Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Purified preparations of recombinant tissue-type plasminogen activator (t-PA) from the recombinant Bowes melanoma cell line TRBM6 were shown to contain multiple species of plasminogen activator. Using a combination of chromatography on Sephadex G25, Sephadex G75 and Heparin Sepharose CL6B we have isolated two fibrinolytically active species, which, under non-reduced SDS PAGE, have apparent Mr = 38,000 and 56,000. Double immunodiffusion studies indicated that both species were closely related to both the t-PA B chain and t-PA itself. N-terminal sequencing identified the Mr = 38,000 species as ala160- t-PA (essentially delta FGKI t-PA) and the Mr = 56,000 species as ser1-tyr2-gln3-glyx-cys51 t-PA (delta F t-PA), the latter probably produced by alternative splicing of the t-PA gene. The pharmacokinetic properties of N,N dimethyl-4-aminobenzoyl (DAB) derivatives of these activators and native t-PA were determined in the guinea pig. Whereas DAB----delta F t-PA showed a similar, rapid plasma disappearance profile to that of DAB----t-PA, DAB----delta FGKI t-PA was cleared significantly slower. These results suggest that a rapid clearance recognition site resides on either the growth factor or kringle 1, or both, domains of t-PA.
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PMID:Isolation, identification and pharmacokinetic properties of human tissue-type plasminogen activator species: possible localisation of a clearance recognition site. 314 86

Two approaches were used to identify and characterize the presence of tissue plasminogen activator (t-PA) in megakaryocytes and platelets. We investigated the fibrinolytic activity of human megakaryocytes (MK) and platelets. The presence of t-PA antigen in megakaryocytes and platelets was demonstrated using immunocytochemical techniques and polyclonal or monoclonal antibodies specific for t-PA. When cells were applied to fibrin plates, lysis zones developed around isolated human megakaryocytes, whereas no fibrinolytic activity appeared when either intact washed platelets or platelet lysate were deposited. After SDS-PAGE of platelet and MK extracts (Triton X-100) immunoblotting and peroxidase staining identified t-PA antigen in several bands. Zymographic analysis of SDS-PAGE carried out on fibrin film overlays identified one or two zones corresponding to free or complexed t-PA. These results indicate that t-PA is present in platelets as well as in the precursor cells, however, in platelets, t-PA may not be immediately available for plasminogen activation and fibrin degradation. From our findings and from previous work of others, it appears that platelets may either activate or inhibit the fibrinolytic system. Therefore the conditions of plasminogen activation by platelet t-PA and plasmin inhibition by platelet alpha 2-antiplasmin or other inhibitors have to be precised before the role of platelets in clot dissolution is understood. The physiological role of platelets in fibrinolysis and clot dissolution remains unclear. In 1953, the antifibrinolytic activity of blood platelets was demonstrated and in the early 1960's a fibrinolytic activity, increasing with platelet concentration in the experimental system, was shown.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tissue plasminogen activator in human megakaryocytes and platelets: immunocytochemical localization, immunoblotting and zymographic analysis. 314 87

The transcriptional and translational signals required for efficient expression of the chloramphenicol acetyltransferase, beta-galactosidase, and tissue plasminogen activator genes, under the control of the polyhedrin promoter in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus, were investigated by SDS-PAGE and RNA dot blot analysis. The recombinant baculoviruses all contained alterations in the leader sequence or 5' proximal coding region of the polyhedrin gene. Highest levels of foreign proteins and polyhedrin-linked mRNAs were observed when portions of the coding sequence of the polyhedrin gene were fused in phase with the foreign gene. Recombinant viruses in which the foreign gene was inserted upstream from the polyhedrin ATG start codon expressed nonfused products but at lower levels than contructs which produced fusion proteins. A corresponding decrease in the levels of mRNAs produced by such constructs was also observed. Some constructs in which the foreign gene was inserted out of phase downstream from the polyhedrin start codon expressed nonfused protein products at low levels but produced polyhedrin-linked mRNA at levels comparable to vectors which produced protein fusions. These data suggest that reinitiation of translation can take place at AUG start codons a short distance downstream from the primary polyhedrin start codon. These results indicate that sequences immediately upstream from the polyhedrin start codon are important for regulation of transcription and that additional sequences near the AUG start codon can have a dramatic influence on the levels of translation observed.
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PMID:Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors. 314 47

The effect of human recombinant tumor necrosis factor (TNF) was studied in vitro on human endothelial cells. TNF (1-1000 pg/ml) induced a dose-dependent increase in PAI level in the supernatant from 6 to 25 U/ml as estimated against urokinase. This effect was time-dependent. It was not suppressed by Polymyxin B thus excluding a possible contribution of an endotoxin contamination. Fibrinoenzymography performed after SDS-PAGE showed that this inhibitor neutralized urokinase and tissue plasminogen activator and gave rise to high molecular weight complexes. TNF (30 micrograms/kg) was also injected in rat. Blood fibrinolytic activity determined 4 hr later was decreased as estimated by the prolongation of the euglobulin clot lysis time from 37 to 188 min. Fibrinoenzymographic profile of the plasma was then characterized by a fainting of the tPA lysis band but the capacity of plasma to neutralize urokinase was not significantly modified. These results suggest that TNF could alter the fibrinolytic balance by stimulating PAI production at the endothelial level. This might be of importance in synergy with the TNF-induced procoagulant activity for promoting vascular occlusion of tumor capillaries.
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PMID:Tumor necrosis factor (TNF) stimulates plasminogen activator inhibitor (PAI) production by endothelial cells and decreases blood fibrinolytic activity in the rat. 314 62

This report describes the development and use of functional immunoradiometric assays that distinguish the activity of beta-migrating endothelial-type plasminogen activator inhibitor (PAI-1) from that of placental-type plasminogen activator inhibitor (PAI-2). These assays are based upon the binding of PAI-1 and PAI-2 to immobilized single-chain tissue-type plasminogen activator (tPA) and to immobilized urokinase (UK), respectively. The extent of binding of each PAI is quantified by incubating the PAI-PA complex first with rabbit antiserum specific for the individual PAI and then with 125I-labeled goat antirabbit IgG. In control experiments, the assays were shown to be sensitive, dose-dependent over a wide range, and specific for each PAI. These assays were employed to establish the PAI profile of a variety of human cells. Neither PAI-1 nor PAI-2 could be detected in Bowes melanoma cells or in a renal adenocarcinoma cell line (ACHN), while the histiocytic lymphoma cell (U-937) produced only PAI-2. Five cell lines, including two that were previously shown to contain one or the other PAI (e.g., umbilical vein endothelial cells and a fibrosarcoma cell line, HT-1080) in fact contained both PAIs. The cells containing both PAIs were studied in more detail. In each case, SDS treatment of CM was shown to enhance PAI-1 activity (by converting the latent form of this inhibitor into its active form) and to destroy PAI-2 activity. Various compounds including interleukin 1, dexamethasone, and phorbol myristate acetate were found to selectively influence the cellular production of one PAI without concomitantly affecting the production of the other, suggesting that the synthesis of these inhibitors is not coordinately regulated.
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PMID:Detection of both type 1 and type 2 plasminogen activator inhibitors in human cells. 325 67

Human plasminogen activator inhibitor-1 (PAI-1) was purified from the conditioned medium of endotoxin-stimulated umbilical vein endothelial cell cultures by combinations of zinc-chelate-Sepharose chromatography, gel filtration on Sephacryl S-300 and immunoadsorption on an insolubilized murine monoclonal antibody (MA-7D4). The final product was obtained with a recovery of approximately 20% from conditioned medium containing about 3 micrograms/ml PAI-1. The yield of PAI-1 was 15-100 micrograms/umbilical cord, depending on the culture and harvest conditions. SDS gel electrophoresis revealed a main band with Mr = 46,000 both under reducing and non-reducing conditions. On gel filtration on Sephacryl S-300, however, the material was separated in two fractions, one eluting at the void volume, which contains active PAI-1, and one with Mr = 46,000 containing inactive material that could be reactivated with 12 M urea. SDS gel electrophoresis of the isolated high-Mr fraction revealed several bands including a main 46,000-Mr component, which reacted with anti-(PAI-1) antibodies on immunoblotting and neutralized tissue-type plasminogen activator (t-PA). The active high-Mr fraction and the reactivated low-Mr fraction of PAI-1 inhibited t-PA very rapidly with an apparent second-order rate constant of (1.5-4) x 10(7) M-1 s-1. The cDNA of endothelial cell PAI-1 was cloned and expressed in Chinese hamster ovary cells. The translation product, purified from conditioned medium of transfected cells, also revealed a high-Mr and a low-Mr fraction on gel filtration, which were indistinguishable from the natural proteins by physicochemical, immunochemical and functional analysis. On reduced SDS gel electrophoresis, the high-Mr fraction was separated into the Mr-46,000 low-Mr PAI-1 and two other components with Mr 65,000 and one barely entering the gel. When reactivated low-Mr PAI-1 was added to plasma, PAI activity and PAI-1 antigen eluted with an apparent Mr greater than or equal to 300,000 on gel filtration, indicating that active PAI-1 complexes with one or more binding proteins in plasma.
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PMID:Purification and characterization of natural and recombinant human plasminogen activator inhibitor-1 (PAI-1). 326 88

Ten human ovarian carcinoma cell lines (A121, A121[as], Caov-3, Caov-4, NIH:OVCAR-3, OVCA 420, OVCA 429, OVCA 432, OVCA 433, and SK-OV-3) were examined for secretion of plasminogen activators (PAs) using a chromogenic PA assay and SDS-PAGE zymography. PA activity was detected in conditioned media from all 10 cell lines. PA levels secreted by the 10 individual lines in a 24-hr period spanned a large range, with the extremes being 8 and 5244 milliPloug units (mPU)/10(6) cells for SK-OV-3 and OVCA 420 cells, respectively. Secreted PAs were identified as urokinase (UK)-like or tissue plasminogen activator (tPA)-like using dual criteria of comigration with UK or tPA standards on SDS-PAGE zymography and fibrin-dependence characteristics. Using both criteria, all 10 cell types produced UK-like activity, while tPA-like activity was produced by only 5 of the lines: A121[as], Caov-3, NIH:OVCAR-3, OVCA 429, and OVCA 433. Two additional cell lines produced PA activities that were tPA-like if judged by only one of the two criteria. Thus, Caov-4 cells produced a PA which comigrated with tPA, yet displayed no fibrin-dependent characteristics. Conversely, SK-OV-3 cells produced a fibrin-dependent PA, yet a band comigrating with tPA was not seen on SDS-PAGE zymography. Two lines derived from primary and ascitic sites from the same patient (A121 and A121[as], respectively) produced PAs with markedly different characteristics. Thus, PA produced by A121 cells was 100% UK-like, while that produced by A121[as] cells was greater than 90% tPA-like. Also, the total PA activity secreted by A121 cells was four times that secreted by A121[as] cells. In addition to bands comigrating with UK or tPA, all of the cell lines except Caov-3 and NIH:OVCAR-3 displayed higher molecular weight PA activities suggestive of the SDS-stable complexes between PAs and PA inhibitors reported in other cell types. While our results indicate that PA production may be a general characteristic of ovarian carcinoma cells in culture, individual patterns of UK and tPA production appear to be complex and vary from cell line to cell line. The precise characteristics of PA production in a given cell line may therefore depend on currently unidentified characteristics of the original tumor.
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PMID:Plasminogen activator secretion by established lines of human ovarian carcinoma cells in vitro. 341 Mar 41

Cultured human endothelial cells produce increased levels of PA-I when stimulated with IL-1. The stimulatory effect was elicited by both alpha (pI 5) and beta (pI 7) IL-1 molecules. The effect was both dose and time-dependent, plateau reached after 8 h. The PA-I measured in cell extracts was SDS-resistant with an apparent m.w. of 53 Kd and inhibited both tissue-type plasminogen activator (t-PA) and urokinase (UK). No change in t-PA antigen level was detected with any of the IL-1 preparations tested.
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PMID:Enhancement by interleukin-1 (IL-1) of plasminogen activator inhibitor (PA-I) activity in cultured human endothelial cells. 349 Feb 60

Human foreskin microvascular endothelial cells synthesize and release tissue-type plasminogen activator (t-PA) in similar amounts as do endothelial cells from umbilical cord artery and vein. Human thrombin increases the production of t-PA by these cells, which could be visualized from 8 h after addition of 0.1-5 units/ml thrombin by fibrin autography after SDS polyacrylamide gel electrophoresis of the endothelial cell conditioned media. Thrombin also increased the secretion of t-PA antigen. Together with t-PA, human microvascular cells release urokinase-type plasminogen activator (u-PA) antigen and endothelial cell-type PA inhibitor, PA inhibitor-1, which were both demonstrated by specific immunoprecipitation from radiolabeled endothelial cell conditioned medium. Thrombin increases the release of u-PA antigen, but no u-PA activity could be demonstrated. Thrombin induced a two-fold stimulation of the synthesis and secretion of PA inhibitor-1 antigen. At 0.1 unit/ml thrombin also an increase in PA inhibitor activity was found. At high concentrations of thrombin a decrease of PA inhibitor activity was found, due to the conversion of the active 46 kD PA inhibitor-1 into a 42 kD product without PA inhibitor activity. Our data indicate that interaction of thrombin with microvascular endothelial cells will shift the balance between t-PA, u-PA and PA inhibitor-1, and thus affects the regulation of fibrinolysis.
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PMID:Effect of thrombin on the production of plasminogen activators and PA inhibitor-1 by human foreskin microvascular endothelial cells. 349 79

We have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein. It encodes almost all of the protein B chain and part of the 3' untranslated region. We have used this clone to screen bacteriophage lambda and cosmid libraries of human genomic DNA. Several related genomic clones were isolated. One of these, a cosmid clone, carried approx. 40 kb of human DNA. Mapping experiments indicate that the region containing the protein-coding exons is approx. 20 kb in length. The cosmid, containing the t-PA gene and the aminoglycosyl-3'-phosphotransferase dominant-selection marker, was introduced into mouse L cells. Approximately half of the transformants were shown to produce human t-PA. We demonstrated that the fibrinolytic t-PA activity could be specifically quenched by anti-t-PA antibody and that the recombinant t-PA was of similar size (by SDS-polyacrylamide gel electrophoresis) to the t-PA produced by the human Bowes melanoma cell line. Our results suggest that the cosmid clone carries the whole t-PA coding region together with the regulatory elements necessary for its expression.
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PMID:Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells. 383 98


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