Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recombinant tissue-plasminogen activator (r-tPA), expressed in Escherichia coli cells in an aggregated form, was solubilized with a strong chaotrope in the absence of any reducing agent. The solubilized molecule was reactivated by a procedure that was developed to mimic the physiological conditions optimal for the functional folding and activity of the native protein. The use of partially purified fibrinogen, as a source of fibrin (the effector), is shown to facilitate the reactivation process and increase its yield by at least a factor of two. The yield of the process is also shown to be particularly dependent on the recombinant protein concentration. At a concentration level of 3-3.7 mg r-tPA/L in the reactivation mixture, up to a 90% yield of activity was obtained. Purification of the activated form of r-tPA was achieved with a two-step column-chromatography scheme. This included a gel filtration step on a Sephadex G-50 column followed by an affinity chromatography step on a lysine-sepharose column. The product was composed of roughly equal amounts of one-chain and two-chain t-PA. The feasibility of using a two water-soluble polymeric phase system, with a centrifugal partition chromatography (CPC), in scaling up the reactivation process or the purification step was also evaluated.
 ...
PMID:Effector-assisted refolding of recombinant tissue-plasminogen activator produced in Escherichia coli. 138 Jul 90

In previous publications [e.g. Voskuilen, Vermond, Veeneman, Van Boom, Klasen, Zegers & Nieuwenhuizen (1987) J. Biol. Chem. 262, 5944-5946] we have shown that fibrin(ogen) chain fragment A alpha-(148-160) contains a site that contributes to the acceleration of Glu-plasminogen activation by tissue-type plasminogen activator (t-PA). In contrast with fibrin, this peptide, however, does not enhance the rate of mini-plasminogen activation. Therefore, possibly more stimulatory sites than A alpha-(148-160) are present in fibrin. In the present investigation we have localized a possible second type of stimulatory site in the fibrin(ogen) molecule. A whole CNBr digest of fibrinogen was applied to a Bio-Gel P-2 column run in water, pH 4. Two peaks with stimulatory activity were observed, one at the void volume and one between the void volume and the total volume. The former contained the previously described stimulating fragment FCB-2 [which comprises A alpha-(148-160)]; the latter had not been observed before and was characterized further. The stimulating material in the low-M(r) fraction of the Bio-Gel P-2 column was precipitated at pH 8.3 in a virtually pure form. It has a high tryptophan content, and an M(r) of 6500 as assessed by SDS/PAGE. On reduction, a main band of M(r) 2500 is seen, plus a weakly staining band of M(r) 4000. These properties plus the amino acid sequence data identify the fragment as FCB-5. FCB-5 consists of two chains, i.e. gamma-(311-336) and gamma-(337-379), linked by a single disulphide bond between Cys-gamma-326 and Cys-gamma-339. Both these chains and the disulphide bond appear to be essential for rate enhancement. FCB-5 enhances the activation rates of Glu-, mini- and micro-plasminogen, with all five kringles, only kringle V and without kringles respectively. FCB-5 binds t-PA, but none of the plasminogen forms binds to FCB-5. This indicates that the rate enhancements induced by FCB-5 are due to an effect on t-PA.
 ...
PMID:Localization in the fibrinogen gamma-chain of a new site that is involved in the acceleration of the tissue-type plasminogen activator-catalysed activation of plasminogen. 156 67

In the normal stomach of male rats marked differences in plasminogen activator activity (PAA) and plasmin inhibition (PI), but not in plasminogen activator inhibition (PAI), were noted among cardiac area, body and pyloric region. Chronic ethanol consumption (for 15 or 30 days) at the concentration of 6% or 12% in the drinking water induced an increase in PAA in the pyloric region and the body of the stomach (the higher concentration after 15 days and both concentrations after 30 days). The response was time- and dose-dependent. At the cardiac area no change of PAA was noted. Ethanol at both concentrations induced after 30 days a decreased PAI in the pyloric region and the body of the stomach, which was expressed against u-PA, but not against t-PA. A decreased PI was noted at both concentrations of ethanol after 30 days only in the pyloric region. Therefore, changes in PAA, PAI and PI after chronic ethanol consumption were dependent on the concentration, the period of the consumption and the area along the gastric wall.
 ...
PMID:Enhancement of plasminogen activator activity in the gastric wall after chronic ethanol consumption. 182 83

The trypsin inhibitor DE-3 from Erythrina caffra (ETI) belongs to the Kunitz-type soybean trypsin inhibitor (STI) family and consists of 172 amino acid residues with two disulphide bridges. The amino acid sequence of ETI shows high homology to other trypsin inhibitors from the same family but ETI has the unique ability to bind and inhibit tissue plasminogen activator. The crystal structure of ETI has been determined using the method of isomorphous replacement and refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement. The refined model includes 60 water molecules and 166 amino acid residues, with a root-mean-square deviation in bond lengths from ideal values of 0.016 A. The crystallographic R-factor is 20.8% for 7770 independent reflections between 10.0 and 2.5 A. The three-dimensional structure of ETI consists of 12 antiparallel beta-strands joined by long loops. Six of the strands form a short antiparallel beta-barrel that is closed at one end by a "lid" consisting of the other six strands coupled in pairs. The molecule shows approximate 3-fold symmetry about the axis of the barrel, with the repeating unit consisting of four sequential beta-strands and the connecting loops. Although there is no sequence homology, this same fold is present in the structure of interleukin-1 alpha and interleukin-1 beta. When the structure of ETI and interleukin-1 beta are superposed, the close agreement between the alpha-carbon positions for the beta-strands is striking. The scissile bond (Arg63-Ser64) is located on an external loop that protrudes from the surface of the molecule and whose architecture is not constrained by secondary structure elements, disulphide bridges or strong electrostatic interactions. The hydrogen bonds made by the side-chain amide group of Asn12 play a key role in maintaining the three-dimensional structure of the loop. This residue is in a position corresponding to that of a conserved asparagine in the Kazal inhibitor family. Although the overall structure of ETI is similar to the partial structure of STI, the scissile bond loop is displaced by about 4 A. This displacement probably arises from the fact that the structure of STI has been determined in a complex with trypsin but could possibly be a consequence of the close molecular contact between Arg63 and an adjacent molecule in the crystal lattice.
 ...
PMID:Crystal structure of a Kunitz-type trypsin inhibitor from Erythrina caffra seeds. 198 76

The kinetic behavior of fibrin clot lysis as induced by tissue-type plasminogen activator (t-PA) was studied using proton magnetic resonance (PMR) and a release assay of fibrin labeled with technetium-99m isotope (99mTc). The lysis pattern of the preformed clot was examined as a function of gradual changes in the amounts of added t-PA and deactivated t-PA. The behavior of fibrinolysis was found to depend strongly on the amount of t-PA in the assay, which markedly affects the lysis rate of fibrin. The changes induced by the lysis were reflected in pronounced prolongation of the transverse relaxation time. The PMR and the radioisotope release measurements point to the possibility that at least two steps are involved in the mechanism of lysis. The PMR seems to be associated with structural features of the clot and reflects the liberation of compartmentalized water from the clot, while the 99mTc analysis reflects the further fragmentation of fibrin.
 ...
PMID:Structural changes in fibrin clot associated with the proteolytic activity induced by tissue type plasminogen activator. An NMR study. 249 19

The clot uptake of labeled active and inhibited t-PA was compared. The most efficient inhibition was obtained with diisopropyl fluorophosphate (DFP) after 4 h incubation at room temperature. Enzyme activity was followed by fibrin-plate assay, radioactivity-release technique and proton magnetic resonance (PMR). The novel PMR method developed by us is sensitive to the effect of as low as nanogram amounts of t-PA on the interaction between the fibrin and the compartmentalized water trapped in the clot. Binding of labeled enzyme to fibrin-coated plates showed that the deactivation by DFP did not impair the affinity of t-PA for fibrin. A rapid binding of 125I-labeled t-PA to the clot occurred, which reached a maximum in 30 min and declined with time. This pattern was explained by consecutive clot binding and lysis. The binding of DFP-t-PA to the clot differed markedly from that of the active protein; 2 h post-incubation the uptake of DFP-t-PA was more than double that of the untreated t-PA. Parallel measurements in clots prepared from human blood showed a qualitatively similar trend. The biodistribution of radiolabeled t-PA in mice was similar for the active and inhibited forms. Blood activity reached 10% of the injected dose within 10 min. DFP-t-PA may prove to be a useful reagent for in-vivo localization of thrombi.
 ...
PMID:Clot uptake of labeled active and inhibited tissue plasminogen activator. 249 63

Tissue plasminogen activator labeled with radioactive iodine (125I-tPA) was immobilized on vascular prostheses chemically modified with a thin coating of water-insoluble surfactant, tridodecylmethylammonium chloride (TDMAC). Surfactant-treated Dacron, polytetrafluoroethylene (PTFE), silastic, polyethylene and polyurethane bound appreciable amounts of 125I-tPA (5-30 micrograms 125I-tPA/cm2). Upon exposure to human plasma, the amount of 125I-tPA bound to the surface shows an initial drop during the first hour of incubation, followed by a slower, roughly exponential release with a t 1/2 of approximately 75 hours. Prostheses containing bound tPA show fibrinolytic activity as measured both by lysis of clots formed in vitro, and by hydrolysis of a synthetic polypeptide substrate. Prior to incubation in plasma, tPA bound to a polymer surface has an enzymic activity similar, if not identical to that of the native enzyme in buffered solution. However, exposure to plasma causes a decrease in the fibrinolytic activity of both bound tPA and enzyme released from the surface of the polymer. These data demonstrate that surfactant-treated prostheses can bind tPA, and that these chemically modified devices can act as a slow-release drug delivery system with the potential for reducing prosthesis-induced thromboembolism.
 ...
PMID:Binding of tissue plasminogen activator to vascular grafts. 250 90

Since streptokinase and urokinase became available for clinical use, numerous attempts have been made to improve these useful thrombolytic agents. To decrease its antigenicity, streptokinase has been fragmented or coupled to human plasminogen or polyethylene glycols. With a plasmin B chain-streptokinase complex a more potent agent was obtained. To prolong their half-life, streptokinase and urokinase were immobilized with water-soluble carriers. Coupling urokinase with fibrin-specific antibodies increases its thrombolytic efficacy, at least in vitro. The only thrombolytic agents with a relative fibrin specificity available for clinical purposes are tissue-type plasminogen activator and single chain urokinase-type plasminogen activator. Mutants and hybrids of these molecules are being constructed and may further improve their fibrin specificity and therapeutic potential.
 ...
PMID:The search for the ideal thrombolytic agent. 295 14

Since streptokinase and urokinase became available for clinical use, numerous attempts have been made to improve these useful thrombolytic agents. In order to decrease its antigenicity, streptokinase has been fragmented or coupled to human plasminogen or polyethylene glycols. With a plasmin B-chain-streptokinase complex a more potent agent was obtained. To prolong their half-life, streptokinase and urokinase were immobilized with water-soluble carriers. Coupling urokinase or tissue-type plasminogen activator with fibrin-specific antibodies increases its thrombolytic efficiency, at least in vitro. The only thrombolytic agents with a relative fibrin-selectivity presently available for clinical purposes are tissue-type plasminogen activator and single chain urokinase-type plasminogen activator. Structural manipulation allows to design and produce mutant proteins with specific deletions, additions or substitutions of non-protease domains. Also chimaeric molecules combining fragments of tissue-type plasminogen activator and single chain urokinase-type plasminogen activator have already been constructed. These modifications of natural molecules may further improve their fibrin-specificity and therapeutic potential.
 ...
PMID:[Recent developments in fibrinolysis]. 327 56

The effects of different kinds of acute stress on collagen-induced whole blood platelet aggregation and fibrinolysis in relation to blood serotonergic measures were studied. In rats water-immersion restraint stress resulted in a shortening of euglobulin clot lysis time (ECLT), an increase in tissue plasminogen activator (tPA) activity with a concurrent fall in its inhibitor activity. Footshock caused rather a suppression in fibrinolysis with a prolongation of ECLT and a decline in tPA activity as well as a reduction in whole blood platelet aggregation induced by collagen. Serotonin (5-HT) level, a marker of a severity of stress, increased after footshock application with a concomitant rise in its major metabolite-5-hydroxyindoleacetic acid (5-HIAA). This indicates an enhanced 5-HT metabolism. Following water-immersion restraint stress 5-HT and 5-HIAA levels did not differ from controls. In both groups of stressed animals an inverse correlation between tPA activity and blood serotonin was observed. Our data indicate that these types of stress may influence either fibrinolysis or peripheral serotonergic mechanism in different ways. Acute and severe stress such as footshock by causing an impairment in fibrinolysis and a rise in 5-HT may contribute to the pathogenesis of thrombosis and henceforth to the development of atherosclerosis.
 ...
PMID:Stress-dependent changes in fibrinolysis, serotonin and platelet aggregation in rats. 751 40


1 2 3 4 5 6 7 8 9 Next >>