Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

K1K2Pu, a recombinant t-PA/u-PA chimera with increased thrombolytic potency in animal models of venous and arterial thrombosis, which consists of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA), was produced and conditioned for use in patients. Chinese hamster ovary cells were transfected with an expression plasmid containing the K1K2Pu cDNA, high producer cell lines were selected and scaled up in 800 cm2 roller bottles, and 350 ml conditioned cell culture medium was harvested 3 to 7 times at 2 to 5 day intervals. Batches of 21 +/- 4 liter (mean +/- SD, n = 28) containing 1.8 +/- 0.6 mg/l of K1K2Pu related antigen were purified by chromatography on Copper chelate-Sepharose and immunoadsorption on an insolubilized murine monoclonal antibody (MA-1C8). Yields were 8.6 +/- 3.4 mg K1K2Pu per batch with a specific activity of 83,000 +/- 44,000 IU/mg. The final material, obtained at a concentration of approximately 0.7 mg/ml, was dialyzed against 0.3 M NaCl, 0.02 M Tris-HCl buffer, pH 7.5, containing 0.01% Tween 80 and 10 KIU/ml aprotinin. It was homogeneous on SDS-PAGE, contained 6.5 +/- 6.9 percent two chain material and the contamination with murine monoclonal antibody was less than 0.1 percent. After filtration of pools of 3 to 5 selected batches on 0.22 microns Millipore filters the material was sterile and virus free by routine screening; it was obtained at a concentration of approximately 0.5 mg/ml with a specific activity of 110,000 +/- 16,000 IU/mg (mean +/- SD, n = 3) and an endotoxin content of 0.5 to 7 units/mg. Bolus injection at a dose of 1 mg/kg in mice did not produce weight loss within 8 days. Thus, this material appears to be suitable for the investigation on a pilot scale of the pharmacokinetic and thrombolytic properties of K1K2Pu in patients with thromboembolic disease.
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PMID:K1K2Pu, a recombinant t-PA/u-PA chimera with increased thrombolytic potency, consisting of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA). Purification in centigram quantities and conditioning for use in man. 163 5

The concept of the haemostatic balance was reviewed, and its potential role in the regulation of tissue repair and the pathogenesis of thrombotic processes was surveyed. Physiological activation of coagulation appears to be dominated by effects of degenerated and injured cells of the vascular wall causing local release of thromboplastin and exposition of activating surfaces. Inhibition of coagulation impairs its progression and the non-thrombogenic nature of the normal endothelium is chiefly caused by the binding of inhibitory components (antithrombin-III, protein C) to specific receptor sites. Physiological activation of fibrinolysis appears to be triggered by and limited to the fibrin because of a specific affinity to fibrin of plasminogen and plasminogen activators. Systemic activation of fibrinolysis is prevented by primary (alpha 2-antiplasmin) and secondary (alpha 2-macroglobulin, alpha 1-antitrypsin) plasmin inhibitors. A plasminogen binding protein (histidine-rich glycoprotein), plasmin inhibitors and activator inhibitors appear to contribute to the regulation of the initial phase of fibrinolysis. A deviation from normal of the dynamic balance, regulating fibrin formation and resolution, may lead to a haemorrhagic and/or a thrombophilic state. Described were the optimization of selected methods for assessment of variables involved in the haemostatic balance. An overestimation of plasminogen concentrations in plasma may occur in patients with elevated levels of fibrinogen or fibrin degradation products, when using assays based on the activation of plasminogen by streptokinase followed by the hydrolysis of a synthetic chromogenic substrate. This source of error could be eliminated by presence of fibrinogen in excess in the plasminogen assay, thereby securing maximum stimulation of the plasminogen-streptokinase complex. The presence of cryoglobulin in plasma interferes with the assessment in euglobulins of plasminogen activator activities. Experiments indicate that tissue-type plasminogen activator adsorb cryoglobulins and that a cold-promoted activation of the factor XII-dependent proactivator system of fibrinolysis is related to the presence of cryoglobulins. Experiments supported the existence of an as yet not characterized factor XII-dependent proactivator. Strictly optimized procedures for the preparation of euglobulins for the accurate determination of plasminogen activators were recommended. The determination of plasminogen activator inhibition in plasma was optimized and simplified. The amidolytic assay of antithrombin-III was shown to be influenced by adsorption to laboratory utensils and aggregation of thrombin. This error could be corrected by protection with additives (Tween 80, polyethyleneglycol 6,000), which also improved the solubility of the chromogenic substrates in aqueous media. The role of thrombosis in myocardial infarction was reviewed.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The haemostatic balance in groups of thrombosis-prone patients. With particular reference to fibrinolysis in patients with myocardial infarction. 219 35

The plasminogen activator secreted by a cultured human melanoma cell line was purified and compared with urokinase and with tissue plasminogen activator from human uterus. The purification procedure consisted of chromatography on zinc chelate-agarose, concanavalin A-agarose, and Sephadex G-150 in the presence of 0.01% (v/v) Tween 80. The purified material was obtained from the culture medium with a yield of 46% and a purification factor of 263. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one main band with a molecular weight of about 72,000, and in the presence of reducing agents, two bands of 33,000 and 39,000. Addition of the protease inhibitor Aprotinin to the culture media and column buffers yielded a one-chain plasminogen activator with a molecular weight of about 72,000. One molecule of activator reacted with about one molecular of [3H]diisopropylfluorophosphate. The melanoma plasminogen activator and the uterine tissue plasminogen activator appeared to be very similar on dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis, and amidolytic properties. Both activators bound to fibrin clots, while urokinase did not. In immunodiffusion, as well as in quenching experiments of the fibrinolytic activities, the melanoma plasminogen activator appeared to be immunologically identical with the uterine tissue plasminogen activator, but unrelated to urokinase. All these findings indicate that the plasminogen activator secreted by human melanoma cells in culture is very similar to, or identical with, the plasminogen activator found in normal tissue, but different from urokinase.
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PMID:Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. 678 58

The plasminogen activator secreted by a cultured rat brain tumor cell line (RT4-71-1) (Imada M. and Sueoka N., Develop. Biol. 69, 97-107, 1978) was purified by chromatography on zinc chelate-agarose, concanavalin A-agarose and Sephadex G-150 in the presence of 0.01% (vol/vol) Tween 80. Aprotinin was added to the culture medium to a concentration of 20 KIU per ml and to the buffers in the first two chromatographic steps to a concentration of 10 KIU per ml. Approximately 90 microgram purified material was obtained from 11 of culture medium with a yield of 39% and a purification factor of 200. Sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence of reducing agents showed one main band with Mr of about 60,000, and a minor band with Mr about 30,000. Fibrinolytic activity was associated with the main band. The rat brain tumor plasminogen activator bound to a fibrin clot to a similar extent as human tissue plasminogen activator, whereas urokinase did not bind. In quenching experiments of the fibrinolytic activities the purified rat brain tumor plasminogen activator appeared to be immunologically related to the human tissue plasminogen activator but unrelated to urokinase.
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PMID:Purification and characterization of the plasminogen activator secreted by a rat brain tumor cell line in culture. 719 1

Nanoparticle drug carriers consist of solid biodegradable particles in size ranging from 10 to 1000 nm (50-300 nm generally). They cannot freely diffuse through the blood-brain barrier (BBB) and require receptor-mediated transport through brain capillary endothelium to deliver their content into the brain parenchyma. Polysorbate 80-coated polybutylcyanoacrylate nanoparticles can deliver drugs to the brain by a still debated mechanism. Despite interesting results these nanoparticles have limitations, discussed in this review, that may preclude, or at least limit, their potential clinical applications. Long-circulating nanoparticles made of methoxypoly(ethylene glycol)- polylactide or poly(lactide-co-glycolide) (mPEG-PLA/PLGA) have a good safety profiles and provide drug-sustained release. The availability of functionalized PEG-PLA permits to prepare target-specific nanoparticles by conjugation of cell surface ligand. Using peptidomimetic antibodies to BBB transcytosis receptor, brain-targeted pegylated immunonanoparticles can now be synthesized that should make possible the delivery of entrapped actives into the brain parenchyma without inducing BBB permeability alteration. This review presents their general properties (structure, loading capacity, pharmacokinetics) and currently available methods for immunonanoparticle preparation.
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PMID:Drug transport to brain with targeted nanoparticles. 1571 62

Polysorbate 80 (Tween 80) has been widely used as an emulsifier with excellent effects in nanoparticles technology for biomedical applications. This work was thus triggered to synthesize poly(lactide)/Tween 80 copolymers with various copolymer blend ratio, which were synthesized by ring-opening polymerization and characterized by 1H NMR and TGA. Nanoparticles of poly(lactide)/Tween 80 copolymers were prepared by the dialysis method without surfactants/emulsifiers involved. Paclitaxel was chosen as a prototype anticancer drug due to its excellent therapeutic effects against a wide spectrum of cancers. The drug-loaded nanoparticles of poly(lactide)/Tween 80 copolymers were then characterized by various state-of-the-art techniques, including laser light scattering for particles size and size distribution, field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) for surface morphology; laser Doppler anemometry for zeta potential; differential scanning calorimetry (DSC) for the physical status of the drug encapsulated in the polymeric matrix; X-ray photoelectron spectrometer (XPS) for surface chemistry; high performance liquid chromatography (HPLC) for drug encapsulation efficiency; and in vitro drug release kinetics. HT-29 cells and Glioma C6 cells were used as an in vitro model of the GI barrier for oral chemotherapy and a brain cancer model to evaluate in vitro cytotoxicity of the paclitaxel-loaded nanoparticles. The viability of C6 cells was decreased from 37.4 +/- 4.0% for poly(D,L-lactide-co-glycolic acid) (PLGA) nanoparticles to 17.8 +/- 4.2% for PLA-Tween 80-10 and 12.0 +/- 5.4% for PLA-Tween 80-20 copolymer nanoparticles, which was comparable with that for Taxol at the same 50 microg/mL drug concentration.
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PMID:In vitro investigation on poly(lactide)-Tween 80 copolymer nanoparticles fabricated by dialysis method for chemotherapy. 1660 31

The purpose of this work was to study the effect of organic solvent and surfactant type on the in vitro release behavior in general and on the burst release in particular of beta-estradiol from PLA/PLGA microspheres. Also the effect of these variables on the encapsulation efficiency was investigated. The microspheres were prepared by solvent evaporation technique using dichloromethane (DCM), ethyl acetate (EtAc), tetrahydrofuran (THF), chloroform (CHCl3) or acetone (AC) as organic solvent and polyvinyl alcohol (PVA), Tween 80, sodium lauryl sulfate (SLS) or benzalkonium chloride (BKCI) as surfactant. The obtained microspheres were tested for encapsulation efficiency and in vitro drug release using 50% methanol/buffer pH 7.4 as dissolution medium. EtAC and PVA formulations showed the highest encapsulation efficiency and the lowest burst release. These microspheres were further characterized for particle size distribution, SEM and zeta potential. The results suggested that these materials could be starting materials to prepare a beta-estradiol biodegradable controlled delivery system.
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PMID:beta-Estradiol biodegradable microspheres: effect of formulation parameters on encapsulation efficiency and in vitro release. 1702 Jan 54

The chemical and physical stability of polymeric nanoparticles is poor in aqueous suspensions, and the drying of these particles is often problematic. In the present study, the stability of freeze-dried low molecular weight poly(L-lactic acid) (PLA) nanoparticles was enhanced by adding glucose and/or lactose to the formulation as cryo- and lyoprotectants, respectively. Also the effect of an extra stabilizer, Tween 80, was studied. The best freeze-dried PLA nanoparticle formulations were achieved, when glucose and lactose were added in combination so that the amount of lactose was double the amount of glucose. With this combination the redispersion of high-quality nanoparticles (homogenous particle dispersion with original size and without aggregates) was achieved. The addition of Tween 80 further improved the quality of freeze-dried PLA nanoparticles by facilitating the redispersion of the lyophilized cake into optimal nanoparticles.
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PMID:Freeze-drying of low molecular weight poly(L-lactic acid) nanoparticles: effect of cryo- and lyoprotectants. 1704 25

The aim of this work was to develop a stable injectable formulation of the antimalarial drug halofantrine (Hf) based on nanocapsules (NC) prepared from biodegradable polymers with Miglyol 810N as the oily core. Poly(D,L-lactide) PLA and its copolymers with poly(ethyleneglycol) (PLA-PEG) were used together with the surfactants poloxamer 188 and lecithin to yield NC with different surface properties. Highly efficient loading of the free base form of Hf was obtained; zeta potential measurements indicated that a part of the associated Hf was at the NC surface, interacting with the lecithin. NC were 150-250 nm in diameter and more stable on storage than nanoemulsions formed from oil and lecithin without polymer. The most stable NC, showing minimal size changes and flocculation, were those with a high density of 20-kDa PEG chains covalently grafted at the surface. Hf release from NC occurred mainly by partition with the external medium. In PBS, even when Tween 80 was added, release was limited to 20% of the total content, whatever the formulation. Addition of serum to the medium allowed complete and rapid release from PLA NC stabilized with adsorbed poloxamer 188, because of the high affinity of Hf for lipoproteins. However, the presence of covalently grafted PEG chains at the surface limited release by providing a hydrophilic steric barrier at the particle surface. A dense coverage with long PEG chains provided the best reduction of release. Such systems could constitute a long-circulating intravenous formulation of Hf for treating severe malaria.
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PMID:Surface-modified and conventional nanocapsules as novel formulations for parenteral delivery of halofantrine. 1704 36

PAI-749 is a potent and selective synthetic antagonist of plasminogen activator inhibitor 1 (PAI-1) that preserved tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) activities in the presence of PAI-1 (IC(50) values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophore-tagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent K(d) (254 nM) was similar to the IC(50) (140 nM) for PAI-NBD119 inactivation. PAI-749 analogs displayed the same potency rank order for neutralizing PAI-1 activity and perturbing PAI-NBD119 Fl; hence, binding of PAI-749 to PAI-1 and inactivation of PAI-1 activity are tightly linked. Exposure of PAI-1 to PAI-749 for 5 min (sufficient for full inactivation) followed by PAI-749 sequestration with Tween 80 micelles yielded active PAI-1; thus, PAI-749 did not irreversibly inactivate PAI-1, a known metastable protein. Treatment of PAI-1 with a PAI-749 homolog (producing less assay interference) blocked the ability of PAI-1 to displace p-aminobenzamidine from the uPA active site. Consistent with this observation, PAI-749 abolished formation of the SDS-stable tPA/PAI-1 complex. PAI-749-mediated neutralization of PAI-1 was associated with induction of PAI-1 polymerization as assessed by native gel electrophoresis. PAI-749 did not turn PAI-1 into a substrate for tPA; however, PAI-749 promoted plasmin-mediated degradation of PAI-1. In conclusion, PAI-1 inactivation by PAI-749 using purified components can result from a dual mechanism of action. First, PAI-749 binds directly to PAI-1, blocks PAI-1 from accessing the active site of tPA, and abrogates formation of the SDS-stable tPA/PAI-1 complex. Second, binding of PAI-749 to PAI-1 renders PAI-1 vulnerable to plasmin-mediated proteolytic degradation.
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PMID:Neutralization of plasminogen activator inhibitor I (PAI-1) by the synthetic antagonist PAI-749 via a dual mechanism of action. 1762 79


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