[Direct activator effect of streptokinase on plasminogen. Studies on plasminogen activation in relation to the concentration of urokinase and streptokinase].
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Animals↗
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Plasminogen-fibrinogen complex formation as a prelude to fibrinogenolysis. A density-gradient ultracentrifugation study of radioiodinated systems involving urokinase, plasminogen, and fibrinogen.
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Animals↗
[Contribution to the study of plasminogen. II. Purification of human plasminogen by exclusion chromatography on Shepadex G-200].
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Blood Proteins↗
[Contribution to the study of plasminogen. I. Purification of bovine plasminogen].
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Animals↗
Preparation of human plasminogen suitable as substrate for quantitative determination of plasminogen activators.
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Chromatography, Gel↗
[Recombinant murine cell lines, transformed by various vectors based on bovine type 1 papillomavirus and expressing human tissue plasminogen activator. II. Analysis of cell lines, producing recombinant tissue plasminogen activator].
We have characterized a number of recombinant cell lines established with BPV1 and Lx1 (containing duplication of LCR-E6-E7 sequence) vectors on the basis of C127 cells. It had been shown that Lx1 based vectors possess the higher number of intracellular copies than analogous vectors on the basis of wtBPV, and most part of them is integrated into the host genome. Using various concentrations of heavy metal salts we have developed the optimized procedure for induction of recombinant tPA synthesis which is controlled by the mouse MT1 promoter. A 8-fold increase of rtPA concentration was reached in the course of induction. It had been shown that native and non-glucosylated forms of recombinant and human tPA are identical in their properties.
Animals↗
A basic study on theglobal coagulation and fibrinolysis of hyperlipaemic and atherosclerotic patients.2. Plasminogen activator, plasminogen, a2-macroglobulin in atherosclerosis and endogenous hypertriglyceridaemia.
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Adult↗
Interaction of staphylokinase with different molecular forms of plasminogen.
In order to obtain more information on the mechanism of plasminogen activation by staphylokinase (STA), we have studied the interaction between recombinant STA (STAR) and different molecular forms of human plasminogen, including Glu-plasminogen (native moiety), Lys-plasminogen (partially degraded moiety) and low-molecular-mass (LMM) plasminogen (moiety lacking kringles 1-4). Addition of 2 microM STAR to 0.4 microM Glu-plasminogen, Lys-plasminogen or LMM plasminogen resulted in the generation of proteolytic activity towards the chromogenic substrate D-Val-Leu-Lys-NH-PhNO2 (S-2251) corresponding to the exposure of 1 active center/plasminogen molecule. Complex formation was associated with conversion of the one-chain plasminogen moieties to two-chain plasmin, and with quantitative conversion of Glu-plasminogen to Lys-plasmin. The stoichiometry of the plasminogen-STAR complex, determined by binding of the complex to Lys-Sepharose and measurement of residual STAR, was found to be equimolar. The plasminogen-STAR complexes were inhibited by alpha 2-antiplasmin with second-order rate constants of 2.4 +/- 0.17 x 10(6) M-1 s-1 for Glu-plasminogen, 2.4 +/- 0.21 x 10(6) M-1 s-1 for Lys-plasminogen and 9.4 +/- 1.5 x 10(4) M-1 s-1 for LMM plasminogen. Glu-plasmin-STAR, Lys-plasmin-STAR and LMM plasmin-STAR had comparable catalytic efficiencies (kcat/Km) for the activation of Glu-plasminogen (0.24-0.29 microM-1 s-1), Lys-plasminogen (0.57-0.79 microM-1 s-1) or LMM plasminogen (0.11-0.16 microM-1 s-1). In a human plasma milieu in vitro STAR, Glu-plasmin-STAR, Lys-plasmin-STAR and LMM-plasmin-STAR were equally effective for the lysis of 125I-fibrin-labeled human plasma clots [50% clot lysis in 2 h (EC50) with 11-13 nM test compound] and equally fibrin-selective (residual fibrinogen levels of 72-84% after 2 h at EC50). Our results thus confirm that plasminogen and STAR form a 1:1 stoichiometric complex in which plasminogen is converted to plasmin and Glu-plasminogen to Lys-plasmin. The lysine-binding sites in kringles 1-4 of plasminogen are not required for the complex formation with STAR, nor for the enzyme activity of the complex with STAR in purified systems and in a human plasma milieu. The lysine-binding sites are, however, important for the rate of the inhibition of the complexes by alpha 2-antiplasmin.
Enzyme Activation↗
Retinoic acid upregulates the plasminogen activator system in human epidermal keratinocytes.
The activation of the proteolytic plasminogen activator system is important for the re-epithelialization of skin wounds. Keratinocytes synthesize and secrete the urokinase-type plasminogen activator, which binds to its specific receptor on keratinocytes. Receptor-bound urokinase-type plasminogen activator efficiently activates cell surface bound plasminogen. This results in pericellular proteolysis, which facilitates keratinocyte migration. Urokinase-type plasminogen activator activity is specifically controlled by plasminogen activator inhibitor-1 and -2. As retinoids have been reported to accelerate epithelialization of skin wounds in animal studies and clinical settings, we investigated the effects of all-trans retinoic acid on the plasminogen activator system in human epidermal keratinocytes. As tested in a chromogenic plasminogen activation assay, incubation with 10 microM all-trans retinoic acid caused a marked induction of cell-associated plasminogen activity after 24 h, and this induction was blocked by neutralizing anti-urokinase-type plasminogen activator antibodies, but not anti-tissue-type plasminogen activator antibodies. All-trans retinoic acid lead to a strong increase in urokinase-type plasminogen activator (enzyme-linked immunosorbent assay) and urokinase-type plasminogen activator receptor cell surface expression (flow cytometry) after 24 h. At this time-point, tissue-type plasminogen activator and plasminogen activator inhibitor-1 and -2 proteins were not or only slightly increased. Northern blot analyses revealed that all-trans retinoic acid caused an early and short-lived increase of plasminogen activator inhibitor-1, but a prolonged induction of urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA levels. Collectively, these data suggest that all-trans retinoic acid activates the plasminogen activator system in human epidermal keratinocytes by differentially regulating activating and inhibiting components. The activation of the plasminogen activator system may be one mechanism by which all-trans retinoic acid exerts beneficial effects in cutaneous wound healing.
Cells, Cultured↗
Loss of cell viability dramatically elevates cell surface plasminogen binding and activation.
The plasminogen activation cascade is focused at the cell surface by virtue of the presence of plasminogen and plasminogen activator receptors. We have utilized flow cytometric plasminogen (plg) binding and activation assays to examine both plasminogen binding and activation on the surface of specific subpopulations of U937 cells (viable, apoptotic, and dead cells). A direct relationship was found to exist between cell viability (propidium iodide uptake) and the magnitude of lysine-dependent plasminogen binding, with apoptotic and dead subpopulations of cells binding up to 100-fold more plasminogen than viable cells. Despite the high level of lysine-dependent plasminogen binding on dead cells, plasminogen activation was minimal due to low levels of cell-surface urokinase plasminogen activator. Plasminogen activation readily occurred on the surface of apoptotic cells because of a dramatic increase in both lysine-dependent plasminogen binding and endogenous urokinase plasminogen activator. These results indicate that colocalization of plasminogen and urokinase plasminogen activator are paramount for plasminogen activation to proceed on the cell surface. Our data also strongly implicate the involvement of the plasminogen activation cascade in apoptosis, especially on urokinase plasminogen activator-expressing cell types. The current study clearly supports the important role of flow cytometry in cellular plasminogen binding and activation studies.
Amino Acid Chloromethyl Ketones↗
Expression pattern of the urokinase-plasminogen activator system in rat DS-sarcoma: role of oxygenation status and tumour size.
The urokinase plasminogen activator system plays a central role in malignant tumour progression. Both tumour hypoxia and enhancement of urokinase plasminogen activator, urokinase plasminogen activator-receptor and plasminogen activator inhibitor type 1 have been identified as adverse prognostic factors. Upregulation of urokinase plasminogen activator or plasminogen activator inhibitor type 1 could present means by which hypoxia influences malignant progression. Therefore, the impact of hypoxia on the expression pattern of the urokinase plasminogen activator system in rat DS-sarcoma in vivo and in vitro was examined. In the in vivo setting, tumour cells were implanted subcutaneously into rats, which were housed under either hypoxia, atmospheric air or hyperoxia. For in vitro studies, DS-sarcoma cells were incubated for 24 h under hypoxia. Urokinase plasminogen activator and urokinase plasminogen activator-receptor expression were analysed by flow cytometry. Urokinase plasminogen activator activity was measured using zymography. Plasminogen activator inhibitor type 1 protein levels in vitro and in vivo were examined with ELISA. PAI-1 mRNA levels were determined by RT-PCR. DS-sarcoma cells express urokinase plasminogen activator, urokinase plasminogen activator-receptor, and plasminogen activator inhibitor type 1 in vitro and in vivo. The urokinase plasminogen activator activity is enhanced in DS-sarcomas compared to normal tissues and rises with increasing tumour volume. The oxygenation level has no impact on the urokinase plasminogen activator activity in cultured DS-sarcoma cells or in solid tumours, although in vitro an increase in plasminogen activator inhibitor type 1 protein and mRNA expression after hypoxic challenge is detectable. The latter plasminogen activator inhibitor type 1 changes were not detectable in vivo. Hypoxia has been demonstrated to contribute to the upregulation of some components of the system in vitro, although this effect was not reproducible in vivo. This may indicate that the serum level of plasminogen activator inhibitor type 1 is not a reliable surrogate marker of tumour hypoxia.
Animals↗
Depletion of plasminogen in vitro or during thrombolytic therapy limits fibrinolytic potential.
Thrombolytic therapy frequently induces a "lytic state" associated with a decrease in plasma plasminogen concentration that could limit therapeutic efficacy. We therefore investigated the influence of soluble plasminogen concentration on in vitro lysis of retracted whole-blood clots in plasma from normal subjects and from patients undergoing thrombolytic therapy. With recombinant tissue plasminogen activator (1000 ng/ml) or two-chain urokinase plasminogen activator (250 U/ml), minimal clot lysis occurred in normal plasma depleted of plasminogen by lysine Sepharose chromatography. Clot lysis induced by two-chain urokinase plasminogen activator increased progressively in normal plasma at initial plasminogen concentrations between 0.06 to 6 U/ml, whereas maximum lysis with recombinant tissue plasminogen activator occurred between 0.5 U/ml and 1 U/ml and was less at lower and higher concentrations of plasminogen. Incubation of whole-blood clots in normal plasma with recombinant tissue plasminogen activator resulted in little change in plasminogen concentration during 6 hours, with a constant rate of clot lysis. Incubation with two-chain urokinase plasminogen activator, however, caused a rapid decrease in plasminogen concentration and a corresponding decrease in lysis rate; lysis rate was restored after repletion with purified plasminogen. The effect of in vivo activator-induced plasminogen depletion on in vitro clot lysis rates was tested with plasma obtained from patients 90 to 120 minutes after they had received 30 mg of acylated plasminogen-streptokinase activator complex that showed depletion of plasminogen to 14% +/- 2%. These plasma samples produced only 4% +/- 1% in vitro clot lysis during 4 hours but lysis increased progressively after repletion with 1, 2, and 4 U/ml plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)
Blood Coagulation↗
Coordinate expression of urinary-type plasminogen activator and its receptor accompanies malignant transformation of the ovarian surface epithelium.
OBJECTIVE: Because elevated expression and cell surface association of urinary-type plasminogen activator have been linked to invasive potential in certain tumor types, we examined the expression of urinary-type plasminogen activator and urinary-type plasminogen activator receptor in ovarian epithelial carcinoma tissues and cells as compared with normal ovarian epithelium. STUDY DESIGN: Monoclonal antibodies specific for urinary-type plasminogen activator and urinary-type plasminogen activator receptor were used for immunohistochemical staining of tissues and cells to assess expression of these antigens in frozen sections of normal and tumor tissue. Substrate zymography was used to detect plasminogen activator activity in ovarian carcinoma ascites and in conditioned media of cultured cells, whereas a Western blot assay was used to identify urinary-type plasminogen activator receptor in cultured cells. RESULTS: Normal ovarian epithelium expressed urinary-type plasminogen activator receptor (4/4 positive) but little or no urinary-type plasminogen activator (0/4 positive), whereas epithelial ovarian carcinomas frequently expressed urinary-type plasminogen activator (4/8 positive) in conjunction with urinary-type plasminogen activator receptor (7/9 positive). High levels of urinary-type plasminogen activator were detected in 15 of 19 samples of ascites. DOV 13, OVCA 420, OVCA 429, OVCA 432, and OVCA 433 cell lines secreted urinary-type plasminogen activator in variable quantities, whereas normal ovarian epithelial cells did not secrete any detectable plasminogen activator. Urinary-type plasminogen activator receptor had similar levels of expression in all cancer cell lines and normal ovarian epithelium. CONCLUSION: Overexpression of urinary-type plasminogen activator is associated with malignant transformation of the ovarian epithelium. Increased cell surface proteolysis mediated by urinary-type plasminogen activator bound to cell surface urinary-type plasminogen activator receptor may contribute to metastatic behavior in ovarian carcinoma.
Carcinoma↗
On the reliability of plasminogen measurement employing the proactivator-activator converting method.
A simple plasminogen determination method is presented. It is based upon the conversion of plasminogen into activator by large and constant amounts of streptokinase. The activator contained in a standard coagulum consisting of bovine fibrin, streptokinase, and a 1:40 dilution of human plasma converts the plasminogen adsorbed on bovine fibrin into plasmin. Lysis of the test coagulum is hereby induced. The speed of such lysis is limited by the concentration of the activator incorporated in the test coagulum. The variable component of the activator being human plasminogen, the speed of lysis is directly dependent upon the concentration of human plasminogen in the standard coagulum. Using the thromboelastograph according to Hartert in recording the test clot lysis times, this method of plasminogen determination was shown to be a simple and quick procedure. The standard deviation ranged from +/- 13,2 tp 68%, depending upon the plasminogen value to be measured (lower rates of error were attached to high, and higher rates of error to low, plasminogen concentrations). The biological variation of plasminogen values in a group of 26 men aged from 40 to 65 years was calculated to be +/- 21%. Both plasminogen and plasmin, its activated form, were exchangeable in the test, i.e. plasminogen determinations performed by activator assay did not differentiate between plasminogen and plasmin. There was no influence by varying anti-SK titers in the plasma up to a circulating antibody content of 2 million. Furthermore, plasma antiplasmins did not affect the plasminogen measuring system. Plasminogen tested by activator assay displayed values closely related to those achieved by immunochemical methods. Plasminogen measurements were performed in patients undergoing streptokinase and urokinase infusion treatment. 5,000 u streptokinase per hour, as well as 270,000 CTA-u urokinase per hour, infused over a period of 2 days produced a fall in plasminogen down to 30-60% of normal. In contrast, 100,000 u streptokinase per hour lowered the plasminogen concentration down to values of below 1%. The foregoing data indicate that plasminogen measurement, according to the principles outlined here (activator assay), may be regarded as a valuable and reliable method for the routine control of streptokinase and urokinase therapy.
Adult↗
Zymogen-activation kinetics. Modulatory effects of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid and poly-D-lysine on plasminogen activation.
The kinetics of plasminogen activation catalysed by urokinase and tissue-type plasminogen activator were investigated. Kinetic measurements are performed by means of a specific chromogenic peptide substrate for plasmin, D-valyl-L-leucyl-L-lysine 4-nitroanilide. Two methods are proposed for the analysis of the resulting progress curve of nitroaniline formation in terms of zymogen-activation kinetics: a graphical transformation of the parabolic curve and transformation of the curve for nitroaniline production into a linear progress curve by the addition of a specific inhibitor of plasmin, bovine pancreatic trypsin inhibitor. The two methods give similar results, suggesting that the reaction between activator and plasminogen is a simple second-order reaction at least at plasminogen concentrations up to about 10 microM. The kinetics of both Glu1-plasminogen (residues 1-790) and Lys77-plasminogen (residues 77-790) activation were investigated. The results confirm previous observations showing that trans-4-(aminomethyl)cyclohexane-1-carboxylic acid at relatively low concentrations enhances the activation rate of Glu1-plasminogen but not that of Lys77-plasminogen. At higher concentrations both Glu1- and Lys77-plasminogen activation are inhibited. The concentration interval for the inhibition of urokinase-catalysed reactions is shown to be very different from that of the tissue-plasminogen activator system. Evidence is presented indicating that binding to the active site of urokinase (KD = 2.0 mM) is responsible for the inhibition of the urokinase system, binding to the active site of tissue-plasminogen activator is approx. 100-fold weaker, and inhibition of the tissue-plasminogen activator system, when monitored by plasmin activity, is mainly due to plasmin inhibition. Poly-D-lysine (Mr 160 000) causes a marked enhancement of plasminogen activation catalysed by tissue-plasminogen activator but not by urokinase. Bell-shaped curves of enhancement as a function of the logarithm of poly-D-lysine concentration are obtained for both Glu1- and Lys77-plasminogen activation, with a maximal effect at about 10 mg/litre. The enhancement of Glu1-plasminogen activation exerted by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid is additive to that of poly-D-lysine, whereas poly-D-lysine-induced enhancement of Lys77-plasminogen activation is abolished by trans-4-(aminomethyl)cyclohexane-1-carboxylic acid. Analogies are drawn up between the effector functions of poly-D-lysine and fibrin on the catalytic activity of tissue-plasminogen activator.
Aprotinin↗
Plasminogen activator and its inhibitor in cancer patients treated with tumor necrosis factor.
BACKGROUND: We noted the presence of plasma fibrin degradation products in patients treated with recombinant human tumor necrosis factor (TNF) in a phase I trial. PURPOSE: To further define this observation, we investigated the effects of TNF on the fibrinolytic system in patients entered in the same trial. METHODS: In the 14 patients studied, fibrinolytic parameters were measured by analyzing blood samples for tissue plasminogen activator and inhibitor at 0, 1, 2, 4, 6, and 18-24 hours after initiation of TNF treatment. We used a chromogenic substrate method to determine activity of plasminogen activator and its inhibitor and an enzyme-linked immunosorbent assay (ELISA) to determine levels of antigen (tissue-type plasminogen activator). Molecular weight was determined by zymographic assay. RESULTS: TNF treatment was associated with tissue-type plasminogen activator induction within 1 hour of TNF initiation. The plasminogen activator produced was consistent with tissue-type plasminogen activator derived from endothelium as evidenced by molecular weight analysis and ELISA. Moreover, induction of plasminogen activator inhibitor occurred following the release of tissue-type plasminogen activator, and our data suggest a dose-response effect for TNF. At high doses (i.e., 200 and 240 micrograms/m2), there was a more rapid and prolonged release of plasminogen activator inhibitor, which had an inverse relationship with the level of antigenic tissue-type plasminogen activator. Zymographic analysis showed urokinase-type plasminogen activator activity in 13 of 14 patients. In three patients, simultaneous measurements of white blood cells and tissue-type plasminogen activator revealed a temporal association between the TNF-associated rapid granulocytopenia at 30 minutes after TNF initiation and release of tissue-type plasminogen activator antigen. CONCLUSIONS: The results suggest a positive association between TNF and rapid induction of plasminogen activator activity that is consistent with an endothelial product. It is possible that, at high doses, TNF may interact directly with vascular endothelium, leading to rapid and prolonged production of plasminogen activator inhibitor. There was a dose-response effect between TNF and release of tissue-type plasminogen activator. The release of tissue-type plasminogen activator was preceded by granulocytopenia, which may indicate an association between a proposed TNF-induced granulocyte-endothelial interaction in vivo and release of tissue-type plasminogen activator. IMPLICATIONS: These findings demonstrating the effects of TNF on the fibrinolytic system can be analyzed further in experimental systems to determine the implications for use of this agent as a biological response modifier in cancer therapy.
Amino Acid Sequence↗
A battery of monoclonal antibodies that induce unique conformations to evolve cryptic but constitutive functions of plasminogen.
Two groups of anti-plasminogen monoclonal antibodies, whose epitope was either in the kringle 1 + 2 + 3 domain (F3P2, F11P5, F11P6, and F12P18) or the kringle 5 domain (F1P6 and F12P16), were isolated and their effects on the conformation of plasminogen were explored. All antibodies except F1P6 had 3- to 10-fold higher affinity toward Lys-plasminogen than Glu-plasminogen. F1P6 exhibited a comparable affinity to Glu- and Lys-plasminogen. Among these, only F11P5 binding was inhibited by epsilon-amino-nu-caproic acid (EACA) in a concentration-dependent manner, with half maximal inhibition at 3 mM. From a competition assay, we concluded that the epitopes of F11P5, F11P6, and F12P18 should be very close, and located at or near the low affinity lysine binding site on the kringle 2 + 3. These three antibodies dramatically enhanced the binding of Glu-plasminogen to the other antibodies, except to F1P6. Interestingly, F3P2, whose non-overlapping epitope was in the kringle 2 + 3 domain, also augmented the binding of Glu-plasminogen to the other antibodies. In contrast, we did not observe enhanced binding of Lys-plasminogen to one antibody in the presence of the other antibodies, and the binding of Glu-plasminogen to these antibodies did not increase in the presence of 10 mM EACA. In the presence of these antibodies, including F1P6, Glu-plasminogen bound more efficiently to immobilized degraded fibrin, with a binding profile similar to Lys-plasminogen. All antibodies except F1P6 enhanced the conversion rate of plasminogen to plasmin remarkably. Taken together, we propose that these two groups of monoclonal antibodies can dissociate the intramolecular interactions of Glu-plasminogen and induce the conformational transition of Glu-plasminogen to Lys-plasminogen. In addition, the kringle 2 + 3 and kringle 5 structures of Glu-plasminogen liganded with EACA are distinct from the Lys-plasminogen structure.
Animals↗
Plasminogen activators in normal tissue and carcinomas of the human oesophagus and stomach.
Carcinogenesis in the human colon is associated with a marked increase of urokinase type plasminogen activator and a decrease of tissue type plasminogen activator. This study was performed to determine the concentrations of urokinase type plasminogen activator and tissue type plasminogen activator in normal tissue and carcinomas along the upper part of the gastrointestinal tract. Activity and antigen levels of both activators were determined in homogenates of endoscopically obtained biopsies from normal and carcinomatous tissues. Although the concentrations of tissue type plasminogen activator and urokinase type plasminogen activator in normal squamous epithelium of the oesophagus were low compared with those in columnar epithelium from the stomach, the urokinase type plasminogen activator/tissue type plasminogen activator antigen ratio of the different locations showed hardly any difference. Significant but heterogeneous increases were found in urokinase type plasminogen activator concentrations of biopsy specimens originating from carcinomas of both epithelial cell types. A decrease in tissue type plasminogen activator concentrations, as found in human colon carcinomas, could only be shown in carcinomas of columnar epithelium origin but not in squamous cell carcinomas of the oesophagus. The increase of urokinase type plasminogen activator and urokinase type plasminogen activator/tissue type plasminogen activator antigen ratio and the decrease of tissue type plasminogen activator in the carcinomas did not show a significant correlation with known prognostic determinants as differentiation grade, TNM classification, intestinal metaplasia, inflammation, and ulceration. The heterogeneous increase of urokinase type plasminogen activator in oesophageal and stomach carcinomas, together with the recently described association of urokinase type plasminogen activator in tissue extracts of breast carcinomas with aggressiveness and prognosis, may be relevance to prognostic studies, may be of relevance to prognostic studies in oesophageal and gastric cancer.
Duodenum↗