Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new fluorogenic peptide substrate for plasmin, 7-(N-succinoylalanylphenylalanyl-lysylamido)-4-methylcoumarin trifluoroacetate salt, was prepared that can be used in a simple and direct assay. The results obtained by the assay method are linear over a wide range of enzyme concentrations and sensitive enough to detect as little as 10(-5) CTA units of plasmin. By making use of the inhibitor Trasylol and the differences in kinetic constants, plasmin can be specifically assayed even in the presence of the plasminogen activator thrombin, as well as in culture fluids from HeLa cells.
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PMID:A new fluorogenic substrate for plasmin. 16 7

The question whether single-chain urokinase-type plasminogen activator (Sc-uPA) possesses an enzymatic activity has been a subject of intense investigation for a number of years but still remains unresolved. Recent studies from several laboratories suggest that Sc-uPA or its plasmin-resistant mutants obtained by site-directed mutagenesis possess significant, albeit low, amidolytic and plasminogen activator activities, ranging from 0.1% to 1% of that observed for two-chain urokinase (Tc-uPA). In an effort to characterize these putative intrinsic activities, Sc-uPA was repeatedly treated with dansyl-Glu-Gly-Arg chloromethyl ketone (dansyl-EGRck) or diisopropyl fluorophosphate (DFP) (0.1-0.25 mM added thrice over a period of 24 h at 0 degrees C). This treatment exhaustively inactivated the Tc-uPA contaminant but did not affect Sc-uPA, as evidenced by the lack of significant incorporation of radiolabeled inhibitor in Sc-uPA and full activation of the inhibitor-treated Sc-uPA by plasmin. Assayed in the presence of excess DFP or dansyl-EGRck to ensure trapping of any Tc-uPA generated in the assay mixture, Sc-uPA (84 micrograms/mL, 10,500 latent units/mL) did not elicit any detectable cleavage of the chromogenic substrate S-2444 (detection limit 0.1 unit of Tc-uPA/mL). However, if the Tc-uPA inhibitors were removed prior to assay, a trace amount of amidolytic activity invariably reappeared in the Sc-uPA preparation. Incorporation experiments with [3H]DFP suggested that the appearance of this amidolytic activity was due to formation of Tc-uPA. Plasminogen activator assay of DFP- and dansyl-EGRck-treated Sc-uPA (0.45-2.25 microM), performed in the presence of these inhibitors and Trasylol (10 microM) to ensure entrapment of any Tc-uPA or plasmin generated in the reaction mixture, showed no significant cleavage of 125I-labeled plasminogen (detection limit 0.1 nM). However, if dansyl-EGRck and DFP were removed from the inhibitor-treated Sc-uPA and the assay was performed in the presence of Trasylol alone, there was significant cleavage of 125I-plasminogen due to contamination by Tc-uPA. Fibrin, a positive effector of plasminogen activation by Tc-uPA or Sc-uPA preparations in the absence of DFP and dansyl-EGRck, did not promote cleavage of plasminogen or S-2444 by Sc-uPA in the presence of the Tc-uPA inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Single-chain urokinase-type plasminogen activator does not possess measurable intrinsic amidolytic or plasminogen activator activities. 182 71

Activity of peritoneal plasminogen activator and its regulation by dextran and other macromolecules that clinically suppress postoperative adhesions was studied. Plasminogen activator activity was assayed by a two-stage globinolytic assay that monitors formation of plasmin, as well as by cleavage of a chromogenic peptide substrate (S-2444) in the presence of aprotinin (Trasylol). Plasminogen activator activity was located on the outer surface of human peritoneum. Incubation of peritoneal tissue with buffer in vitro (conditioning) prompted release of plasminogen activator into the conditioning medium. The released plasminogen activator formed a single band on sodium dodecyl sulfate-gel electrophoresis at an apparent molecular weight of 174,000 and was markedly suppressed by antiserum raised against human melanoma tissue-type plasminogen activator. Nonspecific proteolytic activity did not accumulate in the medium during conditioning. The presence of dextran 80 during conditioning of peritoneum reversibly suppressed tissue-bound plasminogen activator activity and reduced plasminogen activator activity in the spent medium. A similar inhibition of peritoneal plasminogen activator was induced by dextran 500, methyl cellulose, and polyvinylpyrrolidone. Dextran, when added to the medium after conditioning, had no direct inhibitory effect on plasminogen activator activity. Dextran did not induce peritoneal production of inhibitor(s) of trypsin, chymotrypsin, or urokinase. On the basis of these findings, two possible mechanisms for the effect of viscous polymers in the reduction of adhesion formation are proposed. These mechanisms consider the importance of peritoneal tissue-type plasminogen activator for removal of fibrin clots and suggest that polymer coating either prevents the shedding of plasminogen activator into the abdominal cavity or reduces the access of fibrin clots to the serosal surfaces.
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PMID:Effect of viscous macromolecules on peritoneal plasminogen activator activity: a potential mechanism for their ability to reduce postoperative adhesion formation. 245 68

In five patients with venous thromboembolic disease treated with recombinant tissue-type plasminogen activator (rt-PA), there was a marked increase in the mean concentrations of fibrinopeptide A (from 0.6 to 5.9 nM; P less than 0.0001) and desarginine fibrinopeptide B (from 5.6 nM to 24.1 nM; P less than 0.01) 30 min after a bolus of rt-PA (0.6 mg/kg). Thrombin was unlikely to be responsible because the levels of desarginine fibrinopeptide B exceeded those of fibrinopeptide A and the changes occurred despite concomitant heparin therapy. The purpose of this study therefore, was to determine whether rt-PA directly releases the fibrinopeptides from fibrinogen. Incubation of rt-PA with heparinized plasma or purified fibrinogen resulted in time and dose-dependent release of both fibrinopeptide A and B. Contaminating thrombin was not responsible for this activity by the following criteria: the rate of rt-PA mediated fibrinopeptide B release was considerably faster than that of fibrinopeptide A, and fibrinopeptide release was unaffected by heparin, hirudin, or a monospecific antithrombin IgG. Aprotinin also had no effect on fibrinopeptide release, indicating that this activity was not plasmin mediated. Fibrinopeptide release was shown to be due to rt-PA because this activity was completely blocked by a monoclonal antibody against the enzyme. Further, the specificity of rt-PA for the thrombin cleavage sites on fibrinogen was confirmed by the demonstration that rt-PA released fibrinopeptide A or fibrinopeptide B from fibrinopeptide A or B-containing substrates, respectively. These studies thus demonstrate that (a) rt-PA releases fibrinopeptides A and B from fibrinogen thereby indicating that this enzyme is not specific for plasminogen, and (b) plasma fibrinopeptide A and desarginine fibrinopeptide B levels are not specific markers of thrombin action on fibrinogen in patients receiving rt-PA.
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PMID:Human tissue-type plasminogen activator releases fibrinopeptides A and B from fibrinogen. 314 81

Confluent cultures of bovine aortic endothelial cells (BEC) were found to secrete both tissue type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA). Exposure of the cultures to increasing concentrations of dexamethasone resulted in a time and concentration dependent inhibition of cellular and secreted u-PA. Complete inhibition of u-PA production and secretion was found at dexamethasone concentrations of 10(-8) molar or higher. Several distinct PA forms with molecular weights ranging from 10,000-20,000 to greater than 200,000 were found in the conditioned medium of untreated BEC cultures. After addition of dexamethasone (10(-7) molar) to the culture medium the PAs with molecular weights of 117,000, 58,000, 47,000, were absent suggesting that they were u-PAs, whereas the PAs with molecular weights of greater than 200,000 and 75,000 remained unchanged suggesting their t-PA origins. The PAs with lower molecular weights of 35,000, 28,000 and 10,000 to 20,000 were most likely generated from the higher molecular weight forms by limited proteolysis since they were absent when the medium was conditioned in the presence of the protease inhibitor Trasylol. The two PA types may therefore be independently regulated in BEC.
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PMID:Glucocorticoids inhibit plasminogen activator production by endothelial cells. 642 Sep 27

Line 10 guinea pig carcinoma cells cultured in serum-free medium for 4 hr elaborate plasminogen activator (PA) activity that remained in the supernatant after ultracentrifugation (100,000 X g, 90 min). PA activity in line 10 conditioned medium occurred in both active and cryptic forms. The vast majority of active PA adsorbed to lysine-Sepharose and could be eluted at low pH as several activities that electrophoresed in the Mr 50,000 to 80,000 range on nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A small amount of active PA, running in the Mr 50,000 to 60,000 region, and cryptic PA did not adhere to lysine-Sepharose. Treatment of lysine-Sepharose-nonadherent fractions with catalytic amounts of plasmin or trypsin induced substantial new PA activity that adsorbed to lysine-Sepharose, bound [3H]diisopropylfluorophosphate, and that electrophoresed as several bands of activity with molecular weights from 50,000 to greater than 100,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of additional interest, the amount of active PA measured in conditioned medium was substantially increased when certain protease inhibitors, tranexamic acid, epsilon-aminocaproic acid, or Trasylol, were included during culture.
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PMID:Cryptic and active plasminogen activators secreted by line 10 tumor cells in culture. 668 8

The plasminogen activator secreted by a cultured human melanoma cell line was purified and compared with urokinase and with tissue plasminogen activator from human uterus. The purification procedure consisted of chromatography on zinc chelate-agarose, concanavalin A-agarose, and Sephadex G-150 in the presence of 0.01% (v/v) Tween 80. The purified material was obtained from the culture medium with a yield of 46% and a purification factor of 263. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one main band with a molecular weight of about 72,000, and in the presence of reducing agents, two bands of 33,000 and 39,000. Addition of the protease inhibitor Aprotinin to the culture media and column buffers yielded a one-chain plasminogen activator with a molecular weight of about 72,000. One molecule of activator reacted with about one molecular of [3H]diisopropylfluorophosphate. The melanoma plasminogen activator and the uterine tissue plasminogen activator appeared to be very similar on dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis, and amidolytic properties. Both activators bound to fibrin clots, while urokinase did not. In immunodiffusion, as well as in quenching experiments of the fibrinolytic activities, the melanoma plasminogen activator appeared to be immunologically identical with the uterine tissue plasminogen activator, but unrelated to urokinase. All these findings indicate that the plasminogen activator secreted by human melanoma cells in culture is very similar to, or identical with, the plasminogen activator found in normal tissue, but different from urokinase.
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PMID:Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. 678 58

Clonal mouse skeletal muscle cells which differentiate in culture and form synapses with neuronal cells were found to secrete high levels of protease activity as measured with an 125I-fibrin assay. The secreted proteolytic activity was more than 90% dependent upon the presence of plasminogen in the medium, and had a pH optimum at 7 to 8. This activity was not inhibited by n-ethylmaleimide, pepstatin, EDTA, or EGTA. At millimolar concentrations, greater than 90% inhibition was obtained with either soybean trypsin inhibitor, epsilon aminocaproic acid, Trasylol, or leupeptin. Almost complete inhibition occurred with 1 mM diisopropylfluorophosphate suggesting the presence of a serine residue at the catalytic site. In contrast to the high levels of secreted activity, a lower steady-state level of cell-associated protease activity was detected in cell lysates. The high level of plasminogen activator secreted into the medium of cultured muscle cells suggests a role for such extracellular protease activity in myogenesis during development and remodeling following muscle injury. Such information may be useful in understanding the initial degeneration of neuromuscular contacts in experimental and pathologic denervation.
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PMID:Plasminogen activator: the major secreted neutral protease of cultured skeletal muscle cells. 704 Apr 26

The plasminogen activator secreted by a cultured rat brain tumor cell line (RT4-71-1) (Imada M. and Sueoka N., Develop. Biol. 69, 97-107, 1978) was purified by chromatography on zinc chelate-agarose, concanavalin A-agarose and Sephadex G-150 in the presence of 0.01% (vol/vol) Tween 80. Aprotinin was added to the culture medium to a concentration of 20 KIU per ml and to the buffers in the first two chromatographic steps to a concentration of 10 KIU per ml. Approximately 90 microgram purified material was obtained from 11 of culture medium with a yield of 39% and a purification factor of 200. Sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence of reducing agents showed one main band with Mr of about 60,000, and a minor band with Mr about 30,000. Fibrinolytic activity was associated with the main band. The rat brain tumor plasminogen activator bound to a fibrin clot to a similar extent as human tissue plasminogen activator, whereas urokinase did not bind. In quenching experiments of the fibrinolytic activities the purified rat brain tumor plasminogen activator appeared to be immunologically related to the human tissue plasminogen activator but unrelated to urokinase.
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PMID:Purification and characterization of the plasminogen activator secreted by a rat brain tumor cell line in culture. 719 1

Hyperfibrinolysis during orthotopic liver transplantation (OLT) has been attributed to high plasma levels of tissue plasminogen activator (t-PA). This study investigated the contribution of urokinase plasminogen activator (u-PA) to hyperfibrinolysis and the effects of high-dose perioperative aprotinin (Trasylol) on fibrinolytic activation. Plasma samples were collected before, during, and after OLT in fifty five patients receiving either high dose aprotinin or placebo in a randomized double-blind trial. t-PA antigen and u-PA antigen and activity levels were increased preoperatively compared with normal controls (P < 0.05). Hyperfibrinolysis was seen during the anhepatic phase as shown by shortened euglobulin clot lysis times (ECLT) and an increase in D-dimer titers. t-PA levels peaked on reperfusion and fell at the end of the operation, and u-PA levels did not increase during OLT, but showed a decrease at the end of the operation. With aprotinin treatment, t-PA levels were lower on graft reperfusion than the placebo group (P < 0.05), but there was no difference in u-PA antigen or activity levels between groups. Fibrinolytic inhibition during OLT by aprotinin was demonstrated by prolonged ECLT (P < 0.05), reduced D-dimer levels (P < 0.05), and an increase in antiplasmin activity (P < 0.05). This study showed that the main antifibrinolytic action of aprotinin is as an antiplasmin agent with some effect on t-PA-but not u-PA-mediated fibrinolysis.
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PMID:Fibrinolytic activity during orthotopic liver transplantation with and without aprotinin. 752 49


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