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

A hybrid hybridoma (FU1-74), secreting a bispecific monoclonal antibody (bs mAb), was obtained by fusion of a murine hybridoma secreting a monoclonal antibody (mAb) specific for human fibrin with a murine hybridoma secreting a mAb against urokinase-type plasminogen activator (u-PA). The bs mAb (MA-FU1-74), purified to homogeneity from mouse ascitic fluid, migrated as a single band with apparent Mr 150,000 on nonreduced SDS-PAGE and had an affinity for both human fibrin (Ka = 2 x 10(7) M-1) and for u-PA (Ka = 10(8) M-1) comparable to that of the mAbs obtained from the respective parental hybridomas. MA-FU1-74 did not influence the enzymatic activity of two-chain u-PA (tcu-PA) towards plasminogen or towards a chromogenic substrate. The complex of MA-FU1-74 with recombinant single chain u-PA (rscu-PA) or with tcu-PA (urokinase) enhanced the fibrinolytic potency of the plasminogen activator towards clotted human plasma 20-fold and 5-fold, respectively. In a hamster pulmonary embolism model, the rscu-PA/MA-FU1-74 complex had a 13- to 17-fold increased thrombolytic potency (percent lysis per mg/kg u-PA administered) relative to that of rscu-PA. The specific thrombolytic activity (percent lysis per microgram/ml steady state plasma level of u-PA antigen) of the complex was, however, not significantly different from that of rscu-PA. The complex of rscu-PA with the parental anti-u-PA mAb (MA-UK1-3) had only a 2-fold enhanced thrombolytic potency relative to that of rscu-PA and had a 5-fold decreased specific thrombolytic activity. The plasma clearance rates of the complexes of rscu-PA with both MA-FU1-74 and MA-UK1-3 were about 10-fold lower than that of rscu-PA. In a rabbit jugular vein thrombosis model, the rscu-PA/MA-FU1-74 complex had a 4-fold enhanced thrombolytic potency, an unchanged specific thrombolytic activity and 20-fold reduced plasma clearance. In both animal models, the rscu-PA/MA-FU1-74 complex did not cause more extensive systemic activation of the fibrinolytic system than rscu-PA. It is concluded that the bispecific anti-fibrin/anti-u-PA mAb MA-FU1-74 targets u-PA to the fibrin clot, resulting in a significantly enhanced thrombolytic potency of the plasminogen activator.
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PMID:Enhancement of clot lysis in vitro and in vivo with a bispecific monoclonal antibody directed against human fibrin and against urokinase-type plasminogen activator. 179 14

The purity, composition and in vitro fibrinolytic activity of four commercially available fibrinolytic agents, alteplase (recombinant tissue plasminogen activator, rt-PA, Actilyse; CAS 105857-23-6), streptokinase, urokinase and anistreplase (ansioyl-plasminogen-streptokinase activator-complex, APSAC), have been compared in this investigation. The fibrinolytic activity was measured in an in vitro thrombolytic assay. In this assay a human blood thrombus is dissolved in an environment of human plasma. This assay is representative for the in vivo situation, where plasminogen activation is also a limiting step in thrombolysis. In the in vitro thrombolytic assay alteplase is about 10 times more effective in clot lysis than either streptokinase or urokinase and more than 300 times more active than anistreplase. In addition, the ratio of active ingredient to total protein content in the preparations was analysed by RP-HPLC, SDS-PAGE, GPC-HPLC and amino acid analysis. The portion of active ingredient per total protein was 99.9% for alteplase, 55% for anistreplase, 20% for urokinase and 1% for streptokinase. This demonstrates that alteplase is the only fibrinolytic agent tested which is essentially free of protein additives of human origine and potential contaminants associated therewith. The superior purity of alteplase compared to the other fibrinolytics was confirmed by SDS-PAGE, RP-HPLC, and HPLC-GPC. Significant levels of aggregates were detected in streptokinase and urokinase preparations, whereas alteplase and anistreplase were essentially free of aggregates. These data demonstrate that there are significant differences in composition, purity and in vitro activity between different fibrinolytic agents.
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PMID:Quality aspects of fibrinolytic agents based on biochemical characterization. 181 Feb 68

Prourokinase is a plasminogen activator of 411 amino acids which displays a clot-lysis activity through a fibrin-dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction. The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain. In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichia coli as inclusion bodies: prourokinase and a deletion derivative (delta 125-prourokinase) in which 125 amino acids of the N-terminal region have been removed. Deletion of this sequence brings to higher refolding yields and faster kinetics (first-order rate constant of renaturation of 0.57 h-1 for delta 125-prourokinase and 0.25 h-1 for prourokinase). Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain. When applied to pure, non-glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure. Lower yields (15% and 30%, respectively, for prourokinase and delta 125-prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria. After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160,000 and 250,000 IU/mg protein, respectively, for prourokinase and delta 125-prourokinase. As with prourokinase, the deletion mutant delta 125-prourokinase displays a zymogenic nature, being activated by plasmin to the active two-chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile.
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PMID:Efficient renaturation and fibrinolytic properties of prourokinase and a deletion mutant expressed in Escherichia coli as inclusion bodies. 184 67

A recombinant deletion mutant of the 155-amino acid form of human basic fibroblast growth factor (bFGF), lacking amino acid residues 27-32 (Lys-Asp-Pro-Lys-Arg-Leu), was expressed in Escherichia coli and purified to homogeneity by heparin-Sepharose affinity chromatography. When maintained in the presence of an equimolar concentration of soluble heparin, the bFGF mutant (M1-bFGF) is as potent as bFGF in stimulating cell proliferation in normal and transformed fetal bovine aortic endothelial cells, in adult bovine aortic endothelial cells, and in NIH 3T3 fibroblasts. However, under the same experimental conditions, M1-bFGF is at least 100 times less efficient than bFGF in stimulating plasminogen activator (PA) production in endothelial cells, as assayed by chromogenic PA assay, SDS/PAGE zymography, and Northern blot analysis of urokinase-type PA mRNA. In the presence of heparin, M1-bFGF binds to bFGF plasma membrane receptors present on endothelial cells in a manner undistinguishable from bFGF. It also induces the same tyrosine phosphorylation pattern when added to NIH 3T3 cells. The data suggest that the PA-inducing activity of bFGF may depend upon a functional domain that differs from those involved in the mitogenic activity of the growth factor and that the binding of bFGF to its plasma membrane receptor may not be sufficient to induce urokinase-type PA production in endothelial cells.
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PMID:A six-amino acid deletion in basic fibroblast growth factor dissociates its mitogenic activity from its plasminogen activator-inducing capacity. 184 69

The possible carcinogenicity of insoluble chromium (VI) compound, PbCrO4, in human cells has been tested using a nontumorigenic human osteosarcoma cell line (HOS, TE 85). Electron microscopic studies show that PbCrO4 is phagocytosed by HOS cells and accumulates within the vacuoles in the cytoplasm. A number of cell lines have been isolated following multiple treatment of HOS cells with PbCrO4. These cell lines are morphologically different from HOS cells, form anchorage-independent colonies in soft agar and form quickly regressing small tumor nodules in athymic nude mice. The cellular and secreted plasminogen activator (PA) levels of 5 cell lines isolated after PbCrO4 treatment are increased up to 8 fold and up to 10 fold respectively as compared to untreated HOS controls. SDS-PAGE analysis in the presence of copolymerized substrates is consistent with increase in 55 kDa urokinase-type PA (u-PA) and 68 kDa tissue-type PA (t-PA). These results show that PbCrO4 treatment leads to stable phenotypic changes indicative of the transformation of HOS cells.
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PMID:Induction of morphological transformation, anchorage-independent growth and plasminogen activators in non-tumorigenic human osteosarcoma cells by lead chromate. 188 37

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

The plasminogen activator was purified to the extent of 150-fold from 20,000 x g supernatant of Yoshida ascites Sarcoma by ammonium sulphate precipitation at 33% saturation followed by affinity chromatography on p-aminobenzamidine-Sepharose 4B. The specific activity of the purified activator was 10,260 IU/mg expressed in terms of International units of urokinase, the known activator of plasminogen. The activator was homogeneous by polyacrylamide slab gel electrophoresis with an apparent molecular weight 75 kDa by gel filtration on Sephadex G-100. Analysis by SDS-polyacrylamide gel electrophoresis under reducing conditions, revealed the presence of two subunits of about 48 and 29 kDa. The activator displayed binding preference to fibrin and was immunologically distinguishable from urokinase, indicating that it could be of non-urokinase origin. The preparation further revealed similarity to standard tissue plasminogen activator with respect to fibrin binding and immunological cross reactivity.
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PMID:Isolation and purification of plasminogen activator from Yoshida ascites Sarcoma of rats. 190 68

A protein factor which stimulated [3H]thymidine uptake into free hepatocytes prepared from normal mouse liver was detected in the ascitic fluid of gynecological cancer patients. The factor was subsequently further purified from the ascitic fluid of an endometrial cancer patient by DEAE-Sephacel, Sephadex G-150 and Phenyl-Sepharose CL-4B column chromatographies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single protein band of 54,000 Da, designated tentatively as 54K ascitic protein (54K-AP). 54K-AP was similar to human alpha 1-antitrypsin (alpha 1-AT) in terms of SDS-PAGE and immunological behavior, but was slightly different in terms of amino acid sequence and isoelectric point. Although 54K-AP inhibited the activities of bovine trypsin and alpha-chymotrypsin as did human alpha 1-AT, 54K-AP inhibited the plasminogen activator released from human endometrial cancer Ishikawa cells more efficiently than alpha 1-AT. Because, in contrast to normal serum, the serum from the endometrial cancer patients stimulated [3H]thymidine uptake into hepatocytes, the possibility arises that 54K-AP could be produced by the cancer host as a defence mechanism against the cancer.
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PMID:Characterization of a 54 kDa, alpha 1-antitrypsin-like protein isolated from ascitic fluid of an endometrial cancer patient. 190 55

Interleukin-1 (IL-1) release from monocyte-macrophages (Mo) appears dependent on pericellular proteolysis mediated by plasmin. Thus plasminogen activator inhibitors (PAI) which bind the serine proteases responsible for the conversion of plasminogen to plasmin, may inhibit IL-1 release from Mo. We have examined the effect of purified PAI from a hepatoma cell line Hep G2, on IL-1 release from Mo with secondary effects on lymphocyte proliferation in vitro. Fast acting inhibitors of both urokinase (u-PA) and tissue plasminogen activator (two chain t-PA) were noted in harvest fluids of Hep G2 cells. These inhibitors were stable at pH 3 but lost activity at 45 degrees C. They were SDS-stable and migrated with Mr53 and 104 kDa. These properties conformed to characteristics of type-1 plasminogen activator inhibitor (PAI-1). Partially purified PAI-1 added to human Mo cultured on 125I fibrin layer both in the presence and absence of plasminogen inhibited secretion of IL-1 by Mo in response to LPS. This effect, however, did not correlate with the inhibition of plasminogen dependent fibrinolysis. This suggested a degree of sequestration and inaccessibility of membrane bound u-PA of LPS activated Mo to PAI-1. PAI-1, in addition, inhibited mitogen stimulated peripheral blood mononuclear cell (PBMC) proliferation at similar concentration ranges. This effect was abrogated by the addition of specific antisera to PAI-1. PAI-1 may be released as part of an acute phase response. In addition to influencing fibrinolysis, PAI-1 may constitute a negative feedback pathway on Mo IL-1 release and subsequent immune activation in vivo.
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PMID:Monocyte-macrophage release of IL-1 is inhibited by type-1 plasminogen activator inhibitors. 196 70

The molecular interactions involved in the fibrin-mediated stimulation of plasminogen activation by tissue-type plasminogen activator (t-PA) were studied using natural human plasminogen (nPlg) and rPlg-Ala740, a recombinant human plasminogen in which the catalytic site is destroyed by mutagenesis of the active site Ser740 to Ala. Using this rPlg-Ala740 moiety, the dissociation constant of the interaction between plasminogen and CNBr-digested fibrinogen was determined to be 0.40 microM. In addition, conversion of 125I-labeled single chain plasminogen to two chain plasmin by single chain recombinant t-PA (rt-PA) in the absence or the presence of CNBr-digested fibrinogen was quantitated on reduced SDS-gel electrophoresis, combined with autoradiography and radioisotope counting of gel bands. In the absence of fibrin, the activation rate of nPlg and rPlg-Ala740 by single-chain rt-PA was comparable. In the presence of fibrin, however, the activation rate of rPlg-Ala740 was about 20-fold lower than that of nPlg. These results with rPlg-Ala740 may be explained by an impaired formation of the stable cyclic ternary complex between plasminogen, t-PA and fibrin, which mediates the fibrin stimulation of plasminogen activation by t-PA or, alternatively, by impaired conversion of single chain rt-PA to two chain rt-PA at the fibrin surface.
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PMID:On the molecular interactions between fibrin, tissue-type plasminogen activator and plasminogen. 210 98


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