UNIPROT:P00750 - FACTA Search

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Query: UNIPROT:P00750 (PLA)
16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The hormonal regulation of two plasminogen activators, tissue-type plasminogen activator (t-PA) and urokinase (u-PA), was studied both in 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma and in DMBA-induced rat mammary dysplasia. t-PA activity in DMBA-mammary carcinoma was decreased markedly by oophorectomy and recovered upon estradiol administration to reach the maximum level at 12 hr. In contrast to its effect on DMBA-mammary carcinoma, estradiol had no effect on t-PA activity in DMBA-mammary dysplasia. Furthermore, DMBA-mammary carcinoma cells in primary culture displayed similar estrogen-dependency in production of t-PA, while t-PA production in DMBA-mammary dysplasia cells was not under the control of estradiol in vitro. Moreover, estrogen-stimulated production of u-PA activity was not observed in DMBA-mammary carcinoma cells or DMBA-mammary dysplasia cells both in vivo and in vitro. Taken together, these results suggest that estrogen stimulates the production of t-PA but not u-PA and that this estrogen dependency of t-PA is limited to malignant DMBA-mammary tumor cells.
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PMID:Specific stimulation by estradiol of tissue-type plasminogen activator production in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor cells. 147 14

K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717-19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6-15-fold higher than that of rscu-PA. Conversion of 1 microM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0 +/- 0.15 nmol.min-1.nmol plasmin-1 and 0.85 +/- 0.074 nmol.min-1.nmol plasmin-1, compared to 14 +/- 2.3 nmol.min-1.nmol plasmin-1 and 18 +/- 2.6 nM.min-1.nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 59-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Biochemical characterization of single-chain chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody and single-chain urokinase. 148 77

Nine healthy volunteers and 23 patients with various types of von Willebrand disease were studied before and after DDAVP infusion. We investigated the behaviour of factor VIII/von Willebrand factor measurements, and of tissue plasminogen activator and urokinase-type plasminogen activator. In mild von Willebrand disease the increase of both plasminogen activators was similar to that seen in normal controls. A different fibrinolytic behaviour was found in the type I platelet low and in the type III von Willebrand disease patients. An impaired and absent fibrinolytic response to DDAVP was seen in the former and in the latter von Willebrand disease, respectively. A close relation between either u-PA and t-PA or von Willebrand factor was observed. The possibility of a linkage among these three proteins was discussed.
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PMID:Urokinase-type plasminogen activator release after DDAVP in von Willebrand disease: different behaviour of plasminogen activators according to the synthesis of von Willebrand factor. 152 8

We have previously shown that alpha-thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase-type (u-PA) and tissue-type plasminogen activator (t-PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI-1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L-type voltage-sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin-stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10(-8) M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1. Both thrombin-stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 microM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP-inactivated thrombin which binds the thrombin receptor and gamma-thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u-PA, t-PA, and PAI-1 synthesis although to a lesser extent than alpha-thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by alpha-thrombin in glomerular epithelial cells: 1) Ca2+ influx through a dihydroperidine-sensitive calcium channel, which seems critical for mitogenesis; 2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u-PA, t-PA, and PAI-1; 3) protein kinase C activation by other phospholipid breakdown can stimulate u-PA, t-PA, and PAI-1 synthesis but not mitogenesis.
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PMID:Thrombin signal transduction mechanisms in human glomerular epithelial cells. 153 79

Transforming growth factor-beta (TGF beta) is the most potent known inhibitor of keratinocyte growth. Pericellular proteolytic activity is usually high in proliferating and malignant cells and decreased in resting or growth-arrested cells. We have therefore analyzed the effects of TGF beta 1 on the production of plasminogen activator activity by normal human keratinocytes and a mouse keratinocyte cell line under serum-free conditions. The plasminogen activator activity of the culture medium was analyzed using caseinolysis-in-agar and zymography assays, immunoblotting, and Northern hybridization analysis for the plasminogen activators (PA) and PA inhibitor-1 (PAI-1). Alterations of radiolabeled polypeptides were observed in fluorograms of gels. It was found that like in human epidermoid carcinoma cells picomolar concentrations of TGF beta 1 (0.2-20 ng/ml) enhanced total plasminogen activator activity in both keratinocyte cell systems. Zymographic and immunoblotting analyses of the medium indicated that the activator was of the urokinase type (u-PA). Immunoprecipitation and Concanavalin A affinity chromatography of the culture medium indicated that the cells also started to produce PAI-1. Analysis of the pericellular matrix preparations of the keratinocytes showed that PAI-1 is deposited to the pericellular space. Evidently due to elevated u-PA activity PAI-1 was removed from the extracellular matrix more rapidly in TGF beta 1-treated cells than from control cultures. Northern hybridization analysis of human keratinocytes showed that TGF beta 1 rapidly elevated both u-PA and PAI-1 mRNA levels. Comparison of the temporal induction profiles indicated that the mRNA for u-PA increased more slowly but was more persistent than that of PAI-1. Actinomycin D inhibited the induction of both u-PA and PAI-1 mRNA, suggesting that the induction was due to increased transcription. The results suggest that enhanced plasminogen activator activity can be associated with growth inhibition also in nonmalignant cells like cultured human or murine keratinocytes.
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PMID:Enhanced production of plasminogen activator activity in human and murine keratinocytes by transforming growth factor-beta 1. 162 32

K1K2Pu, a recombinant t-PA/u-PA chimera with increased thrombolytic potency in animal models of venous and arterial thrombosis, which consists of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA), was produced and conditioned for use in patients. Chinese hamster ovary cells were transfected with an expression plasmid containing the K1K2Pu cDNA, high producer cell lines were selected and scaled up in 800 cm2 roller bottles, and 350 ml conditioned cell culture medium was harvested 3 to 7 times at 2 to 5 day intervals. Batches of 21 +/- 4 liter (mean +/- SD, n = 28) containing 1.8 +/- 0.6 mg/l of K1K2Pu related antigen were purified by chromatography on Copper chelate-Sepharose and immunoadsorption on an insolubilized murine monoclonal antibody (MA-1C8). Yields were 8.6 +/- 3.4 mg K1K2Pu per batch with a specific activity of 83,000 +/- 44,000 IU/mg. The final material, obtained at a concentration of approximately 0.7 mg/ml, was dialyzed against 0.3 M NaCl, 0.02 M Tris-HCl buffer, pH 7.5, containing 0.01% Tween 80 and 10 KIU/ml aprotinin. It was homogeneous on SDS-PAGE, contained 6.5 +/- 6.9 percent two chain material and the contamination with murine monoclonal antibody was less than 0.1 percent. After filtration of pools of 3 to 5 selected batches on 0.22 microns Millipore filters the material was sterile and virus free by routine screening; it was obtained at a concentration of approximately 0.5 mg/ml with a specific activity of 110,000 +/- 16,000 IU/mg (mean +/- SD, n = 3) and an endotoxin content of 0.5 to 7 units/mg. Bolus injection at a dose of 1 mg/kg in mice did not produce weight loss within 8 days. Thus, this material appears to be suitable for the investigation on a pilot scale of the pharmacokinetic and thrombolytic properties of K1K2Pu in patients with thromboembolic disease.
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PMID:K1K2Pu, a recombinant t-PA/u-PA chimera with increased thrombolytic potency, consisting of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA). Purification in centigram quantities and conditioning for use in man. 163 5

The goal of the present study was to assess the relative importance of receptor-bound and secreted plasminogen activator urokinase (u-PA) in generating cell-surface plasmin and fostering destruction of normal tissue by tumor cells. We first showed that active site-inhibited u-PA could displace endogenous u-PA from the surface of the human colon adenocarcinoma cell line HCT 116. We then prepared expression vectors for u-PA and for a mutant molecule in which the codon for the active site serine residue was changed to encode alanine. Expression of non-functional mutant u-PA decreased the level of cell-bound active u-PA by more than 95% via a mechanism that involved competition for receptor sites. Decreased cell-surface u-PA activity was associated with a decrease in cell-bound plasmin activity to undetectable levels, suggesting that receptor-bound u-PA plays an important role in the generation of plasmin on the cell surface. Transfectants that secreted eightfold to 20-fold elevated levels of active wild-type u-PA showed approximately 50% increases in cell-associated u-PA and only twofold to fourfold increases in cell-associated plasmin, suggesting that the role of secreted u-PA in generating cell-surface plasmin activity was relatively minor. In parent cells and both types of transfectants there was a good correlation between the amount of plasmin bound to the tumor cell surface and the extent to which a basement membrane substrate was degraded. These studies show that receptor-bound u-PA provides an efficient mechanism for plasmin generation on the surface of tumor cells, which, in turn, contributes significantly to their degradative potential.
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PMID:Effects of urokinase receptor occupancy on plasmin generation and proteolysis of basement membrane by human tumor cells. 164 83

Adenosine 3',5'-cyclic monophosphate (cAMP) elevation in cultured rat mesangial cells causes urokinase-dependent adhesion loss, stress-fiber fragmentation, and shape change. Thrombin cleaves single-chain urokinase (scu-PA), causing its inactivation, but not two-chain u-PA [tcu-plasminogen activator (PA)] or tissue-type PA. We tested the ability of thrombin to inhibit the effects of cAMP elevation in mesangial cells and inactivate cell-associated scu-PA. In an assay of trypsin-sensitive adhesion, 65.9% of control cells and 5.5% of cells treated with isoproterenol + methylisobutylxanthine (IM) remained adherent. In the presence of 0.01, 0.1, 1.0, and 10.0 unit/ml thrombin, 20.9, 46.6, 50.4, and 53.3%, respectively, of IM-treated cells remained attached. Thrombin also inhibited stress-fiber fragmentation and shape change. The effects of thrombin were blocked by hirudin or antithrombin III plus heparin. Direct zymography in gels containing gelatin and plasminogen revealed loss of a closely spaced pair of PA bands with thrombin treatment (1.0 unit/ml). Hirudin blocked the loss. alpha-Thrombin inactivated by diisopropyl fluorophosphate neither inhibited shape change nor caused loss of the PA bands; however, gamma-thrombin was nearly as active as native alpha-thrombin in both regards. Pretreatment of the cells with as little as 1.0 unit/ml thrombin for 1.0 min caused marked inhibition of shape change and near total loss of the slower migrating u-PA band (of the doublet). The faster migrating band was inhibited less. The results indicate that the slower migrating band represents scu-PA; the nature of the faster migrating band is less certain. Thrombin reversed the adhesion loss and shape change caused by 8-(4-chlorophenylthio)-cAMP and MIX. Thus physiological concentrations of thrombin rapidly inactivate mesangial cell scu-PA and inhibit and reverse cAMP-stimulated adhesion loss and shape change.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of mesangial cell adhesion and shape by thrombin. 165 8

Hyperthermia is a clinical sign of inflammation and constitutes in itself an adaptive defense mechanism. The fibrinolytic system, a highly regulated proteolytic system, is involved in inflammatory processes. Plasminogen activator inhibitor 1 (PAI-1) is the principal inhibitor of the two activators of the fibrinolytic system: tissue- and urokinase-type PAs (t-PA and u-PA). Our present paper provides the first evidence that hyperthermia can directly induce PAI-1. A moderate heat stress, sufficient to induce heat shock protein 70 mRNA approximately 100-fold, resulted in a two- to three-fold increase in functionally active PAI-1 in the conditioned medium of human HT-1080 fibrosarcoma and Hep G2 hepatoma cells. Exposure of these cells to 42 degrees C led to a similar two-fold and two- to five-fold induction of PAI-1 mRNA expression in HT-1080 and Hep G2 cells, respectively, as has been determined by using both oligo d(T) selected and total RNA preparations. These results suggest that the observed increase in PAI-1 accumulation is due to an induction of PAI-1 biosynthesis. Run-on transcription analysis indicates that the induction of PAI-1 biosynthesis by hyperthermia is mediated by a stimulation of PAI-1 gene transcription. No significant effect of hyperthermia was found on t-PA or u-PA at the level of antigen accumulation, mRNA, and gene transcription in human HT-1080 fibrosarcoma cells. These results point to an additional regulatory mechanism of fibrinolysis in the context of inflammation.
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PMID:Induction of plasminogen activator inhibitor 1 biosynthesis by hyperthermia. 165 90

We have compared the cell-specific expression and regulation of the receptor for urokinase-type plasminogen activator (u-PAR) by transforming growth factor beta type 1 (TGF-beta 1) in 10 human cell lines derived from both normal and neoplastic tissues. The basal expression of u-PAR mRNA as well as its response to TGF-beta 1 varied strongly between different cell lines; however, five out of the 10 cell lines responded to TGF-beta 1 by an increase in the u-PAR mRNA level. Among these, A549 cells were selected for a detailed elucidation of the molecular mechanism involved in TGF-beta 1 regulation of u-PAR mRNA expression. TGF-beta 1 caused an early increase in u-PAR mRNA level, with a maximal 15-fold enhancement after 24 h of treatment. This was paralleled by an increase in u-PAR protein as detected by crosslinking studies with radiolabeled ligand, and also resulted in an increase in cell surface plasmin generation. The protein synthesis inhibitor cycloheximide also increased the level of u-PAR mRNA in a time-dependent fashion and when both cycloheximide and TGF-beta 1 were used, an additive effect was seen. Nuclear run-on experiments demonstrated only a moderate (3-fold) increase in the u-PAR gene transcription rate after exposure of the cells to TGF-beta 1 for 3 h compared with a 12-fold increase in the mRNA level. TGF-beta 1 also caused an increase of both u-PA and PAI-1 antigens, while there was no detectable effect on t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Urokinase-receptor biosynthesis, mRNA level and gene transcription are increased by transforming growth factor beta 1 in human A549 lung carcinoma cells. 165 20

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