Search PubMed⌕ Search

Biomedical subjects

D Collen

Publications and source records attributed to D Collen.

At least 613 records · Page 34Linked to original sources
PubMed1982

Messenger RNA for human tissue plasminogen activator.

A human melanoma cell line (Bowes), which secretes extrinsic (tissue-type) plasminogen activator, was used as a source for the preparation of mRNA for extrinsic plasminogen activator. The cells were lysed and total RNA was extracted with the phenol method. A poly(A)-rich RNA fraction was isolated by affinity chromatography on oligo(dT)-cellulose. This preparation was translated by oocytes into (a) protein(s) that had biological activity in the assay for extrinsic plasminogen activator. On sucrose gradient centrifugation the translatable fraction of the RNA migrated with a sedimentation coefficient of approximately 19 S, a value compatible with the molecular weight of extrinsic plasminogen activator (70 000). The translation product was characterized as being similar to or identical with authentic extrinsic plasminogen activator by the following criteria: (a) serological cross-reactivity with purified extrinsic plasminogen activator in neutralization and immunoprecipitation reactions; (b) plasminogen dependency of fibrinolytic activity and (c) apparent molecular weight of 70 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis.

Animals↗
PubMed1982

Thrombolysis with human extrinsic (tissue-type) plasminogen activator in dogs with femoral vein thrombosis.

Extrinsic (tissue-type) plasminogen activator (plasminogen activator) was isolated either as a single-chain or as a two-chain molecule from the culture medium of a human melanoma cell line. The thrombolytic activity of both molecular forms of activator was investigated in beagle dogs with an experimental femoral vein thrombosis and compared with that of urokinase. The 125I-fibrinogen-labeled thrombus was formed in an isolated 4-cm segment of the vein, aged for 30 min, and the thrombolytic substances were infused over a 4-h period. The degree of thrombolysis was measured 2 h later as the difference between the injected and recovered 125I. In six control animals with a saline infusion the extent of thrombolysis was 16.3 +/- 3.8% (mean +/- SEM), in five dogs receiving 100,000 IU urokinase, 17.4 +/- 3.7% (P less than 0.4) and in four dogs with 1,000,000 IU urokinase 40.6 +/- 4.8% (P less than 0.001). Infusion of 100.000 IU single-chain plasminogen activator in five dogs resulted in 3.5 +/- 7.8% lysis (P less than 0.05) and of 100,000 IU two-chain plasminogen activator in five dogs in 60.1 +/- 10.8% (P less than 0.001). Infusion of 300,000 IU one-chain plasminogen activator yielded 57.5% lysis and of the same amount of two-chain plasminogen activator 72.9%. Significant activation of plasminogen, consumption of alpha 2-antiplasmin, and fibrinogen breakdown in plasma was only observed in animals receiving the high doses of urokinase but not in the saline, plasminogen activator, or the low-dose urokinase groups. It is thus concluded that in this thrombosis model human extrinsic plasminogen activator has a higher specific thrombolytic effect that urokinase. Plasminogen activator also appears to induce thrombolysis without systemic fibrinolytic activation and fibrinogen breakdown.

Animals↗
PubMed1981

Studies on the specific fibrinolytic effect of human extrinsic (tissue-type) plasminogen activator in human blood and in various animal species in vitro.

Human extrinsic (tissue-type) plasminogen activator (EPA) was highly purified from the culture fluid of a human melanoma cell line, both as a one-chain or as a two-chain molecule. Its specific fibrinolytic effect on human whole blood clots or plasma clots with different degrees of fibrin crosslinking was evaluated in an in vitro system, composed of a 125I-fibrin labeled clot, hanging in circulating human plasma. After infusion of EPA (30 IU per ml over 3 hrs), non-crosslinked clots lysed more extensively (75-100 percent in 5 hrs) than totally-crosslinked clots (50-65 percent), and no difference was found between one-chain or two-chain EPA. The extent of lysis of totally-crosslinked human or animal plasma clots hanging in autologous plasma induced by EPA varied markedly form one species ot the other. When 90 IU of EPA were infused over 3 hrs, crosslinked human plasma clots dissolved for over 95 percent within 5 hrs. Under comparable conditions, the degree of lysis was 80 percent in primate plasma (cynomolgus fascicularis), 60 percent in cat and rabbit plasma, 30 percent in dog plasma and only 10 percent in rat plasma. Systemic activation of the fibrinolytic system in the circulating plasmas was minor and dose-dependent in all species, but complete fibrinogen breakdown was not observed in any species following infusion of up to 90 IU EPA per ml plasma. It is concluded that the human system is more susceptible to EPA induced fibrinolysis than the other animal systems which were investigated, and that even totally-crosslinked clots can be lysed after infusion of EPA.

Animals↗
PubMed1981

Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture.

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.

Amino Acids↗
PubMed1981

Comparison of the relative fibrinogenolytic, fibrinolytic and thrombolytic properties of tissue plasminogen activator and urokinase in vitro.

The relative fibrinogenolytic, fibrinolytic and thrombolytic properties of human tissue plasminogen activator and human urokinase were compared in purified systems, in whole human plasma and in a system composed of a radioactive human blood clot (125I-fibrinogen) hanging in circulating human plasma. The human tissue plasminogen activator was highly purified from the culture fluid of a human melanoma cell line. In purified systems composed of fibrinogen of fibrin, plasminogen and alpha 2-antiplasmin as well as in whole plasma, tissue plasminogen activator digested fibrin without degrading fibrinogen significantly. Urokinase did not have this specific fibrinolytic effect. In the circulating plasma system, the degree of fibrinolysis was proportional to the amount of activator added, tissue plasminogen activator being about 10 times more efficient than urokinase. In addition, tissue plasminogen activator appeared to cause negligible fibrinogen degradation. Tissue plasminogen activator still induced significant thrombolysis at a concentration of 10 IU per ml whereas no effect of urokinase was observed at 20 IU per ml. Infusion of 100 IU (1 microgram) of tissue plasminogen activator per ml resulted in moderate activation of the fibrinolytic system as judged from a decrease of plasminogen and alpha 2-antiplasmin to 40-50 percent. Nevertheless, extensive fibrinolysis (50 to 80 percent of radioactivity released after 12 hrs) and only very limited fibrinogenolysis were observed. An equivalent amount of urokinase (100 IU per ml) only induced approximately 15 percent lysis in 12 hrs. At higher concentrations of urokinase (260 IU per ml or more) extensive activation of the fibrinolytic system was obtained as evidenced by a depletion of plasminogen, alpha 2-antiplasmin and fibrinogen. This was associated with extensive fibrinolysis (approximately 60 percent after 12 hrs). It is concluded that human tissue plasminogen activator is a more specific and effective fibrinolytic-thrombolytic agent than human urokinase.

Endopeptidases↗
PubMed1981

Neutralization of human extrinsic (tissue-type) plasminogen activator in human plasma: no evidence for a specific inhibitor.

Human extrinsic plasminogen activator (EPA), highly purified from a melanoma cell culture fluid is inactivated in human plasma with a half-life (t 1/2) of 90-105 min. Gel filtration on Ultrogel AcA 34 of mixtures of 125I-labeled EPA and human plasma, incubated at 37 degrees C, revealed the progressive formation of two radioactive components, one with an apparent Mr of 150,000 and one eluting at the void volume. The component with an Mr of 150,000 was identified as consisting at least in part of EPA-alpha 2-antiplasmin complex since: 1) it reacted with antibodies against alpha 2-antiplasmin, but not with antibodies against the other known plasma protease inhibitors, and 2) formation of this component was strongly reduced in plasma specifically depleted in alpha 2-antiplasmin or when the active site of EPA was blocked. The component eluting at the void volume was identified as consisting at least in part of EPA-alpha 2-macroglobulin complex since: 1) it only reacted with antibodies against these two proteins and 2) was not formed in plasma depleted in alpha 2-macroglobulin or when the active site of EPA was blocked. In purified systems alpha 2-antiplasmin inhibited one-chain EPA with a rate constant of 60 M-1s-1 and two-chain EPA with a rate constant of 130 M-1s-1, which corresponds to a t 1/2 in plasma of 180 min or 90 min, respectively. alpha 2-Macroglobulin inhibited one-chain EPA with a rate constant of 15 M-1s-1 and two-chain EPA with a rate constant of 30 M-1s-1, which corresponds to a t 1/2 in plasma of 4 or 2 hrs. All these findings taken together indicate that EPA is slowly neutralized in human plasma primarily by alpha 2-antiplasmin and to a lesser extent by alpha 2-macroglobulin. There appears to be no specific inhibitor in human plasma, which would inactivate EPA either rapidly or to a significant extent.

Chromatography, Gel↗