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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Vascular endothelial cells undergo morphological and functional changes at sites of cell-mediated immune responses which may serve to promote the pathogenesis of inflammation. These changes, described as "endothelial cell activation" can be invoked by a variety of cytokines which include interleukin I (IL-1), tumor necrosis factor (TNF), and lipopolysaccharide (LPS). We report here on the regulation of the plasminogen activator (PA) proteolytic system by human recombinant TNF alpha in short term cultures (less than 4 passages) of human umbilical vein endothelial cells (HUVECs). TNF alpha treatment of HUVECs enhanced the production of 55 kDa urokinase (u) PA activity and uPA antigen by fourfold, in a concentration dependent manner (5-100 U/ml), following a 24 h treatment as determined by PA zymography and micro-ELISA assays, respectively. This response was specific for uPA since, no change in extracellular tissue type PA activity and tPA antigen levels were noted under analogous conditions. A similar 4-fold increase in the de novo synthesis of [35S]-methionine radiolabeled uPA was observed by immunoprecipitation following a 24 h TNF treatment. The induction of uPA by TNF was inhibited by actinomycin D and cycloheximide implying the necessity of RNA and protein synthesis, respectively. The effect of TNF could not be prevented by the addition of IL-1 neutralizing antibodies. Therefore, it is unlikely that TNF acts through the induction of IL-1 secretion. Time course studies using PA zymography indicate that within 8 h after TNF exposure, a 2-fold increase in uPA activity above untreated basal levels was observed. Upregulation of extracellular uPA production in HUVECs following TNF treatment suggests yet a new aspect of cellular and interstitial PA regulation in endothelium during inflammation and angiogenesis.
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PMID:Tumor necrosis factor induction of urokinase-type plasminogen activator in human endothelial cells. 180 6

A GnRH agonist (5-50 micrograms) is capable of inducing ovulation in PMSG-primed hypophysectomized immature rats, as is the case in hCG-induced ovulation, but 2-4 h earlier than hCG. GnRH-induced ovulation is effectively blocked by the concomitant administration of the GnRH-antagonist which failed to interfere with hCG-induced ovulation, indicating that GnRH and its agonists do not share a receptor with LH/hCG. Like hCG, GnRH is also capable of inducing tissue type (tPA), but not urokinase type (uPA) PA. The plasminogen activator activity in ovarian homogenates and the granulosa and theca-interstitial cells increase in a time-dependent manner, reaching maximum levels just prior to ovulation. Similar to hCG, GnRH also increases tPA activity in cumulus-oocyte complexes in a time-dependent fashion.
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PMID:A comparative study on involvement of tPA activity in ovulation induced by hCG and GnRH agonist in hypophysectomized rats. 180 39

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

Human glomerular epithelial cells (GECs) in culture synthesize single-chain, urokinase-type plasminogen activator (SC-uPA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor 1 (PAI-1) and possess specific membrane-binding sites for u-PA. Using purified 125I-alpha thrombin, we demonstrate here the presence of two populations of specific binding sites for thrombin on GECs (1.Kd = 4.3 +/- 1.0 x 10(-10) M, 5.4 +/- 1.4 x 10(4) M sites per cell, 2. Kd = 1.6 +/- 0.5 x 10(-8) M, 7.9 +/- 1.8 x 10(5) sites per cell). Purified human alpha thrombin promoted the proliferation of GECs and induced a time- and dose-dependent increase of SC-uPA, t-PA, and PAI-1 antigens released by GECs. Thrombin-mediated increase in antigen was paralleled by an increase in the levels of corresponding u-PA and PAI-1 messenger RNA. In contrast, thrombin decreased u-PA activity in conditioned medium. This discrepancy between u-PA antigen and u-PA activity was explained by a limited proteolysis of SC-uPA by thrombin, leading to a two-chain form detected by immunoblotting and that could not be activated by plasmin. Thrombin also decreased the number of u-PA binding sites on GECs (p less than 0.05) without changing receptor affinity. Hirudin inhibited the binding and the cellular effects of thrombin, whereas thrombin inactivated by diisopropylfluorophosphate had no effect, indicating that both membrane binding and catalytic activity of thrombin were required. We conclude that thrombin, through specific membrane receptors, stimulates proliferation of GECs and decreases the fibrinolytic activity of GECs both at the cell surface and in the conditioned medium. These results suggest that thrombin could be involved in the pathogenesis of extracapillary proliferation and persistency of fibrin deposits in crescentic glomerulonephritis.
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PMID:Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells. 184 34

In an earlier publication (Harvey, et al. (1982) J. Biol. Chem., 257, 5645-5651) the discovery of a family of unusually large molecules with plasminogen activator activity in the conditioned medium of a human lung cancer cell line was reported. These molecules are related to urokinase (uPA) by functional and immunological criteria. We have now purified two representatives of this glycoprotein family of Mr 900,000 (PA900) and Mr 660,000 (PA660). While these could be fractionated into subspecies exhibiting size and charge differences, reduction yielded in all cases two predominant chains of 70 and 40 kDa, respectively. Since the amino acid composition of the subfractions was identical, we conclude that the heterogeneity is due to demonstrated differences in glycosylation. The amino acid composition of the unreduced species and of the major reduced chains differed from that of 55 kDa uPA. These enzymes are active toward the substrate, plasminogen, as well as toward the uPA-specific synthetic substrate, Spectrozyme UK, and these activities are inhibitable by diisopropylphosphorofluoridate (DFP). Treatment of PA660 with [3H]DFP resulted in the incorporation of 1.4 mol of DFP into 1 mol of enzyme, suggesting the presence of a single active site. The label was quantitatively recovered in a 21 kDa fragment in a reduction experiment. This fragment also demonstrated immunological reactivity with antiurokinase. It is postulated that PA660 is composed of five or six pairs of the 70 and 40 kDa chains, and of a single uPA-like entity. All of these chains are linked by disfulfide bonds. Whether larger portions of uPA are also present in this molecule, is not yet clear. By electron microscopy, PA900 shows a filamentous structure, while PA660 is predominantly globular. The occurrence of large uPA-like activators in extracts of human colon carcinomas that crossreact with monospecific antibody against uPA, is discussed.
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PMID:Characterization of a family of high-molecular-weight plasminogen activators secreted by a lung tumor cell line. 185 26

In this study, we have evaluated the effects of four different thrombolytic agents, including Streptokinase from Hoechst and from Kabivitrum, Urokinase from Abbott and tissue plasminogen activator (t-PA) from Genetech, on platelet-rich plasma clots and platelet aggregation. At concentrations lower than 50 ugs/ml, t-PA had no inhibitory effect on clot retraction or platelet aggregation induced by weak or potent agonist. At a higher concentration (greater than 100 ugs/ml), t-PA specifically antagonized the action of thrombin on clot formation and platelet aggregation. Streptokinase (Kabivitrum) potentiated the action of weak agonists on platelet aggregation, but the same agent from Hoechst had no negative or positive influence. None of the drugs tested had an adverse effect on platelet function at suggested therapeutic levels. None of the thrombolytic agents were capable of dissociating preformed clots made from platelet-rich plasma. However, all of them caused lysis of whole blood clots. Also, prior incubation of plasma alone or platelet-rich plasma with any of the agents prevented subsequent clot formation. The studies demonstrate that thrombolytic drugs at therapeutic concentrations do not affect platelet function adversely. They have a potent effect on whole blood clots, but not on clots from platelet-rich plasma. Therefore, platelets may play a critical role in determining the degree of reperfusion and the frequency of reocclusion following treatment with thrombolytic agents in vivo.
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PMID:Influence of thrombolytic agents on human platelet function. 186 14

Plasminogen activation on the cell surface is regulated by a variety of modulators which balance surface-bound plasminogen activators (PAs) and plasminogen activator inhibitors (PAIs). In this study, we developed as assay system to assess modulation of cell-associated plasminogen activation. Plasmin generation by endogenous plasminogen activators was measured with a combination of exogenously added plasminogen and a chromogenic substrate, S-2251, in the presence of living cells. A cell surface PA activity was quantitated by adopting a rate of plasmin generation. We used HT-1080, a human fibrosarcoma cell line, as representative of cells which have both PAs and PAIs on their cell surface. A basal level of cell surface PA activity was specifically reduced by anti-urokinase-type PA IgG and enhanced by anti-PAI-1 IgG, suggesting that the basal level is determined by a balance between uPA and PAI-1 on the cell surface. We examined effects of dexamethasone and thrombin on cell surface PA activity in the assay system. Dexamethasone appeared to suppress the cell surface PA activity by enhancing de novo synthesis of PAI-1, whereas thrombin suppressed it by inactivating single-chain urokinase-type plasminogen activators. These results indicate that our assay system can be adapted for the screening of various types of PA modulators.
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PMID:An assay system for the modulators of plasminogen activation on the cell surface. 189 67

Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to greater than 95% pure and was fully active. The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 x 10(6) M-1 s-1, and for two-chain tPA, 0.7 x 10(5) M-1 s-1). As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases.
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PMID:Purification and characterisation of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae. 190 Oct 39

Urokinase-type (u-PA) and tissue-type plasminogen activator antigen (t-PA) as well as plasminogen activator-inhibitor activity were determined in seminal plasma and lysates of the respective spermatozoas in 67 ejaculate of males in infertile marriage without genito urinary pathology. U-PA was determined by a competition RIA, t-PA by an ELISA and PAI by a spectrophotometric assay. 15 patients showed normozoospermia, 11 azoospermia and 41 oligoasthenoteratozoospermia (OAT-syndrome). In lysates of spermatozoas, significantly higher levels of both plasminogenactivators and PAI were found in patients with OAT syndrome as compared to those exhibiting normozoospermia. Whereas PAI was absent in the seminal plasma of normozoospermic ejaculate, patients with azoospermia (180 +/- 13 mU/ml.) and OAT-syndrome (60 +/- 5 mU/ml.) showed high PAI levels. The similarly high values of t-PA (190.8-227.8 ng./ml.) and u-PA (19.4-32 ng./ml.) in the same compartment confirm their predominantly prostatic origin and seem to have no influence on the quality of the ejaculate.
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PMID:Fibrinolytic parameters in spermatozoas and seminal plasma. 190 31

The concentrations of cathepsin D (Cath D), urokinase (uPA) and two plasminogen activator inhibitors (PAI-1 and PAI-2) were analysed in the cytosols of 130 human mammary tumours (43 benign tumours and 87 primary and unilateral breast carcinomas). uPA, PAI-1 and PAI-2 levels were measured by antigenic immunoassays and Cath D by immunoradiometric assay. The median levels of the four parameters were significantly higher in the malignant tumours than in the benign ones. Cath D and uPA increases were 4-fold and 5-fold respectively. PAI-1 and PAI-2 increases were much more important, 74-fold and 29-fold respectively. In malignant tumours, median levels of Cath D and uPA did not vary according to classical prognostic factors (histologic grade, presence or absence of axillary lymph nodes, steroid receptors, UICC stage, tumour size, age, and menopausal status). However, PAI-1 decreased in ER+ and PR+ tumours and PAI-2 increased in menopausal women's tumours. When Cath D, uPA, PAI-1 and PAI-2 levels in malignant tumours were compared, positive correlations were found for all combinations. The implication of plasminogen activator inhibitors in the phenomenon was surprising and merits further investigation using tools other than global antigen measurements in tumours.
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PMID:Relationship between cathepsin D, urokinase, and plasminogen activator inhibitors in malignant vs benign breast tumours. 193 18


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