Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Four large-scale batches of Antihemophilic Factor (AHF, factor VIII) were prepared from plasma derived from 4 to 6-day-old blood applying a method developed for preparation of AHF from fresh frozen plasma. The AHF product was 6 to 9-fold concentrated over plasma with 7 to 10-fold purification and a recovery of 100 to 140 factor VIII units per liter of starting plasma. In terms of purity and yield, this is about half that of AHF obtained from fresh frozen plasma. The AHF concentrate was free of detectable thrombin and plasmin and the solubility of the dry product was comparable to that of the product derived from fresh plasma but the hemoglobin content was slightly increased. After further fractionation with polyethylene glycol (PEG 4000), a highly soluble AHF product 100-fold purified, and 30-fold concentrated, was obtained with 60% factor VIII recovery, which corresponds to a final yield of 60 to 85 factor VIII units per liter of starting plasma.
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PMID:Preparation of antihemophilic factor from indated plasma. 95 29

Human and bovine alpha-thrombin cleaved at the B-chain by chymotrypsin generates catalytically competent zeta-thrombins, which are comprised of two noncovalently linked fragments: a 36-(human) or 49-(bovine) residue A-chain linked by a disulfide to B-chain residues B1-148 (zeta 1-thrombin) and B-chain residues B149-259 (zeta 2-thrombin). Human and bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins were prepared by reaction of the active-site histidine (H-B43) and serine (S-B205) with PPACK and PMSF, respectively. Unfolding and dissociation of the noncovalently linked polypeptide chains of either human or bovine D-Phe-Pro-Arg-CH2-zeta- and PhMeSO2-zeta-thrombins in 4.5 M guanidine-HCl and refolding upon 30-fold dilution in 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 0.1% PEG resulted in biphasic generation of catalytic activity. The slow phase was eliminated in the presence of the competitive inhibitor benzamidine-HCl. Unfolding and refolding mixtures of the appropriate inactive precursors generated the active chimeric thrombins bovine zeta 1-thrombin:human zeta 2-thrombin and human zeta 1-thrombin:bovine zeta 2-thrombin. Human zeta 1-thrombin and zeta 2-thrombin were isolated, and, upon recombining, the isolated fragments refolded to generate catalytically competent zeta-thrombin with an active-site content, specific activity toward Chromozym-TH, and a specificity constant (kcat/Km) for FPA release from fibrinogen that were all within 60% of those of native alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catalytically competent human and bovine zeta-thrombin and chimeras generated from unfolded polypeptide chains. 130 87

By radioimmunoassays established on human derived antigens, PAPP-A, PP5 and PP14 immunoreactivity was detected in placental extracts and blood of pregnant baboons. None of the serial dilution curves suggested parallelism between respective human and baboon samples. Based on slopes of regressed logit-log transformed binding data, PAPP-A demonstrated the greatest degree of interspecies immunological crossreactivity. PP14 showed the least conservation of antigenic determinants. Physicochemical characterization on heparin, zinc chelate and bovine thrombin affinity matrices could not distinguish human from baboon-derived antigens. As in the human, baboon PAPP-A and PP5 were not detected in blood of male or non-pregnant animals. PP14 was detected in baboon follicular fluid, and only PP5 immunoreactivity was measured in culture media of baboon embryos. Of the three antigens, PAPP-A was detected in pregnant baboons at about 61 days gestation, that is, 4 weeks before PP5 and PP14. With the exception of PP14 which attained peak concentration at 118 days of pregnancy, PAPP-A and PP5 concentrations were greatest at term. In conjunction with physicochemical and immunological criteria, these physiological kinetics clearly support a role for developing a baboon model to serve for further studies into feto-maternal signals, particularly antigens such as PAPP-A and PP5.
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PMID:A baboon model for pregnancy-associated antigens (PAPP-A, PP5, PP14). 169 92

Adsorption loss of thrombin may produce considerable systematic error when quantitative evaluation of its reaction with antithrombin III is based on measurements of changes in enzyme activity with time. At 25 degrees C, pH 7.8 (0.1 M NaCl, 0.01 M TRIS, 0.01 M HEPES) glass, siliconized glass, polypropylene and polystyrene surfaces adsorb thrombin in a similar manner. Its adsorption is a saturation reaction reaching an equilibrium after about three hours. At initial concentrations of enzyme convenient for assay with chromogenic substrate (10 nM or less) a large fraction of activity is lost. Binding of alpha thrombin to polypropylene is very tight. The association constants (Ka) were found to be 12.6 X 10(9) M-1 and 4.0 X 10(9) M-1 for human and bovine alpha thrombin respectively. The adsorbed enzyme has hardly any ability to hydrolyze chromogenic substrate. A small part of its activity can be recovered by displacement with PEG 6000, more when the substrate is present. The addition of non-ionic detergents and continuous stirring allows recovery of up to 30% of the enzyme activity and accelerates this process. Complete and long term (more than 20 hours at 25 degrees C) prevention of adsorption loss of human alpha and bovine alpha and beta thrombin at pH between 7.0-8.3 is achieved by the addition of 0.1% PEG 6000 to the buffer and use of tubes precoated with PEG 20,000. Alternative use of non-ionic detergents (BRIJ-35, TRITON X-100) to modify the solvent also appears effective but their possible interference with reactions studied should be borne in mind.
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PMID:Reaction of thrombins with human antithrombin III. I. Enzyme activity losses unrelated to the inhibitory reaction and their prevention. 375 Feb 77

The rate of thrombin inhibition by AT III depends upon the molecular form (alpha, beta, gamma) and species origin of the enzyme. The following apparent second order rate constants (.1000/M.s) were established alpha human 11.24 +/- 0.8; alpha bovine 7.46 +/- 0.27; beta bovine 6.49 +/- 0.34 and gamma human thrombin 2.80 +/- 0.11, 25 degrees C, pH = 7.80, 0.01 M TRIS, 0.01 M HEPES buffer, 0.0025 M EDTA, 0.3 M NaCl, 1 mg/mL PEG 6000. Using these values, the concentration of active AT III in an unknown sample can be calculated from the measured apparent first order rate constant in moles/liter instead of relative units. In contrast to the reactions in the absence of heparin, in the presence of high affinity heparin, the differences between various forms of thrombin are more pronounced and the shape of the progress curves, as well as rates, are highly dependent on the ionic strength. In the presence of heparin, measurement of the rate of inhibition under pseudo first order conditions can be made only when the NaCl concentration is at least 0.3 M. The significance of the presented data for designing a functional assay of AT III is discussed.
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PMID:Reaction of thrombins with human antithrombin III: II. Dependence of rate of inhibition on molecular form and origin of thrombin. 375 46

Concentrated fibrinogen was prepared from whole blood by cryoprecipitation or chemical precipitation and combined with thrombin to make fibrin glue (FG). Surgical applications of FG include control of bleeding, adhesion of tissues, and sealing of tissue defects. The purpose of this study was to compare cryoprecipitation (cryo) of fibrinogen to precipitation using ethanol, ammonium sulfate (AS), and poly(ethylene glycol) (PEG). Our results suggest that AS precipitation is as effective as cryo in yielding fibrin glues with high bond strengths and is more effective than ethanol and PEG precipitation. In addition, the volume of FG per milliliter of plasma is greater after AS precipitation than after a single freeze-thaw cycle. It is concluded that AS is an efficient means for preparing FG from autologous blood.
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PMID:Preparation of fibrin glue: a study of chemical and physical methods. 749 8

The thrombocyte is the avian equivalent of the mammalian blood platelet and is involved in hemostasis through a fibrinogen-mediated process. Although fibrinogen has been implicated as a molecular bridge between activated cells during aggregation, the location of this molecule and its receptor on thrombocytes has not been characterized. Pigeon fibrinogen, isolated from plasma by precipitation with PEG-1000 and purified over Sepharose 4B, was used to study receptor-ligand interaction. Separation of pigeon fibrinogen on SDS-PAGE resulted in three peptides of molecular mass 62, 55, and 47 kDa, which were comparable to those of human fibrinogen. The role of fibrinogen and its receptor in thrombocyte function was established by turbidimetric aggregation using thrombin as an agonist under conditions requiring Ca2+ and fibrinogen. Maximum response occurred using 3 mM Ca2+ and 100 micrograms/ml fibrinogen. Fibrinogen-dependent aggregation was inhibited by an anti-GPIIb antibody, verifying a role for fibrinogen receptors in thrombocyte function. Fibrinogen-gold conjugates were used to describe receptor and ligand localization on aggregated cells. Computer reconstruction was used to verify relocalization of fibrinogen receptors following activation. Fibrinogen distribution changed from a dispersed state in preactivated cells to focal localization at points of cell contact and along pseudopods following activation. This selective positioning of fibrinogen suggests that a functional relocalization of the receptor occurs upon thrombocyte activation, and this relocation facilitates the role of fibrinogen as a molecular bridge. These studies establish similarities between the avian and the human systems and document the conserved nature of the hemostatic process.
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PMID:Localization of fibrinogen during aggregation of avian thrombocytes. 760 Dec 70

The dipeptide Mtr-Asp-D-Adf-Pip 10 represents a potent thrombin inhibitor. In comparison to NAPAP, 10 exhibited improved tolerability and a longer half-life in vivo, i.e., 20 +/- 5 min. We have coupled aminopolyethyleneglycolmonomethylether of various molecular weights to the carboxyl moiety of 10 and evaluated their biological properties. First, Mtr-Asp-OBut was coupled to the amino group of the PEG employing TOTU as an activating agent. This was followed by the removal of the OBut protecting group and coupling of D-Adf-Pip using TOTU as well. The PEG-bound thrombin inhibitors showed inhibition constants vs. thrombin in the subnanomolar range, i.e., they were more active than the parent molecule 10. Moreover, the pegylated inhibitors exhibited a longer lasting effect in vivo. In rats the half-life of Mtr-Asn (PEG10000-OMe)-D-Adf-Pip 14 was determined to be 63 min. Mtr-Asn(PEG10000-OMe)-D-Adf-Pip 14 showed a half-life of 120 min in pigs. It could be concluded that these PEG-bound thrombin inhibitors may be employed as versatile drugs for parenteral administration in treating thrombotic disorders.
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PMID:Preparation and evaluation of PEG-bound thrombin inhibitors based on 4-amidinophenylalanine. 765 88

The purpose of this study was to develop an Escherichia coli expression system to facilitate study of the structure and function of blood coagulation factor XIII (FXIII) A-chains. We engineered an NcoI site into the full-length FXIII A-chain cDNA and subcloned it into pKK233-2 expression vector. A low level of full-length FXIII A-chain and a 30-kDa FXIII A-chain-related antigen were expressed in the JM 105 strain of E. coli. Protein sequencing of the 30-kDa protein demonstrated that it was synthesized by internal translation starting at either Met474 or Met475. We mutated the internal ribosome-binding sequences from AGGA to TGGT (pKF13A2 construct) and found that it yielded a 30-fold increase in the production of full-length FXIII A-chains. JM105 harboring pKF13A2 produced 20 mg of soluble FXIII A-chains antigen from 1 liter culture in TB medium. The recombinant FXIII A-chain was readily purified to homogeneity through PEG fractionation, Q-Sepharose, and mono-P column chromatography with a 2100-fold increase in specific activity and a yield of 150 to 200 micrograms of FXIII A-chains per liter of culture. The purified FXIII A-chains behaved as a dimer on gel filtration analysis, were thrombin- and calcium-activated, cross-linked fibrin, and bound to fibrin to the same extent as purified plasma FXIII A-chains and recombinant FXIII A-chains purified from yeast. These results document that FXIII A-chains can be readily expressed and purified from E. coli culture and that they retained properties similar to those of purified human factor XIII A-chains.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of recombinant human coagulant factor XIII A-chains expressed in E. coli. 791 44

Reproducible disseminated intravascular coagulation in rabbits was provoked by two intravenous injections 24 hours apart of endotoxin from Salmonella enteritidis. There were mild symptoms of DIC before the second injection which considerably increased thereafter. In detail there was a sharp drop of the platelet count and a considerable diminution of Antithrombin III, of Protein C, Plasminogen and Antiplasmin as well as an appearance of fibrin monomer complexes and an increase of the aPTT. When PEG-hirudin in a single dose of 1 mg/kg BW was given simultaneously with the second injection of endotoxin the following alterations were observed: The drop of the platelet count and of the activities of Antithrombin III, Protein C, Plasminogen and Antiplasmin was significantly less pronounced. The fibrin monomer complexes were lower and the aPTT was less prolonged. The thrombin time, however, as a sign of a direct action of hirudin on thrombin was considerably more prolonged than in the controls. The combined effect of these alterations strongly points in the direction of a favourable influence of PEG-hirudin on the course of DIC. It is of special interest that 6 h after the intravenous injection of PEG-hirudin its full effect on the thrombin time was still detectable. This is apparently due to a longer half-life in circulation of PEG-hirudin than of natural hirudin.
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PMID:The effect of a long-acting recombinant hirudin (PEG-hirudin) on experimental disseminated intravascular coagulation (DIC) in rabbits. 847 80


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