Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasminogen activators initiate the fibrinolytic system by conversion of the proenzyme plasminogen to the active fibrin degrading enzyme plasmin. Plasminogen activator inhibitors inhibit the effects of both plasminogen activators. Uncomplicated pregnancies are accompanied by hypercoagulability and an increased risk of thromboembolic disease. Thrombosis is rare in the first trimester and most events are noted in the last trimester. Therefore, we studied the fibrinolytic system at the end of pregnancy and in the puerperium. Plasma concentrations of urokinase plasminogen activator (u-PA/competitive radioimmunoassay), tissue type plasminogen activator (t-PA/sandwich ELISA) and plasminogen activator inhibitor (PAI/functional assay) were determined in 44 women (age: 24.3 +/- 4.3 years) with normal pregnancy near term. Plasma samples were collected before the onset of labour and 1, 2, 3, 4 and 5 days after delivery. Compared with an age-matched non pregnant control group (8.3 +/- 3.94 U/ml) significantly increased PAI activity (12.13 +/- 4.79 U/ml - p less than 0.005) was measured before delivery with a subsequent significant decrease (8.13 +/- 1.97 U/ml) to normal values on day 1 after delivery; plasma u-PA and t-PA antigen levels remained unchanged. Placental weight and birth weight had no influence on plasma levels of both plasminogen activators.
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PMID:Influence of delivery on plasminogen activator inhibitor activity. 268 64

The tumor-promoting phorbol ester PMA induces changes in the histiocytic human lymphoma cell line U-937 akin to cellular differentiation (Ralph, P., N. Williams, M. A. S. Moore, and P. B. Litcofsky, 1982, Cell. Immunol., 71:215-223) and concomitantly stimulates the biosynthesis of plasminogen activator inhibitor 2 (PAI 2) and of urokinase-type plasminogen activator (u-PA). PAI 2 is found in a nonglycosylated intracellular and a glycosylated secreted form. The former appears to be identical to PAI 2 previously purified from placental extracts and large-scale U-937 cell cultures. The sixfold increase of PAI 2 antigen measured 24 h after PMA treatment in cell extracts and conditioned media is accompanied by an equal increase of active PAI 2 mRNA, whereas the 6 to 13-fold increase of u-PA antigen in the same samples is associated with only a 1.5-fold mRNA increase. The increase of PAI 2, but not of u-PA, biosynthesis requires transcription. A 50-fold molar excess of PAI 2 over u-PA is found in both extracts and conditioned media of PMA-treated cells. PAI 2 represents at least 0.3% of total de novo synthesized protein 24 h after induction with PMA. Thus, PAI 2, but not u-PA, is an abundant product of this precursor analogue of the mononuclear phagocyte lineage, and might represent a new marker for monocyte/macrophage differentiation.
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PMID:Phorbol ester induces the biosynthesis of glycosylated and nonglycosylated plasminogen activator inhibitor 2 in high excess over urokinase-type plasminogen activator in human U-937 lymphoma cells. 310 4

The site of the reaction between plasminogen activators and plasminogen activator inhibitor 1 (PAI-1) was investigated in cultures of human umbilical vein endothelial cells. In conditioned medium from endothelial cells, two forms of a plasminogen activator-specific inhibitor can be demonstrated: an active form that readily binds to and inhibits plasminogen activators and an immunologically related quiescent form which has no anti-activator activity but which can be activated by denaturation. In conditioned medium, only a few percent of PAI-1 is the active form. However, the addition of increasing concentrations of tissue-type plasminogen activator (t-PA) or urokinase to confluent endothelial cells produced a saturable (3.0 pmol/5 x 10(5) cells), dose-dependent increase of the activator-PAI-1 complex in the conditioned medium even in the presence of actinomycin D or cycloheximide. This resulted also in a dose-dependent decrease of the residual PAI activity measured by reverse fibrin autography both in the conditioned medium and cell extracts. Short-time exposure of endothelial cells to a large amount of t-PA caused almost complete depletion of all cell-associated PAI activity. Although there was no detectable PAI activity even after activation of PAI by denaturants or antigen in the culture medium at 4 degrees C without the addition of t-PA, the addition of t-PA at 4 degrees C not only resulted in the formation of 70% of the amount of the t-PA.PAI complex in conditioned medium at 37 degrees C, but also induced PAI-1 antigen in a time and dose-dependent manner in the conditioned medium. Moreover, 125I-labeled t-PA immobilized on Sepharose added directly to endothelial cells formed a complex with PAI-1 in a dose-dependent manner. On the other hand, no detectable complex was formed with PAI-1 when Sepharose-immobilized 125I-labeled t-PA was added to endothelial cells under conditions in which the added t-PA could not contact the cells directly but other proteins could pass freely by the use of a Transwell. All these results suggest that a "storage pool" on the surface of endothelial cells or the extracellular matrix produced by endothelial cells contains almost all the active PAI-1, and reaction between PA and PAI-1 mainly occurs on the endothelial cell membranes, resulting in a decrease of the conversion of active PAI-1 to the quiescent form.
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PMID:Interaction of tissue-type plasminogen activator and plasminogen activator inhibitor 1 on the surface of endothelial cells. 312 83

The effects of sulfonylureas on the production of plasminogen activator (PA) and antiactivator (PAI) were investigated using bovine aortic endothelial cells. All compounds studied stimulated PA release (1.3- to 5.2-fold), with glipizide being the most potent, followed by tolazamide, chlorpropamide, and tolbutamide, in that order, while glyburide was the least effective. Both tissue-type and urokinase-type PA production was enhanced. Studies using metabolic inhibitors indicated that both RNA and protein syntheses are required for the sulfonylurea-mediated stimulation of PA release. In addition to continuous release of the two PAs, there was also a continuous release of a single PAI, which did not show an increase after the sulfonylureas. These results suggest that, in addition to their beneficial effects in the treatment of diabetes mellitus, some sulfonylurea compounds may also have significant thrombolytic effects. These results also suggest that pharmacological enhancement of PA production by vascular endothelial cells may be a promising antithrombotic mechanism.
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PMID:Effects of sulfonylureas on the synthesis and secretion of plasminogen activator from bovine aortic endothelial cells. 312 27

Plasminogen activator inhibitor 1 (PAI-1) was purified from medium conditioned by cultured bovine aortic endothelial cells by successive chromatography on concanavalin A Sepharose, Sephacryl S-200, Blue B agarose, and Bio-Gel P-60. As shown previously for conditioned media (C. M. Hekman and D. J. Loskutoff (1985) J. Biol. Chem. 260, 11581-11587) the purified PAI-1 preparation contained latent inhibitory activity which could be stimulated 9.4-fold by sodium dodecyl sulfate and 45-fold by guanidine-HCl. The specific activity of the preparation following treatment with 0.1% sodium dodecyl sulfate was 2.5 X 10(3) IU/mg. The reaction between purified, guanidine-activated PAI-1 and both urokinase and tissue plasminogen activator (tPA) was studied. The second-order rate constants (pH 7.2, 35 degrees C) for the interaction between guanidine-activated PAI-1 and urokinase (UK), and one- and two-chain tPA are 1.6 X 10(8), 4.0 X 10(7), and 1.5 X 10(8) M-1 S-1, respectively. The presence of CNBr fibrinogen fragments had no affect on the rate constants of either one- or two-chain tPA. Steady-state kinetic analysis of the effect of PAI-1 on the rate of plasminogen activation revealed that the initial UK/PAI-1 interaction can be competed with plasminogen suggesting that the UK/PAI-1 interaction may involve a competitive type of inhibition. In contrast, the initial tPA/PAI-1 interaction can be competed only partially with plasminogen, suggesting that the tPA/PAI-1 interaction may involve a mixed type of inhibition. The results indicate that PAI-1 interacts more rapidly with UK and tPA than any PAI reported to date and suggest that PAI-1 is the primary physiological inhibitor of single-chain tPA. Moreover, the interaction of PAI-1 with tPA differs from its interaction with UK, and may involve two sites on the tPA molecule.
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PMID:Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator. 312 76

Ovarian follicles produce two types of plasminogen activator (tPA and uPA), and their inhibitor (PAI). We have examined the interaction and regulation of these factors in ovarian intact follicles as well as granulosa and theca-interstitial cells. The results show that only tPA, but not uPA, is regulated by the gonadotropins and reaches maximum prior to ovulation. PAI is secreted mainly by theca-interstitial cells and is stimulated also by the gonadotropins. The formation of complexes between PA and PAI completely blocks or decreases PA activity. It is suggested that the interaction between plasminogen activators and their inhibitor in the follicles may play a very important role in maintaining normal ovarian function and the machnism of ovulation.
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PMID:Interaction and regulation of plasminogen activators and their inhibitor in rat follicles during periovulatory periods. 313 65

A new functional assay of PAI-activity in human plasma is described. Hitherto known assays for fast acting PAI have some disadvantages: predilution and/or acidification steps of the sample to afford the required inactivation of alpha-2-antiplasmin (A2-PI). This approach in contrast implicates test performance in presence of chloramine T (CT), an oxidant that destroys plasma antiplasmin activity without impairing significantly the activity of urokinase (u-PA) or plasmin. The following reaction conditions were found optimal: 50 microliter of undiluted plasma, anticoagulated with citrate or EDTA, were first incubated with 1 IU u-PA in a Tris-buffer, pH 8.4 and then with Glu-plasminogen (0.85 mumol/l final), CT (2.5 mmol/l final), tranexamic acid (0.9 mmol/l final) for 5 min. at 37 degrees C. After addition of 0.3 mmol/l of the chromogenic plasmin substrate H-D-Nva-CHA-Lys-pNA (pNA = para nitroanilide) and of NaCl (250 mmol/l final) a linear kinetic with delta A405/t in the range of 0.2/min for normal plasma was recorded. In an endpoint version of the test the chromogenic substrate can be added together with plasminogen resulting an A/t2-kinetic. Dilution studies showed a linear calibration curve from 0 to 14 arbitrary u-PA inhibiting units (AU)/ml plasma. By means of PAI-standard plasmas PAI-capacity values of 20 healthy volunteers (10 males/10 females) (x = 26 years, sigma = 4.2) were determined. They ranged from 0.4 - 6.9 (x = 1.3, sigma = 0.9) AU/ml plasma. Plasma samples containing more than 14 AU/ml were prediluted with PAI-deficient plasma. Intra- and inter-assay coefficients of variation (CV) were determined to be 1.3 +/- 0.6 and 4.3 +/- 0.5%, respectively. The values of this assay correlate well with those obtained by acidification of the samples. However, the possibility of measuring plasma PAI (and PA) activities by means of a simple and direct approach can be considered as an important progress with regard to routine hospital practice. The presented oxidative inactivation of A2-PI mimics the leukocyte attack phase, suggesting that activated leukocytes create a microenvironment of uncontrolled plasmin activity.
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PMID:Determination of plasminogen activator inhibitor (PAI) capacity of human plasma in presence of oxidants: a novel principle. 313 81

Mononuclear phagocytes are known to produce both urokinase-type plasminogen activator (u-PA) and a specific PA inhibitor, PAI-2. In this study we have investigated the effects of calcitriol and PMA-induced differentiation on the comparative expression of PA and PAI in monoblast-like U937 cells. Cells were incubated for 96 h in the presence or absence of 50 nM calcitriol. After transfer into serum-free medium, the cells were cultured for 48 h with PMA (0 to 50 ng/ml). PA and PAI activities of conditioned media and cell lysates were measured with a plasminogen dependent colorimetric assay. Control cells uniformly secreted PAI activity (70.3 +/- 24.9 PAI U/ml), while calcitriol pretreatment induced the cells to secrete PA activity (52.6 +/- 47.2 milli-Ploug unit/ml). PMA induced secretion of PAI activity in calcitriol-pretreated cells to levels 4.6- to 8.3-fold greater than controls (p less than 0.05). Parallel effects on PA and PAI activities were seen in cell lysates. To determine how changes in the expression of u-PA and PAI-2 might account for these effects, mRNA for u-PA and PAI-2 were assessed by Northern blot analysis. Calcitriol induced an increase in u-PA mRNA with a marked reduction in PAI-2 mRNA. PMA alone induced modest increases in both mRNA species. In calcitriol pre-treated cells, PMA induced a moderate increase in u-PA mRNA and a marked increase in PAI-2 mRNA. We conclude that agonist-specific differentiation of U937 cells modulates the expression of PA and PAI activities by altering the proportionate biosynthesis of u-PA and PAI-2 proteins. The ability of mononuclear phagocytes to control plasminogen activation at inflammatory foci may therefore be contingent on the independent regulation of u-PA and PAI-2 gene expression.
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PMID:Modulation of urokinase-type plasminogen activator and plasminogen activator inhibitor-2 expression by U-937 mononuclear phagocytes. Effects of 1 alpha, 25-dihydroxyvitamin D3 and phorbol ester. 313 63

The effect of human recombinant tumor necrosis factor (TNF) was studied in vitro on human endothelial cells. TNF (1-1000 pg/ml) induced a dose-dependent increase in PAI level in the supernatant from 6 to 25 U/ml as estimated against urokinase. This effect was time-dependent. It was not suppressed by Polymyxin B thus excluding a possible contribution of an endotoxin contamination. Fibrinoenzymography performed after SDS-PAGE showed that this inhibitor neutralized urokinase and tissue plasminogen activator and gave rise to high molecular weight complexes. TNF (30 micrograms/kg) was also injected in rat. Blood fibrinolytic activity determined 4 hr later was decreased as estimated by the prolongation of the euglobulin clot lysis time from 37 to 188 min. Fibrinoenzymographic profile of the plasma was then characterized by a fainting of the tPA lysis band but the capacity of plasma to neutralize urokinase was not significantly modified. These results suggest that TNF could alter the fibrinolytic balance by stimulating PAI production at the endothelial level. This might be of importance in synergy with the TNF-induced procoagulant activity for promoting vascular occlusion of tumor capillaries.
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PMID:Tumor necrosis factor (TNF) stimulates plasminogen activator inhibitor (PAI) production by endothelial cells and decreases blood fibrinolytic activity in the rat. 314 62

This report describes the development and use of functional immunoradiometric assays that distinguish the activity of beta-migrating endothelial-type plasminogen activator inhibitor (PAI-1) from that of placental-type plasminogen activator inhibitor (PAI-2). These assays are based upon the binding of PAI-1 and PAI-2 to immobilized single-chain tissue-type plasminogen activator (tPA) and to immobilized urokinase (UK), respectively. The extent of binding of each PAI is quantified by incubating the PAI-PA complex first with rabbit antiserum specific for the individual PAI and then with 125I-labeled goat antirabbit IgG. In control experiments, the assays were shown to be sensitive, dose-dependent over a wide range, and specific for each PAI. These assays were employed to establish the PAI profile of a variety of human cells. Neither PAI-1 nor PAI-2 could be detected in Bowes melanoma cells or in a renal adenocarcinoma cell line (ACHN), while the histiocytic lymphoma cell (U-937) produced only PAI-2. Five cell lines, including two that were previously shown to contain one or the other PAI (e.g., umbilical vein endothelial cells and a fibrosarcoma cell line, HT-1080) in fact contained both PAIs. The cells containing both PAIs were studied in more detail. In each case, SDS treatment of CM was shown to enhance PAI-1 activity (by converting the latent form of this inhibitor into its active form) and to destroy PAI-2 activity. Various compounds including interleukin 1, dexamethasone, and phorbol myristate acetate were found to selectively influence the cellular production of one PAI without concomitantly affecting the production of the other, suggesting that the synthesis of these inhibitors is not coordinately regulated.
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PMID:Detection of both type 1 and type 2 plasminogen activator inhibitors in human cells. 325 67


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