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

Neutrophils (PMN) are important in the cellular phase of blood fibrino lytic activity (FA). The contribution of monocytes (MC), which have FA, is unclear. To determine the relative roles of these cells to activity in normal blood, we examined, by solid phase radiofibrin assay, FA of normal blood and plasma, and of purified PMN and MC, with and without plasminogen (PLG), mini-plasminogen (mPLG), the other major elastase fragment of PLG, or autologous plasma. PMN alone (0.5 x 10(6)/ml) had striking activity (292 +/- 25 SEM ng fibrin lysed/h; n = 10 normal subjects) while MC alone (0.5 x 10(6)/ml) had mean FA of 32 +/- 4 ng/h, which could be accounted for by contaminating PMN (36 +/- 8 ng/h). Thus, in a 1 h assay (when cellular FA accounts for 70-80% of FA in whole blood), normal numbers of MC (0.5 x 10(6)/ml) had no detectable FA when assayed with PLG or normal plasma. With longer assay times (2-6 h), PLG-dependent (plasminogen activator, PA) activity was demonstrated with mixtures of MC and PLG or plasma. This PA activity was released into the medium and required prior contact of MC and an intact, soluble PLG molecule for PA activity to be detected in medium (suggesting a PLG-MC triggering mechanism), since activity was reduced or absent when MC were exposed to mPLG, the other major elastase fragment of PLG, or solid phase PLG. Exposure of MC to solid phase fibrin did not result in PA release. MC PA activity was little affected by cycloheximide pretreatment, indicating preformed rather than newly synthesized PA. By SDS-PAGE and fibrin zymography, MC extracts revealed a single PA band with features of pro-urokinase (single chain urinary-type PA): Mr 55,000, inhibition by antiurokinase antibody (but not by anti-tPA), and resistance to inhibition by DFP. By ELISA assay, approximate normal monocyte content of this PA (as Mr 55,000 urokinase) was 0.03 fg (3.3 x 10(8) molecules) per cell.
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PMID:Fibrinolytic activity of normal human blood monocytes. 292 5

A proteinase inhibitor has been isolated from human colorectal adenocarcinomas by extraction with a low-ionic-strength buffer and a combination of Con A-Sepharose, Sephadex G-200, DEAE-cellulose and chromatofocusing steps. The preparation appeared to be homogeneous upon gel exclusion chromatography and SDS-polyacrylamide gel electrophoresis and had an estimated molecular weight of 66,000. The inhibitor was able to bind and inhibit urokinase, plasmin, trypsin, tissue plasminogen activator and thrombin. The binding appeared to be stoichiometric and relatively fast. The isoelectric point of the protein was 4.6-4.7. The inhibitor did not crossreact with antisera elicited against alpha 2-macroglobulin, alpha 2-antiplasmin, antithrombin III or C1-inhibitor, but it did crossreact with an antiserum against alpha 1-antitrypsin in double immunodiffusion. The antiserum only partially attenuated the activity of the inhibitor. Whereas alpha 1-antitrypsin completely inhibited the amidolytic activity of elastase, the tumor inhibitor had no effect on elastase under the same conditions.
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PMID:Isolation and partial characterization of a proteinase inhibitor from human colorectal adenocarcinoma. 293 82

A hybrid human cDNA was constructed by splicing of a cDNA fragment of tissue-type plasminogen activator (t-PA), encoding 5'-untranslated, the pre-pro region and amino acids Ser1-Thr263, with a cDNA fragment of urokinase-type plasminogen activator (u-PA), encoding amino acids Leu144-Leu411. The cDNA fragments were obtained from full length t-PA cDNA, cloned from Bowes melanoma poly(A)+ mRNA, and from full length u-PA cDNA, cloned from CALU-3 lung adenocarcinoma poly(A)+ mRNA. The hybrid (t-PA/u-PA) cDNA was expressed in Chinese hamster ovary cells and the translation product purified from the conditioned cell culture media. On SDS-gel electrophoresis under reducing conditions, the protein migrated as a single band with approximate Mr 70,000. On immunoblotting, it reacted both with rabbit antisera raised against human t-PA and against human u-PA. The urokinase-like amidolytic activity of the protein was only 320 IU/mg but increased to 43,000 IU/mg after treatment with plasmin, which resulted in conversion of the single-chain molecule (t-PA/scu-PA) to a two-chain molecule (t-PA/tcu-PA). The specific activity of the protein on fibrin plates was 57,000 IU/mg by comparison with the International Reference Preparation for Urokinase. Both the single-chain hybrid (t-PA/scu-PA) and the two-chain plasmin derivative (t-PA/tcu-PA) bound specifically to fibrin, albeit more weakly than t-PA. The t-PA/tcu-PA hybrid had a higher selectivity for fibrin than tcu-PA, measured in a system composed of a whole human 125I-fibrin-labeled plasma clot immersed in human plasma. Both hybrid proteins activated plasminogen directly with Km = 1.5 microM and k2 = 0.0058 s-1 for t-PA/scu-PA and with Km = 80 microM and k2 = 5.6 s-1 for t-PA/tcu-PA. CNBr-digested fibrinogen stimulated the activation of plasminogen with t-PA/tcu-PA (Km = 0.20 microM and k2 = 1.2 s-1). It is concluded that these t-PA/u-PA hybrid proteins combine, at least to some extent, the fibrin-affinity of t-PA with the enzymatic properties of u-PA (either scu-PA or tcu-PA), which in some assays result in improved fibrin-mediated plasminogen activation.
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PMID:Characterization of a fusion protein consisting of amino acids 1 to 263 of tissue-type plasminogen activator and amino acids 144 to 411 of urokinase-type plasminogen activator. 295 60

Platelets were found to provide a surface for activation of plasminogen by the tissue-type plasminogen activator (t-PA) at an optimum concentration and to potentiate the generation of plasmin by the amidolytic method, fibrin lysis time and fibrin plate method. The effect of platelets on amidolytic activity on S-2251 was due to the potentiating effect of plasminogen activation by t-PA, because it was observed only in the presence of both plasminogen and t-PA. Plasmin generation was also evidenced in the SDS-PAGE profile of the supernatant from a mixture containing t-PA and plasminogen with platelets. These findings suggests that the potentiating activity of platelets on plasminogen activation by t-PA in circulation is one of the causes of fibrinogenolysis during fibrinolytic therapy with a high dose of t-PA. Platelets from patients with various diseases showed different potentiating activity on plasminogen activation by t-PA. The assay of this ability of platelets may be a new tool for evaluating their role in the blood fibrinolytic process.
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PMID:Plasminogen activation by tissue plasminogen activator in the presence of platelets. 297 48

Using affinity chromatography on lysine Sepharose 4B, a fast-acting tissue plasminogen activator inhibitor (t-PAI) was partially purified from t-PAI-rich plasma from patients with recurrent DVT. Its inhibition of tissue plasminogen activator (t-PA) was demonstrated in functional assays and its reaction with 125I-t-PA was analyzed by autoradiography following SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). When the t-PAI was mixed with an equimolar concentration of t-PA at 37 degrees C, the half-life of free one-chain and two-chain 125I-t-PA was 1.8 and 0.8 min, respectively. The rate of complex formation between 125I-t-PA and t-PAI was similar both in patient plasma, pregnancy plasma and platelet lysates made from platelet-rich normal, patient and pregnancy plasma. The molecular weights of the complexes between t-PA and the inhibitors in patient plasma and in the different platelet lysates were identical, while that of the inhibitor complex formed in pregnancy plasma was found slightly higher by SDS-PAGE indicating that the pregnancy plasma t-PAI differs from the fast-acting t-PAI found in plasma from thrombotic patients and in platelet lysates.
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PMID:Plasminogen activator inhibitors in plasma and platelets from patients with recurrent venous thrombosis and pregnant women. 308 15

Culture fluid of a monkey kidney cell culture was harvested every two days, for a two week period, in order to obtain urokinase in the zymogen form. Pro-urokinase was isolated by immunoadsorption chromatography and gel filtered on Sephadex G-150, which resulted in three peaks with pro-urokinase activity. SDS-polyacrylamide gel electrophoresis showed that the first peak contained 55 kd pro-urokinase, aggregated with high molecular weight contaminants, whereas the second and third peaks consisted of almost pure 55 kd and 30 kd pro-urokinase, respectively. The latter form represented a relatively unknown and inactive precursor of low molecular weight urokinase, which was, like 55 kd pro-urokinase, activatable with plasmin. In comparison with tissue-type plasminogen activator, 55 kd and 30 kd pro-urokinase only bound weakly to purified fibrin clots and fibrin-sepharose columns. The extent of binding of the two pro-urokinases and their plasmin-activated forms to fibrin-sepharose decreased in the following order: 55 kd pro-urokinase 30 kd pro-urokinase 55 kd urokinase 30 kd urokinase. These results indicate that the two precursors exhibited stronger binding to fibrin-sepharose than the corresponding active enzymes, and the two 55 kd forms exhibited stronger binding than the corresponding 30 kd forms. This indicates the importance of both the zymogen nature and an intact NH2-terminal part of the molecules for binding to fibrin.
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PMID:Characterization and fibrin-binding properties of different molecular forms of pro-urokinase from a monkey kidney cell culture. 308 54

A rapid and high-yield procedure for the purification of single polypeptide tissue-type plasminogen activator (t-PA) from porcine heart tissue has been developed. Delipidated heart tissue was extracted with 0.45 M potassium acetate. The extract was fractionated with ammonium sulfate and purified by a combination of affinity chromatography on heparin-Sepharose CL-6B and gel filtration on Toyopearl HW-55S. The final product had a specific activity of 220,000 IU/mg protein and gave a single protein band (apparent molecular weight; 67,000) in SDS-polyacrylamide gels in the presence or absence of a reducing agent. The increase in specific activity was 3,200-fold, most of which was achieved in the step of heparin-Sepharose chromatography. The yield calculated from the active ammonium sulfate precipitate was about 90% and 500 micrograms or more of the purified enzyme was obtained from 1 kg wet tissue. This procedure may also be useful for the large-scale production of highly purified t-PA from other tissues or tissue culture cells.
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PMID:Rapid and high-yield purification of porcine heart tissue-type plasminogen activator by heparin-sepharose choromatography. 309 93

alpha-Thrombin, DFP-thrombin, and ionophore A23187 induce the rapid release (less than 5 minutes) of a variety of proteins, including t-PA forms (Mr 110 and 70 k, after SDS-PAGE) from primary cultures, and both t-PA and u-PA (Mr 90 and 54 k) from subcultured human HUVECs. All PA activity forms are rapidly decreased in the releasates by some unknown mechanism. gamma-Thrombin does not induce the release of PAs from cultured HUVECs.
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PMID:Rapid release and deactivation of plasminogen activators in human endothelial cell cultures in the presence of thrombin and ionophore A23187. 309 27

Rat adrenal glands were stained immunocytochemically using antibodies against plasminogen activators of the tissue-type (t-PA) and urokinase-type (u-PA). A subpopulation of the cells in the adrenal medulla showed intense cytoplasmic t-PA immunoreactivity, while no u-PA immunoreactivity was detected in any adrenal cells. Fluorescence microscopy of adjacent sections demonstrated that the cells stained for t-PA contained noradrenaline. Analysis with a histochemical fibrin slide technique demonstrated a plasminogen-dependent fibrinolysis in the adrenal medulla. SDS-PAGE of adrenal gland extracts followed by zymography established the molecular weight of this plasminogen activator to be similar to that of rat t-PA. In addition SDS-PAGE followed by immunoblotting with anti-t-PA IgG of adrenal gland extracts revealed one band with an electrophoretic mobility indistinguishable from that found in the zymography. When tissue-sections and immunoblots were incubated with antibodies absorbed with highly purified t-PA no staining was found. In view of the previous finding of t-PA in growth hormone-containing cells of the pituitary gland, these findings substantiate that t-PA can be found in the intact normal organism outside endothelial cells, and further point to t-PA having a function in endocrine cells.
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PMID:Tissue-type plasminogen activator in rat adrenal medulla. 309 16

Recombinant tissue-type plasminogen activator (rt-PA) from cultures of a genetically manipulated Bowes melanoma cell line (TRBM6) was purified in batches of average volume 451 using an autoclavable, reusable, continuous chromatography system comprising zinc chelate-Sepharose CL4B and lysine-Sepharose CL4B. After eight successive purifications the rt-PA was ultrafiltered to yield a preparation containing 4.9 mg protein/ml and 2.7 X 10(6) IU/ml. Analysis by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue R250 showed major protein bands at Mr = 63,000 and 65,000; most of the material was in the 1-chain form. The potential usefulness of a simple, rapid continuous chromatography system that can be operated under aseptic conditions is discussed.
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PMID:Large scale, rapid purification of recombinant tissue-type plasminogen activator. 310 Mar 25


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