Production by Human Tissues in Culture of Immunologically Distinct, Multiple Molecular Weight Forms of Plasminogen Activators*

Bernik, Maria B.; Wijngaards, G.; Rijken, D.C.

doi:10.1111/j.1749-6632.1981.tb29766.x

Cited by 26 publications

(5 citation statements)

References 19 publications

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“…This finding is in good agreement with recent studies from this laboratory showing that virus-transformed murine cells release a plasminogen activator with an approximate Mr of 48 000 in a zymogen form (Skriver et al, 1982). The existence of a zymogen to a human plasminogen activator of the urokinase type has previously been proposed by Bernik et al (1974Bernik et al ( , 1981 and Nolan et al (1977), who found that the plasminogen-activating activity in culture fluid from human cells was enhanced by incubation with trypsin. In those studies, purified enzyme was not available in sufficient amounts to prove the existence of a proactivator.…”

Section: Discussionsupporting

confidence: 92%

Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatography with monoclonal antibody

Nielsen¹,

Hansen²,

Skriver³

et al. 1982

Biochemistry

261

117

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Incorporation of the serine protease active site reagent diisopropyl fluorophosphate (DFP) into a plasminogen activator with an Mr of approximately 52000 released from cultured human glioblastoma cells was strongly enhanced by incubation with plasmin. This observation led to the isolation of an inactive form of the enzyme from serum-free conditioned culture fluid by affinity chromatography on a column of a Sepharose-bound monoclonal antibody raised against urokinase. An 831-fold purification was obtained with a yield of 41%. The purified molecule was homogeneous as evaluated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (NaDodSO4), having one stainable band under nonreducing as well as reducing conditions with an Mr of approximately 52000. It was unable to activate plasminogen, but catalytic amounts of plasmin converted it into active enzyme. After NaDodSO4-polyacrylamide gel electrophoresis, the active enzyme showed one band under nonreducing conditions, but after reduction, two bands with Mr values of approximately 20000 and 32000 were observed. The active enzyme incorporated [3H]DFP into the approximately Mr 32000 band, while no incorporation was observed into the inactive form. These findings show that the Mr 52000 human plasminogen activator exists in a proenzyme form consisting of a single polypeptide chain that by proteolysis between half-cystine residues is converted into the active enzyme consisting of two chains with molecular weights of approximately 20000 and 32000, the active site being on the latter chain. The results are consistent with the active form of the enzyme being identical with the higher molecular weight form of urokinase, and together with recent observations that a murine plasminogen activator is released from sarcoma virus transformed cells as an inactive proenzyme, they suggest that zymogens to plasminogen activators are of more general occurrence.

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Section: Discussionsupporting

confidence: 92%

Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatography with monoclonal antibody

Nielsen¹,

Hansen²,

Skriver³

et al. 1982

Biochemistry

261

117

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“…In addition to EPA, several animal and human tissues in culture appear to produce also another type of plasminogen activator related to urokinase [6]. Furthermore, various plasminogen activators and proactivators (intrinsic and extrinsic) have been identified in blood [I I].…”

mentioning

confidence: 99%

Enhanced fibrinolytic activity during cardiopulmonary bypass in open‐heart surgery in man is caused by extrinsic (tissue‐type) plasminogen activator

Stibbe¹,

Kluft²,

Brommer³

et al. 1984

Eur J Clin Investigation

157

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The nature of the enhanced blood fibrinolytic activity which is known to occur during cardiopulmonary bypass is not understood. We show here that the cause is an increase in extrinsic (tissue-type) plasminogen activator. In six patients, the nature of the enhanced blood fibrinolytic activity that evolved during cardiopulmonary bypass was characterized by differential inhibition using the fibrin plate method and was shown to be C1-inactivator-resistant (extrinsic-activator activity). The C1-inactivator-resistant-activator activity was completely quenched by an antibody against extrinsic (tissue-type) plasminogen activator but not by antiurokinase, proving that the activity was due to the presence of extrinsic (tissue-type) plasminogen activator. The concentration of extrinsic (tissue-type) plasminogen activator increased during cardiopulmonary bypass and disappeared rapidly thereafter. Fibrinogen, plasminogen and alpha 2-antiplasmin were not consumed during cardiopulmonary bypass, while no increase or occasionally a moderate one in fibrinogen degradation products occurred. This is in accord with the property of extrinsic (tissue-type) plasminogen activator which activates plasminogen predominantly at sites where fibrin is present and not in the free circulation.

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“…The observation of an increased cellulax synthesis of PAS as an early rnanifestatioii of the transformation of certain cells by oncogeiiic virus (Unkeless et al, 1973;Jones et al, 1975) and of higher fibrinolytic activity in extracts of primary tumors compared to those of normal tissues (Nagy et al, 1977), have prompted several investigators to study PAS in malignancy. Many tumor cell lines (Astedt and Holmberg, 1976;Hisazumi et al, 1977;Wu et al, 1977;Tucker et al, 1978;Vetterlein et al, 1979Vetterlein et al, , 1980Wu and Yunis, 1979;Wilson et al, 1980;Bernik et al, 1981;Wun et al, 1982b) or crude extracts of carcinoma of the lung , colon or ptostate (Camiolo etal., 1981) were shown to contain UK-related PAS (u-PA) and/or PAS immunologically unrelated to UK. Recently Evers et al (1982) have demonstrated t-PA-related activity in normal and malignant breast tissues.…”

mentioning

confidence: 99%

Characterization of plasminogen activators from normal human breast and colon and from breast and colon carcinomas

Tissot

Hauert

Bachmann

1984

Intl Journal of Cancer

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Triton X-100 and NaSCN extracts of 18 normal breast and colon tissues and of 20 breast and colon carcinomas were fractionated by SDS-PAGE and plasminogen activators (PA) revealed by a zymographic method. Four different lysis bands, corresponding to MWs of 54,000, 68,000, 95,000 and 110,000 were observed. Using immunoadsorption with specific antisera against urokinase (UK) and tissue PA (t-PA), we found that all normal tissue extracts contained free t-PA (68 kd). Some of these revealed, in addition, a complex (110 kd) of t-PA with a 40-kd component. The latter presumably represents the fast-acting specific inhibitor of t-PA and UK. Most carcinoma extracts contained, in addition to the two t-PA-related lysis bands, the UK-related 54 kd PA, and some a 95 kd complex of UK with the 40 kd component. For each extractant, mean total fibrinolytic activity of normal and tumor tissue was comparable when measured on conventional fibrin plates, but breast and colon carcinomas contained higher concentrations of UK-related PA. PA activity was higher in normal and carcinoma NaSCN extracts than in the corresponding Triton X-100 extracts. In general, Triton X-100 but not NaSCN extracts of malignant tissue contained a high concentration of fibrinolytic inhibitors. Mixing experiments revealed that the inhibitory activity was mainly directed against UK. It was abolished by acidification of the carcinoma extracts. The anti-UK inhibitory activity was absent in extracts of normal breast or colon and appears to be different from the 40 kd fast-acting PA inhibitor. These studies show that malignant transformation of breast and colon is accompanied by important changes of the production of a UK-related PA and of an inhibitory activity directed against UK.

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Production by Human Tissues in Culture of Immunologically Distinct, Multiple Molecular Weight Forms of Plasminogen Activators*

Cited by 26 publications

References 19 publications

Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatography with monoclonal antibody

Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatography with monoclonal antibody

Enhanced fibrinolytic activity during cardiopulmonary bypass in open‐heart surgery in man is caused by extrinsic (tissue‐type) plasminogen activator

Characterization of plasminogen activators from normal human breast and colon and from breast and colon carcinomas

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