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1.
Urokinase-type and tissue-type plasminogen activators are essential for in vitro invasion of human melanoma cells 总被引:4,自引:0,他引:4
A Meissauer M D Kramer M Hofmann L J Erkell E Jacob V Schirrmacher G Brunner 《Experimental cell research》1991,192(2):453-459
This study evaluates the contribution of two types of plasminogen activators (PAs; tissue-type PA (tPA) versus urokinase-type PA (uPA) toward the invasiveness of human melanoma cells in a novel in vitro assay. We identified two human melanoma cell lines, MelJuso and MeWo, expressing uPA or tPA as shown at mRNA, protein, and enzyme activity level. MelJuso cells produced uPA as well as plasminogen activator inhibitor-1 (PAI-1). The latter was, however, not sufficient to neutralize the cell-associated or secreted uPA activity. MeWo cells secreted tPA, but the enzyme was not found to be cell-associated. PAI-1 production by these cells was not detectable. Plasminogen activation and fibrinolytic capacity of both cell lines were reduced by anticatalytic monoclonal antibodies specific for the respective type of PA or by aprotinin. In a novel in vitro invasion assay, antibodies to PA as well as aprotinin decreased the invasiveness of both cell lines into a fibrin gel, Matrigel, or intact extracellular matrix. Our results confirm the importance of uPA-catalyzed plasminogen activation in tumor cell invasiveness. Furthermore, we provide evidence that tPA, beyond its key role in thrombolysis, can also be involved in in vitro invasion of human melanoma cells. 相似文献
2.
Plasminogen activator and urokinase are often used as biological markers of cell activation. However, the methods currently used are cumbersome, make no discrimination between tissue-type plasminogen activator and urokinase, and do not allow expression of the results of the overall reaction in International Units. The one-step method described in this paper lacks these drawbacks. Moreover, we propose use of H-D-Val-Phe-Lys-4-nitroanilide as substrate which has a lower Km than the standard H-D-Val-Leu-Lys-4-nitroanilide which is commercially available. Low concentrations of sodium dodecyl sulfate in the reaction mixture dramatically and preferentially accelerate the reaction catalyzed by tissue-type plasminogen activators. Identical results are obtained under kinetic or fixed-time assay conditions using either a photometer or 96-well plate reader. The corresponding formulae are provided. 相似文献
3.
Tissue-type plasminogen activator produced by recombinant DNA technology, has been established as an important thrombolytic agent in the treatment of acute myocardial infarction. New approaches to increase the effectiveness of this agent, including rapid high dose administration are being investigated. Several novel protein engineered variant forms of plasminogen activators have been produced that have increased thrombolytic potency in animal models and offer the potential of a more effective lower dose agent than can be administered clinically as a single bolus intravenous injection. 相似文献
4.
S G Lee N Kalyan J Wilhelm W T Hum R Rappaport S M Cheng S Dheer C Urbano R W Hartzell M Ronchetti-Blume 《The Journal of biological chemistry》1988,263(6):2917-2924
Recent data from several studies have suggested that the non-protease domains in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) determine their biological specificities, including binding to fibrin clots and survival in the circulatory system (Van Zonneveld, A.-J., Veerman, H., and Pannekoek, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4670-4674; Rijken, D. C., and Emeis, J. J. (1986) Biochem. J. 238, 643-646). Structural manipulations (e.g. deletions, additions, or substitutions) in these domains can thus be utilized to maximize the desired biological effects. Using recombinant DNA technology, we constructed a number of hybrid molecules from the t-PA and u-PA genes. In hybrid A, the epidermal growth factor and finger domains of t-PA (residues 1-91) were replaced by the epidermal growth factor and kringle of u-PA (residues 1-131). In hybrids B and C, the u-PA kringle (residues 50-131) was inserted either before (residue 92) or after (residue 261) the double-kringle region of t-PA. All these hybrid PAs containing three kringles were expressed in mouse fibroblast cells (C-127). The hybrid proteins were synthesized in predominantly a single-chain form with molecular weights of 70,000-80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were enzymatically active as assayed by the fibrin-agar plate method. In vitro studies on the binding of hybrid PAs to fibrin showed that hybrid B, like t-PA, possesses affinity toward fibrin, while hybrid A shows lower binding. This suggests that the finger domain, which is not present in hybrid A, plays a role in conferring fibrin affinity to the hybrid PAs. The enzymatic activities of the hybrids were compared with that of recombinant t-PA (rt-PA) expressed in the same vector/host system and found to be similar in activity toward a chromogenic peptide substrate. In addition, plasminogen activation with all the hybrid-PAs, as with rt-PA, was stimulated by fibrin, with the order of activity being rt-PA greater than or equal to hybrid B greater than hybrid C greater than hybrid A. This study shows the feasibility of shuffling functional domain(s) of known specificity in plasminogen activators which may lead to the design of a superior thrombolytic agent. 相似文献
5.
Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators 总被引:12,自引:6,他引:12
Cell extracts and conditioned media (CM) from cultured bovine aortic endothelial cells (BAEs) were fractionated by PAGE in the presence SDS, and plasminogen activator (PA) activity was localized by fibrin autography. Multiple molecular weight forms of PA were detected in both preparations. Cell-associated PAs had Mr of 48,000, 74,000, and 100,000 while secreted PAs showed Mr of 52,000, 74,000, and 100,000. A broad zone of activity (Mr 80,000-100,000) also was present in both cellular fractions. In addition, PAs of Mr 41,000 and 30,000 appeared upon prolonged incubation or repeated freezing and thawing of the samples, and probably represent degradation products of higher molecular weight forms. This complex lysis pattern was not observed when CM was subjected to isoelectric focusing. Instead, only two classes of activator were resolved, one at pH 8.5, the other at 7.6. Analysis of focused samples by SDS PAGE revealed that the activity at pH 8.5 resulted exclusively from the Mr 52,000 form; all other forms were recovered at pH 7.6. The activity of the Mr 52,000 form was neutralized by anti-urokinase IgG but was not affected by antitissue activator IgG indicating that it is a urokinaselike PA. The activities of the Mr 74,000-100,000 forms were not affected by anti-urokinase. They were blocked by antitissue activator suggesting that all the forms in this group were tissue-type PAs. The multiple forms of PA were differentially sensitive to inactivation by diisopropylfluorophosphate (DFP). Treatment of CM with 10 mM DFP for 2 h at 37 degrees C only partially inhibited the 52,000-dalton form. However, it completely inactivated the 74,000-dalton partially inhibited the 52,000-dalton form. However, it completely inactivated the 74,000-dalton PA. The activity of the Mr 100,000 form was not affected by this treatment, or by treatment with 40 mM DFP. Thus, cultured BAEs produce multiple, immunologically distinct forms of PA which differ in size, charge, and sensitivity to DFP. These forms include both urokinaselike and tissue-activator-like PAs. The possibility that one of these forms is a zymogen is discussed. 相似文献
6.
Urokinase-type and tissue-type plasminogen activators have different distributions in cultured bovine capillary endothelial cells 总被引:8,自引:0,他引:8
D Moscatelli 《Journal of cellular biochemistry》1986,30(1):19-29
The cell extracts and conditioned medium from cultured bovine capillary endothelial (BCE) cells were examined to determine the types of plasminogen activator (PA) present in each of these two fractions. The fractions were first analyzed by fibrin autography after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The cell extracts contained two species of PA of Mr 48,000 and 28,000. Multiple forms of PA were detected in the conditioned medium: variable amounts of the Mr 48,000 and 28,000 forms and a broad band of activity with Mr in the range of 67,000-93,000. The major fraction of the Mr 48,000 form was in the cell extract. Treatment of the cells with 12-0-tetradecanoyl phorbol-13-acetate or with a preparation containing angiogenic activity resulted in a proportionate increase in the levels of all forms. The Mr 48,000 form was demonstrated to be a urokinase-like PA, since it was immunoprecipitated with antibodies to urokinase. When conditioned medium or cell extracts from biosynthetically labelled BCE cells were incubated with antiserum to urokinase, the Mr 48,000 form was immunoprecipitated only from the cell extract. The Mr 67,000-93,000 forms were demonstrated to be tissue-type PAs, since they were immunoprecipitated with antibodies to tissue PA. When the same conditioned medium or cell extracts were incubated with antiserum to tissue-type PA, the Mr 67,000-93,000 forms were immunoprecipitated only from the conditioned medium. Therefore, BCE cells are able to produce both tissue-type PA, which is primarily secreted, and urokinase-type PA, which remains primarily cell associated. 相似文献
7.
B Y Karlan A S Clark B A Littlefield 《Biochemical and biophysical research communications》1987,142(1):147-154
A simple and highly sensitive chromogenic microtiter plate assay for plasminogen activators is described. The assay is based on plasmin cleavage of the synthetic tripeptide plasmin substrate H-D-norleucyl-hexahydrotyrosyl-lysine p-nitroaniline, which yields the yellow chromophore p-nitroanilide. Production of the latter compound is then quantitated spectrophotometrically at 405 nm on an ELISA plate reader. Linearity of the assay can be achieved over at least four orders of magnitude in a single experiment (0.01-100 milliPloug units) with appropriate incubation times. Capitalizing on tissue-type plasminogen activator's dependence on fibrin for enzymatic activity, the selective use of soluble fibrin products allows discrimination between urokinase and tissue-type activator. The utility of this aspect of the assay for the analysis of complex samples containing both types of plasminogen activators is demonstrated. 相似文献
8.
Increased activity of plasminogen activators during involution of the rat ventral prostate.
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Plasminogen activator was measured in the ventral prostates of non-castrated, castrated, and androgen-treated rats to determine whether changes in this activity correlated with the process of glandular involution. While the activity was very low in cytosolic extracts from the prostates of non-castrated rats, 2 days following castration the plasminogen activator activity increased in a near-linear fashion such that by day 7 it was 10-fold higher in terms of specific activity (per mg of protein) and cellular concentration (per mg of DNA). During this interval there was a rapid decrease in the cell population of the prostates. Treatment of the 7-day castrated rats with the potent androgen, dihydrotestosterone, both reduced the plasminogen activator activity and restored the cell number in a dose-related manner. Gel electrophoretic analysis revealed two major bands of plasminogen activator activity in the cytosolic extracts from 4- and 7-day castrated rats, plus additional minor bands in samples from 10- and 14-day castrated rats. Approx. 10% of the cellular concentration of plasminogen activator activity was recovered in association with an 18000g pellet fraction from the prostates; this fraction showed less heterogeneity of the plasminogen activator forms as observed by gel electrophoresis. Inhibitor studies indicated that the 18000g pellet fraction from the prostates of non-castrated rats possessed some plasminogen activator inhibitor activity, but the relative concentration of the inhibitor activity was small. We conclude that the involution of the prostate is probably associated with increased synthesis of plasminogen activators through a de-repression process which may involve loss of androgen receptors. 相似文献
9.
We previously demonstrated that tissue-type plasminogen activator (t-PA) specifically bound to its receptor (t-PAR) on human umbilical vein endothelial cells (HUVEC). In addition to analyses of t-PA binding to plasminogen activator inhibitor-1 (PAI-1) in the extracellular matrix (ECM) and to the t-PAR, we further evaluated the binding of three t-PA mutants, deltaFE1X t-PA lacking finger (F), epidermal growth factor-like (E) domains and one sugar chain at Asn177 thus comprising two kringles (K1 and K2) and protease (P) domains, deltaFE3X t-PA with three glycosylation sites deleted at Asn117, 184, and 448, and deltaFEK1 t-PA comprising K2 and P domains without glycosylation. Wild-type t-PA bound to ECM with high affinity, which was completely blocked by anti-PAI-1 IgG. Wild-type t-PA, deltaFE1X t-PA and deltaFEK1 t-PA bound to two classes of binding sites with high and low affinities on monolayer HUVEC. However, all t-PAs bound to a single class of binding site in the presence of anti-PAI-1 IgG. DeltaFEK1 t-PA bound t-PAR maximally among these t-PAs. These results suggested that the high affinity binding of t-PA mainly occurred with PAI-1 on ECM while the low affinity binding was with t-PAR. The deletion of F, E domains and sugar chains had no effect on binding with t-PAR. However, since only K1-missing t-PA (deltaFEK1) exhibited significantly increased binding sites among these t-PAs, it was suggested that the binding to t-PAR was mediated mainly by K2 domain and that the increase of binding was due to direct exposure of K2 domain. 相似文献
10.
Incubation of plasminogen with the subendothelial extracellular matrix (ECM) synthesized by cultured bovine corneal and aortic endothelial cells resulted in generation of fibrinolytic activity, indicated by proteolysis of 125I-fibrin in a time-and dose-dependent manner. Both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) were identified in the ECM by fibrin zymography, immunoblotting, and inhibition of plasminogen activation by anti-u-and anti-t-antibodies. Most of the ECM-resident plasminogen activator (PA) activity did not originate from intracellular PA release occurring when the endothelial cells were lyzed and the ECM exposed, since a comparable amount of PA was associated with the ECM when the cells were lyzed with Triton X-100 or removed intact by treatment with 2 M urea. Active u-PA and t-PA were released from ECM by treatment with heparanase (endo-β-D-), indicating that some of the ECM-resident PA activity is sequestered by heparan sulfate side chains. These results indicate that both u-PA and t-PA produced by endothelial cells are firmly sequestered in an active form by the subendothelial ECM. It is suggested that ECM-resident plasminogen activators participate in sequential matrix degradation during cell invasion and tumor metastasis. PA activity may also function in release of ECM-bound growth factors (i.e., basic fibroblast growth factor) and activation of proenzymes (i.e., prothrombin), resulting in modulation of the ECM growth-promoting and thrombogenic properties. © 1993 Wiley-Liss, Inc. 相似文献
11.
Plasminogen activators convert the proenzyme plasminogen to the active serine protease plasmin by hydrolysis of the Arg560-Val561 peptide bond. Physiological plasminogen activation is however regulated by several additional molecular interactions resulting in fibrin-specific clot lysis. Tissue-type plasminogen activator (t-PA) binds to fibrin and thereby acquires a high affinity for plasminogen, resulting in efficient plasmin generation at the fibrin surface. Single-chain urokinase-type plasminogen activator (scu-PA) activates plasminogen directly but with a catalytic efficiency which is about 20 times lower than that of urokinase. In plasma, however, it is inactive in the absence of fibrin. Chimeric plasminogen activators consisting of the NH2-terminal region of t-PA (containing the fibrin-binding domains) and the COOH-terminal region of scu-PA (containing the active site), combine the mechanisms of fibrin specificity of both plasminogen activators. Combination of t-PA and scu-PA infusion in animal models of thrombosis and in patients with coronary artery thrombosis results in a synergic effect on thrombolysis, allowing a reduction of the therapeutic dose and elimination of side effects on the hemostatic system. 相似文献
12.
13.
B Y Karlan J A Rivero M E Crabtree B A Littlefield 《Molecular endocrinology (Baltimore, Md.)》1989,3(6):1006-1013
Previous studies from our laboratory have demonstrated that OVCA 433 human ovarian carcinoma cells are glucocorticoid responsive by several criteria and contain high affinity, saturable, steroid-specific glucocorticoid receptors. These cells secrete both mammalian plasminogen activators (PAs), urokinase (uPA) and tissue-type PA (tPA). Treatment of OVCA 433 cells with 1 x 10(-7) M dexamethasone (Dex) for 4 days led to 77% and 83% reductions in the extracellular activities of uPA and tPA, respectively, released into serum-free conditioned medium during a 1-h period. Dex treatment led to a 71% decrease in the rate of extracellular uPA antigen accumulation, as determined by enzyme-linked immunosorbent assay, as well as a 73% reduction in steady state uPA mRNA levels. In contrast, Dex treatment led to only a 42% decrease in the rate of extracellular tPA antigen accumulation and a 48% decrease in tPA mRNA levels; such decreases were insufficient to account for the 83% reduction in tPA activity. Thus, while Dex-induced decreases in uPA antigen and mRNA levels accounted for all but 6% of the decrease in uPA activity, a large discrepancy existed between the magnitudes of decreased tPA activity and decreased tPA antigen and mRNA levels. OVCA 433 cells produce both PAI-1 and PAI-2, two specific PA inhibitors. Treatment of cells with 1 x 10(-7) M Dex for 4 days led to a 3.3-fold increase in the rate of extracellular PAI-1 accumulation, with little or no effect on PAI-2 accumulation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator 总被引:6,自引:0,他引:6
The kinetics of the activation of plasminogen by tissue-type plasminogen activator were studied in the presence and the absence of CNBr-digested fibrinogen as a soluble cofactor. Michaelis-Menten kinetics applied and the kinetic parameters obtained were very similar to those previously reported for the activation in the presence of solid phase fibrin (Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919). The affinity of the enzyme for plasminogen dramatically increases in the presence of the soluble cofactor while the catalytic rate constant does not change significantly (KM drops from 83 to 0.18 microM and kcat increases from 0.07 to 0.28 s-1 for tissue-type plasminogen activator of melanoma origin). Fragments containing the lysine-binding sites of plasminogen compete with plasminogen for interaction with CNBr-digested fibrinogen. The dissociation constant of this interaction was found to be 4.5 microM for the high affinity lysine-binding site. No difference was found in the kinetic parameters for the activation of plasminogen by either tissue-type plasminogen activator of melanoma origin or by glycosylated forms of tissue-type plasminogen activator obtained by recombinant DNA technology. The present findings obtained in a homogenous liquid milieu support the previously proposed mechanism of the activation of plasminogen by tissue-type plasminogen activator in the presence of fibrin. This mechanism involves binding of both tissue-type plasminogen activator and plasminogen to fibrin. 相似文献
15.
Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats. 总被引:2,自引:1,他引:2
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In order to assess which part of the tissue-type plasminogen activator (t-PA) molecule should be (genetically) modified to obtain more-slowly-clearing mutants, two-chain t-PA and its isolated heavy and light chains were radiolabelled and injected into rats. The vast majority of t-PA and the heavy chain disappeared from the blood circulation with half-lives of 2.3 and 1.0 min respectively. The clearance of the light chain was biphasic, owing to complex-formation with plasma proteinase inhibitors. The disappearance of di-isopropylphospho-light chain, which has a blocked active site, was nearly monophasic, with a half-life of 5.7 min. Organ distribution studies showed that hepatic clearance constituted the major pathway in all cases. These results strongly suggest that t-PA is recognized by the liver primarily through the heavy chain. 相似文献
16.
M Takeda M Kosaka Y Nishina K Sawada K Matsumoto Y Nishimune 《Journal of cellular biochemistry》1992,49(3):284-289
Sodium butyrate (NaB) can induce teratocarcinoma cell differentiation as retinoic acid (RA). However, the function of these two agents seems to be a little different [Kosaka et al., Exp Cell Res, 192:46-51, 1991]. F9 cells treated with NaB synthesize both tissue-type (tPA) and urokinase-type (uPA) plasminogen activator, though RA induces only tPA production. Urokinase-type PA is demonstrated to exist in association with membrane and to localize its activity to the close environment of the cell surface. This may cause the specific cell morphology and characteristics of differentiated F9 cells induced with NaB. 相似文献
17.
Johannes Kirchheimer Arthur Köller Bernd R. Binder 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,797(2):256-265
Plasminogen activators from prostate tissue were purified to apparent homogeneity by a procedure involving reverse ammonium sulfate gradient solubilization, chromatography on gelatine-Sepharose, gel filtration on Sephadex G-150, and chromatography on Con A-Sepharose as a final step. Two activators were obtained. The predominant one exhibited physicochemical, immunochemical and functional properties indistinguishable from human urinary high molecular weight urokinase. The other one, which amounted to about 20% was immunochemically related to tissue type plasminogen activator and its plasminogen activator activity was enhanced by addition of CNBr-fibrinogen framents in a similar pattern as for the vascular plasminogen activator. The molecular weight, however, and enzymatic activities on the synthetic low molecular weight paranitroanilide substrates were different to vascular plasminogen activator but similar to high molecular weight urinary urokinase. 相似文献
18.
Isolation and characterization of plasminogen activators from hyperplastic and malignant prostate tissue 总被引:2,自引:0,他引:2
Plasminogen activators from prostate tissue were purified to apparent homogeneity by a procedure involving reverse ammonium sulfate gradient solubilization, chromatography on gelatine-Sepharose, gel filtration on Sephadex G-150, and chromatography on Con A-Sepharose as a final step. Two activators were obtained. The predominant one exhibited physicochemical, immunochemical and functional properties indistinguishable from human urinary high molecular weight urokinase. The other one, which amounted to about 20% was immunochemically related to tissue type plasminogen activator and its plasminogen activator activity was enhanced by addition of CNBr-fibrinogen fragments in a similar pattern as for the vascular plasminogen activator. The molecular weight, however, and enzymatic activities on the synthetic low molecular weight paranitroanilide substrates pyro-Glu-Gly-Arg-pNA and H-D-Ile-Pro-Arg-pNA were different to vascular plasminogen activator but similar to high molecular weight urinary urokinase. 相似文献
19.
Barker-Carlson K Lawrence DA Schwartz BS 《The Journal of biological chemistry》2002,277(49):46852-46857
The serine protease tissue-type plasminogen activator (t-PA) initiates the fibrinolytic protease cascade and plays a significant role in motor learning, memory, and neuronal cell death induced by excitotoxin and ischemia. In the fibrinolytic system, the serpin PAI-1 negatively regulates the enzymatic activity of both single-chain and two-chain t-PA (sct-PA and tct-PA). In the central nervous system, neuroserpin (NSP) is a serpin thought to regulate t-PA enzymatic activity. We report that although both sct-PA and tct-PA rapidly form acyl-enzyme complexes with NSP in vitro, the interactions are short-lived, rapidly progressing to complete cleavage of NSP and regeneration of fully active enzyme. All NSP molecules appear to transit through the detectable acyl-enzyme intermediate and progress to completion of cleavage; no subpopulation that functions as a pure substrate was detected. Likewise, all molecules were reactive, with no evidence of a latent subpopulation. The interactions between NSP and t-PA were distinct from those between plasmin and NSP, wherein the same peptide bond was cleaved but there was no evidence of a detectable plasmin-NSP acyl-enzyme complex. The interactions between t-PA and NSP contrast with the formation of long-lived, physiologically irreversible acyl-enzyme complexes between t-PA and PAI-1, suggesting that the physiologic effect of t-PA-NSP interactions may be more complex than previously thought. 相似文献
20.
A new method for determining plasminogen activator levels has been developed. Data are presented which demonstrate measurements of trypsin, plasmin, urokinase, and plasminogen activation. The assay is based on the digestion of N-terminal-blocked protamine and subsequent measurement of the exposed amino groups using the fluorogenic amine reagent, Fluram. The soluble substrate provides an assay which is linear with respect to both time and concentration and which is sensitive enough to allow measurements on a microscale. As little as 1 ng of trypsin, 0.002 CTA units (established by the Committee on Thrombolytic Agents of the NIH) of plasmin, and 0.01 CTA units of urokinase can be detected under the conditions described.Interference with the amine determination due to Fluram-positive material found in biological samples is minimized with the high dilutions attainable with the system. Plasminogen activator in the urine of the female mouse can be detected using 1 μl of urine in a 200-μl test system. 相似文献