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1.
Urokinase as a Multidomain Protein and Polyfunctional Cell Regulator   总被引:4,自引:0,他引:4  
The urokinase type plasminogen activator (urokinase) plays a pivotal role in the regulation of cell adhesion and migration during tissue remodeling. Urokinase not only specifically cleaves plasminogen and converts it into plasmin but also activates intracellular signaling upon binding to certain receptors on the cell surface. The polyfunctional properties of this protein are associated with its three-domain structure as follows: the C-terminal proteolytic domain containing the serine protease active center, the central kringle domain, and the N-terminal domain homologous to epidermal growth factor. This review considers functional properties of urokinase and of its fragments generated on the cell surface as a result of proteolytic processing. This review will discuss the mechanisms of urokinase-mediated regulation of cellular function upon binding to membrane receptors.  相似文献   

2.
We investigated the chemotactic action of PDGF and urokinase on human airway smooth muscle (HASM) cells in culture. Cells were put in collagen-coated transwells with 8-micro m perforations, incubated for 4 h with test compounds, then fixed, stained, and counted as migrated nuclei by microscopy. Cells from all culture conditions showed some basal migration (migration in the absence of stimuli during the assay), but cells preincubated for 24 h in 10% FBS or 20 ng/ml PDGF showed higher basal migration than cells quiesced in 1% FBS. PDGF(BB), PDGF(AA), and PDGF(AB) were all chemotactic when added during the assay. PDGF chemotaxis was blocked by the phosphatidyl 3'-kinase inhibitor LY-294002, the MEK inhibitor U-0126, PGE(2), formoterol, pertussis toxin, and the Rho kinase inhibitor Y-27632. Urokinase alone had no stimulatory effect on migration of quiescent cells but caused a dose-dependent potentiation of chemotaxis toward PDGF. Urokinase also potentiated the elevated basal migration of cells pretreated in 10% FBS or PDGF. This potentiating effect of urokinase appears to be novel. We conclude that PDGF and similar cytokines may be important factors in airway remodeling by redistribution of smooth muscle cells during inflammation and that urokinase may be important in potentiating the response.  相似文献   

3.
Urokinase: a chemotactic factor for polymorphonuclear leukocytes in vivo   总被引:6,自引:0,他引:6  
The effects of injecting urokinase into subdermal air sacs on the back of mice was studied. Urokinase was leukotactic in the concentration range of 2 X 10(-13) to 2 X 10(-15) M. This response was absolutely dependent on the enzyme activity of the serine esterase, but was found to be independent of generation of the chemotactic complement split product C5a. At high doses of urokinase (greater than 2 X 10(-12) M), no cellular infiltration was observed. Injection of 2 X 10(-10) M urokinase i.p. led to the systemic desensitization of mice when challenged in the skin with a lower dose (2 X 10(-14) M) of urokinase. Urokinase desensitization did not alter the ability of mice to respond to the chemical chemotactic factor f-met-leu-phe or to respond to C5a-dependent chemotactic stimuli. Urokinase desensitized mice failed to demonstrate a chemotactic response to nerve growth factor, thrombin, plasmin, or factor X activating enzyme, all of which were chemotactic in non-urokinase pre-treated animals. The results of these studies indicate the presence of three physiologically independent inflammatory pathways in mice: independent of C5 and not influenced by pretreatment with urokinase, independent of C5 and inhibited by pretreatment with urokinase, and dependent on C5 and not influenced by pretreatment with urokinase.  相似文献   

4.
Urokinase activates adhesion of monocytic U937 cells to fibrinogen-coated surface. This effect is due to urokinase proteolytic activity and does not depend on the urokinase binding to its receptor.  相似文献   

5.
We have investigated the interaction of alpha 2-macroglobulin (alpha 2M) with the serine proteinase urokinase, an activator of plasminogen. Urokinase formed sodium dodecyl sulfate stable complexes with purified alpha 2M and with alpha 2M in plasma. These complexes could be visualized after polyacrylamide gel electrophoresis by protein blots using 125I-labeled anti-urokinase antibody or by fibrin autography, a measure of fibrinolytic activity. According to gel electrophoretic analyses under reducing conditions, urokinase cleaved alpha 2M subunits and formed apparently covalent complexes with alpha 2M. Urokinase cleaved only about 60% of the alpha 2M subunits maximally at a mole ratio of 2:1 (urokinase: alpha 2M). Binding of urokinase to alpha 2M protected the urokinase active site from inhibition by antithrombin III-heparin and inhibited, to a significant extent, plasminogen activation by urokinase. Reaction of urokinase with alpha 2M caused an increase in intrinsic protein fluorescence and, thus, induced the conformational change in alpha 2M that is characteristic of its interactions with active proteinases. Our results indicate that both in plasma and in a purified system the alpha 2M-urokinase reaction is functionally significant.  相似文献   

6.
This review considers cellular and molecular mechanisms of the involvement of plasminogen activators in extracellular proteolysis and cell migration and proliferation. The role of plasminogen activators in vascular remodeling in atherosclerosis, restenosis, and angiogenesis is discussed.  相似文献   

7.
The effect of extracellular matrix composition on the location, amount, and activity of cell-associated urokinase-type plasminogen activator was tested using HT-1080 cells adherent to either fibronectin or vitronectin. Specific immunoprecipitation of newly synthesized urokinase indicated that cells adherent to fibronectin synthesized 2-3-fold more urokinase than cells adherent to vitronectin. Complexes of urokinase and plasminogen activator inhibitor type 1 (PAI-1) were detected in cell layers of vitronectin-adherent but not fibronectin-adherent cells. Inhibition of PAI-1 using a neutralizing monoclonal antibody resulted in a 3-fold increase in urokinase enzymatic activity on vitronectin adherent cells. Urokinase activity on fibronectin adherent cells was only slightly increased following PAI-1 neutralization. Examination of both HT-1080 and normal human fibroblast cells by immunofluorescent microscopy localized urokinase-type plasminogen activator to discrete, focal areas underneath cells adherent to vitronectin. Urokinase was not detectable by immunofluorescence on cells adherent to fibronectin. The addition of exogenous prourokinase to locate urokinase receptors on adherent HT-1080 cells indicated that the focal localization of cell-surface urokinase resulted from the clustering of urokinase receptors following adhesion to vitronectin but not fibronectin-coated substrates. These results suggest that vitronectin can contribute to the control of cell-surface plasmin activity by regulating the synthesis of urokinase and directing the localization of urokinase receptors.  相似文献   

8.
The urokinase system, represented by a plasminogen activator of urokinase type (urokinase, uPA), urokinase receptor (uPAR), and inhibitors of plasminogen activator (PAI-1 and PAI-2), plays an important role in the regulation of vascular wall functioning. Urokinase signaling initiates proteolytic cascade and degradation of the extracellular matrix; and also activates intracellular signaling in vascular cells. This study is the first to reveal a urokinase-mediated fundamental mechanism that regulates the growth trajectory and branching morphogenesis of blood vessels. This mechanism may be of particular importance during vessel growth in early embryogenesis and in the adult during tissue regeneration.  相似文献   

9.
Urokinase, a serine protease, catalyzes the conversion of plasminogen to plasmin, which is responsible for dissolution of clots in blood vessels. It is an important drug for treatment of thromboembolic disease. Production of urokinase by mammalian cell culture has the following important steps: synthesis, regulation and secretion. Production and accumulation of this product in a bioreactor is a real challenge for biochemical engineers. Considerable information at molecular level needs to be understood for production of urokinase in order to correlate different parameters, which in turn can maximize the productivity. This information will be highlighted in this review. Moreover, urokinase production is a product-inhibited process. Therefore, in situ urokinase separation strategy is required to operate a bioreactor at its maximum urokinase formation rate. Integrated urokinase production and isolation processes developed recently will also be discussed briefly in this review.  相似文献   

10.
Apart from major molecules involved in angiogenesis, such as growth factors, cytokines and chemokines, much attention is now being paid to guidance molecules that determine growth trajectories of the de novo formation of blood vessels (ephrins and their receptors, semaphorin receptors neuropilins and plexins, Slit receptor Robo, netrin receptor UNC5B, urokinase and its receptor uPAR, T-cadherin). Guidance receptors play an important role in the regulation of vascular growth trajectory during embryogenesis and vascular regeneration in adults. Besides, matrix metalloproteinases (MMPs) and serine proteases (plasmin and urokinase) are also essential for angiogenesis and vascular wall remodeling. Aberrant gene expression profiles or signaling pathways related to the above proteins can lead to various pathologies.  相似文献   

11.
Medium optimization is an important strategy that can lead to several fold increase in the production of proteins in cell culture. However, the usual methods of medium optimization are complex and time consuming. Urokinase is a widely employed thrombolytic drug for the treatment of stroke. We describe here medium optimization for maximizing urokinase production by HT-1080 cells using supplementation of specific amino acids. The new specifically designed method resulted in 240 % increase in urokinase productivity.  相似文献   

12.
The impairment of homocysteine metabolism has been related to several disorders and diseases. Recently, homocysteine has been shown to inhibit key steps of angiogenesis, including endothelial cell proliferation, invasion, and remodeling of the extracellular matrix. Since these are also key steps in tumor invasion and metastasis, it can be hypothesized that homocysteine can also interfere in these processes. Therefore, we studied the effects of homocysteine on tumor proliferation and invasion, as well as on urokinase, a key extracellular matrix-degrading protease, using a model human tumor cell line. This study demonstrates that, in fact, homocysteine inhibits HT-1080 proliferation and invasion, and is a potent inhibitor of tumor cell urokinase expression.  相似文献   

13.
Urokinase was acylated at the active site serine hydroxyl using p-amidinophenyl benzoate or p-nitrophenyl p'-guanidinobenzoate. The enzymatically inactive acyl-urokinase was reactivated in buffer (pH 7.5) or plasma at 37 degrees C with a half-life of 11 min (benzoyl-urokinase) or 10 h (p-guanidinobenzoyl-urokinase). Upon administration (50,000 IU/kg) to rabbits, urokinase was more rapidly eliminated than either acyl-enzyme. The results suggest that urokinase is eliminated via the binding to plasma inhibitors.  相似文献   

14.
15.
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。  相似文献   

16.
Urokinase is a serine protease involved in cancer growth and metastasis. Here we present the first urokinase crystal structure in complex with reversible inhibitors at 2.1 and 2.6 A resolution. These inhibitor complex structures have been obtained from crystals of engineered urokinase type plasminogen activator designed to obtain a crystal form open for inhibitor soaking. The mutant C122S loses its flexible A-chain upon activation cleavage and crystallizes in the presence of benzamidine, which was later displaced by the desired inhibitor. This new soakable crystal form turned out to be of great value in the process of structure-based drug design. The evaluated binding mode of amiloride, and UKI-1D revealed a new subsite of the primary specificity pocket of urokinase that will be employed in the future ligand optimisation process.  相似文献   

17.
Urokinase is a potent fibrinolytic agent. Its good tolerance and the close correlation to the dosage render the treatment well governable and reduce the possible side effects. However, the comparatively high costs have to be taken into account. Up to now, urokinase is an indispensable tool for long-term lysis.  相似文献   

18.
The effects of urokinase, the plasminogen activator of human urine, on the isolated substratum-attached pericellular matrices of cultured human lung fibroblasts were studied in serum-free conditions. Pericellular matrices were prepared from dense cultures of human lung fibroblasts after labelling of the cultures with radioactive glycine. Extraction of the cultures with sodium deoxycholate and hypotonic buffer gave a reproducible pattern of polypeptides when analyzed in polyacrylamide gels. The isolated pericellular matrix was subsequently exposed to affinity chromatography-purified urokinase. Urokinase affected a 66 000-dalton protein but none of the other matrix polypeptides. The appearance of a 62 000-dalton protein that remained attached to the matrix was seen concomitantly suggesting that it was derived from the 66 000-dalton protein. The 66 000-dalton protein is the first known cellular target for urokinase.  相似文献   

19.
The activated magnetic modifier was synthesized from magnetite, alpha, omega-dicarboxymethylpoly(oxyethylene) and N-hydroxysuccinimide (Biochem. Biophys. Res. Commun., 145, 908-914, 1987). Urokinase was directly coupled with the activated magnetic modifier to obtain magnetic urokinase. The magnetic urokinase dispersed in saline and exerted high fibrinolytic activity (13.8 X 10(4) IU/mg protein), and was readily recovered from saline by magnetic force of 250 Oe. By applying magnetic force, the urokinase was attracted at our will and local fibrinolysis was achieved on fibrin gel in a petri dish.  相似文献   

20.
The enzymic properties of urokinase (EC 3.4.21.31) were studied. The kinetic parameters of hydrolysis of 5-oxo-Pro-Gly-Arg-NA were determined in the pH range 5-9, at 25 degrees C and 37 degrees C. The reaction is affected by only one ionizing group of urokinase with pK 7.15 (25 degrees C) and pK 6.82 (37 degrees C). The results indicate that 5-oxo-Pro-Gly-Arg-NA is a good model substrate for studies of the conversion of plasminogen to plasmin. The Km values of the urokinase-catalysed hydrolysis of plasminogen and 5-oxo-Pro-Gly-Arg-NA are of the same order of magnitude. Plasmin catalyses the hydrolysis of 5-oxo-Pro-Gly-Arg-NA, but the Km value is several hundred times that of urokinase. Urokinase is shown not to react with good plasmin substrates, such as Bz-Arg-OEt and D-Val-Leu-Lys-NA, but is linearly competitively inhibited by 6-amino-hexanoic acid and trans-4-aminomethylcyclohexane-1-carboxylic acid.  相似文献   

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