA technology, may constitute a specific clot-selective thrombolytic agent with higher specific activity and fewer side effects than those currently in use.
Les activateurs du plasminogène sont l'objet d'un enjeu biotechnologique considérable. Répartis en deux classes immunologiquement distinctes, les u-PA ou activateurs de type urokinase (dépourvus d'affinité pour la fibrine) et les t-PA ou activateurs de type tissulaire (présentant de l'affinité pour la fibrine), ils sont présents de façon ubiquitaire dans les tissus normaux ou tumoraux d'origine animale ou humaine. Intervenant dans de nombreuses situations physiologiques ou pathologiques, ils maintiennent l'intégrité du système vasculaire et sont impliqués dans les processus de migration cellulaire et de remodelage tissulaire. C'est leur activité fibrinolytique, via la catalyse de la formation de plasmine à partir du plasminogène circulant, qui en font des agents thrombolytiques actuels (urokinase, streptokinase) ou potentiels (t-PA). Les structures primaires de l'urokinase et du t-PA ont été déterminées et ces deux enzymes ont été clonées. En vue d'une utilisation massive dans le traitement d'infarctus du myocarde, de thromboses cérébrales et d'embolies pulmonaires, les efforts de l'industrie pharmaceutique se concentrent actuellement sur l'obtention, par génie génétique, de grandes quantités de t-PA. Cette revue présente les propriétés moléculaires et structurales des activateurs du plasminogène ainsi que les principaux aspects physiologiques, pathologiques et thérapeutiques les concernant.
Considerable interest in plasminogen activators as human thrombolytic drugs has stimulated rapid biotechnologic progresses. These enzymes have been classified in two immunochemically distinct groups: “urokinase-like” activators or u-PA which do not interact with fibrin and “tissue activator-like” activators or t-PA which interact with fibrin. Plasminogen activators are widely distributed in normal and malignant tissues and they are implicated in various physiological and pathological processes. They maintain the functional integrity of the vascular system and their presence may be of importance in tissue remodeling and cell migration. Urokinase and streptokinase are used in human thrombolytic therapy. However, the properties displayed by t-PA suggest that this enzyme may be a superior fibrinolytic agent. The primary structures of urokinase and t-PA are known; both enzymes have been synthesized by DNA technology. In order to produce t-PA in large quantities by gene cloning, intensive studies are conducted by pharmaceutical industries. Clinical trials using t-PA for dissolving thrombi in coronary heart disease, strokes and pulmonary embolism are in progress. This review presents the molecular and structural properties of plasminogen activators, as well as related physiological, pathological and therapeutic aspects.
In vivo kinetics and thrombus accumulation of <sup>67</sup>ga-labeled urokinase
1985, International Journal of Nuclear Medicine and BiologyHighly purified high and low molecular weight urokinase (H-UK and L-UK) were labeled with 67Ga using deferoxamine (DF) as a bifunctional chelating agent. The labeling efficiency was 91.7% for the H-UK, and 90.4% for the L-UK, respectively. The 67Ga labeled UK (67Ga-DF-UK) fully retained the enzymatic activity of the parent UK. Studies on the in vivo behavior of the 67Ga labeled UK in rabbits showed a very rapid blood clearance with half-life of 4 min (67Ga-DF-L-UK) to 8 min (67Ga-DF-H-UK Studies carried out in rabbits with induced thrombi in the femoral vein showed thrombus-to-blood 67Ga-DF-UK activity ratios, 2 h after injection, of 2.00–3.08 for the H-UK, and 0.84–1.65 for the L-UK, respectively, with thrombi aged 4 to 3 days. A dose effect of the 67Ga-DF-H-UK on its thrombus accumulation was observed. Gel chromatographic analysis of plasma samples withdrawn from those animals injected with this radiopharmaceutical revealed a reduction of the 67Ga-DF-UK effectiveness due to complexation with protein inhibitors. This led to formation of high molecular weight complexes which was reflected in the very fast blood clearance. Its implication in thrombus accumulation is discussed. In conclusion, usefulness of DF for labeling UK with 67Ga or 68Ga with no alteration of UK enzymatic properties was demonstrated. The use of 67Ga-DF-UK as a diagnostic or therapeutic radiopharmaceutical is promising.
Plasminogen Activators, Tissue Degradation, and Cancer
1985, Advances in Cancer ResearchThis chapter discusses the role of plasminogen activators in various biological processes. In specific, it describes two types of plasminogen activators—namely, the urokinase-type plasminogen activator (u-PA) and the tissue-type plasminogen activator (t-PA), which are essentially different gene products. The amino acid sequences of these activators and nucleotide sequences of the corresponding cDNAs have largely been determined, and the cDNAs have been cloned using recombinant techniques. A variety of enzymatic as well as immunological assay and detection methods have also been developed that allows a precise quantification of the activators, a distinction between u-PA and t-PA, determination of whether an activator is present in its active or zymogen form, analysis of the kinetics of different steps of the cascade reaction, and immunocytochemical identification of u-PA and t-PA in tissue sections. Much of the studies on plasminogen activators and cancer has been guided by the hypothesis that proteolysis of the components of extracellular matrix, initiated by the release of plasminogen activator from the cancer cells, plays a decisive role for the degradation of normal tissue, and thereby for invasive growth and metastases.
Natural inhibitors of fibrinolysis
1979, Progress in Cardiovascular Diseases