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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Factor XII was purified approximately 14 000-fold from bovine plasma by ammonium sulfate fractionation followed by heparin-agarose, DEAE-Sephadex, CM-cellulose, arginine-agarose, and benzamidine-agarose column chromatography. By this method, about 15 mg of protein was purified from 15 L of plasma with an overall yield of 18%. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal analysis. Bovine factor XII is a glycoprotein with a mol wt of 74 000 as determined by sedimentation equilibrium centrifugation. It contains 13.5% carbohydrate including 3.4% hexose, 4.7% N-acetylhexosamine, and 5.4% N-acetylneuraminic acid. Factor XII is a single polypeptide chain with an NH2-terminal sequence of Thr-Pro-Pro-Trp-Lys-Gly-Pro-?-Lys-His. This sequence is homologous to the reactive-site regions of a number of protease inhibitors. The amino acid sequence of a carboxyl-terminal fragments prepared by cyanogen bromide digestion was found to be Leu-Cys-Ala-Gly-Phe-Leu-Glu-Gly-Gly-Thr-Asp-Ala-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro-Leu-Val-Cys-Glu-Asp-Glu. This sequence is homologous with the active site of a number of plasma serine proteases including thrombin, factor IXa, factor Xa, and plasmin. These data indicate that bovine factor XII is a precursor to a serine enzyme with an inhibitor sequence and a catalytic site located in the same single polypeptide chain.
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PMID:Isolation and characterization of bovine factor XII (Hageman factor). 86 Dec 10

A blood coagulation factor, Factor XIII, was highly purified from bovine fresh plasma by a method similar to those used for human plasma Factor XIII. The isolated Factor XIII consisted of two subunit polypeptides, a and b chains, with molecular weights of 79,000 +/- 2,000 and 75,000 +/- 2,000, respectively. In the conversion of Factor XIII to the active enzyme, Factor XIIIa, by bovine thrombin [EC 3.4.21.5], a peptide was liberated. This peptide, designated tentatively as "activation peptide," was isolated by gel-filtration on a Sephadex G-75 column. It contained a total of 37 amino acid residues with a masked N-terminal residue and C-terminal arginine. The whole amino acid sequence of "Activation peptide" was established by the dansyl-Edman method and standard enzymatic techniques, and the masked N-terminal residue was identified as N-acetylserine by using a rat liver acylamino acid-releasing enzyme. This enzyme specifically cleaved the N-acetylserylglutamyl peptide bond serine and the remaining peptide, which was now reactive to 1-dimethylamino-naphthalene-5-sulfonyl chloride. A comparison of the sequences of human and bovine "Activation peptide" revealed five amino acids replacements, Ser-3 to Thr; Gly-5 to Arg; Ile-14 to Val; Thr-18 to Asn, and Pro-26 to Leu. Another difference was the deletion of Leu-34 in the human peptide. Adsorption chromatography on a hydroxylapatite column in the presence of 0.1% sodium dodecyl sulfate was developed as a preparative procedure for the resolution of the two subunit polypeptides, a or a' chain and b chain, constituting the protein molecule of Factor XIII or Factor XIIIa. End group analyses on the isolated pure chains revealed that the structural change of Factor XIII during activation with thrombin occurs only in the N-terminal portion of the a chain, not in the N-terminal end of the b chain or in the C-terminal ends of the a and b chains. From these results, it was concluded that the activation of bovine plasma Factor XIII by thrombin must be accompanied by a limited proteolysis of the arginyl-glycyl bond located in the N-terminal region of the a chain, liberating the "Activation peptide." The possibility of activating Factor XII with other porteinases was examined using Factor Xa [EC 3.4.21.6], Factor XIIa, kallikreins [EC 3.4.21.8], urokinase [EC 3.4.99.26], trypsin [EC 3.4.21.4], ficin [EC 3.4.22.3], papain [EC 3.4.22.2], and bromelain [EC 3.4.22.4]. Among these enzymes, only bromelain and trypsin showed clear activating effects.
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PMID:On the activation of bovine plasma factor XIII. Amino acid sequence of the peptide released by thrombin and the terminal residues of the subunit polypeptides. 122 22

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type inhibitor that directly inhibits factor Xa and, in a factor Xa-dependent fashion, also inhibits the factor VIIa/tissue factor (TF) catalytic complex. The Kunitz-2 domain in TFPI is needed for the binding and inhibition of factor Xa, while the Kunitz-1 domain appears to be responsible for binding factor VIIa in a quaternary factor Xa-TFPI-factor VIIa/TF inhibitory complex. Human leukocyte elastase (HLE) proteolytically cleaves TFPI between threonine-87 and threonine-88 within the polypeptide that links the Kunitz-1 and Kunitz-2 domains in the TFPI molecule. HLE treatment not only affects the ability of TFPI to inhibit factor VIIa/TF, but also dramatically reduces its inhibition of factor Xa. Both purified HLE and stimulated neutrophils regenerate TF activity from a preformed factor Xa-TFPI-factor VIIa/TF inhibitory complex. Kinetic analysis suggests that HLE cleavage does not effect the affinity of the initial encounter interaction between factor Xa and TFPI, whereas it markedly reduces the affinity of the final factor Xa:TFPI complex with Ki (final) values for untreated and HLE-treated TFPI of 58 pmol/L and 4.4 nmol/L, respectively. Thus, an epitope in the amino-terminal region of TFPI or a conformation of the TFPI molecule that requires the presence of this region is needed in concert with the Kunitz-2 domain to produce optimal inhibition of factor Xa by TFPI.
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PMID:The effect of leukocyte elastase on tissue factor pathway inhibitor. 155 67

About 30% of human plasma protein C is smaller than the predominant form as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has been suggested that this species, referred to as beta protein C, is a degraded molecule. However, beta protein C is secreted in culture by the HepG2 cell line and is present in plasma collected directly into numerous proteinase inhibitors; the percentage of beta protein C does not change with time during culture or after blood collection. Neither thrombin, activated protein C, nor activated factor X converts the alpha form to beta in the presence or absence of calcium and phospholipids. The NH2-terminal sequences of the heavy chains of both forms are identical, and both release the same dodecapeptide and develop a functional active site when cleaved by thrombin. Both also react with antibodies to a synthetic COOH-terminal peptide. Timed digests with N-glycosidase are consistent with the interpretation that beta protein C has three N-linked oligosaccharide chains whereas alpha protein C has four. It is asparagine 329 that is not glycosylated in beta protein C since antibodies to a synthetic peptide based on the sequence around this amino acid react only with beta protein C. This site is unique in having cysteine instead of serine or threonine 2 residues distal. It is likely that the sulfhydryl group can substitute for the usual hydroxyl group as a hydrogen bond acceptor for the glycosylation reaction only until it forms a disulfide bond. The percentage of protein C that is glycosylated at this site may therefore depend at least in part on the rate of disulfide bond formation which may in turn be related to the rate of protein synthesis.
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PMID:Beta protein C is not glycosylated at asparagine 329. The rate of translation may influence the frequency of usage at asparagine-X-cysteine sites. 169 79

Cancer procoagulant (CP) is a cysteine proteinase found in a variety of malignant cells and tissues and in human amnion-chorion tissue. It initiates coagulation by activating factor X. However, the amino acid sequence of the substrate protein that determines the cleavage site of cysteine proteinases is different from that of the serine proteinases that normally activate factor X, such as factor IXa, VIIa and Russell's Viper Venom (RVV). Therefore, it was of interest to determine the site of cleavage of human factor X by CP. Purified CP was incubated with purified factor X and the reaction mixture was electrophoresed on a 10% Tris-tricine SDS-PAGE gel. The proteins were electroeluted on to a polyvinylidene difluoride (PVDF) membrane, and stained with Coomassie blue. The heavy chain of activated factor X was cut out of the PVDF membrane and sequenced with an Applied Biosystems 477A with on-line HPLC. The primary cleavage sequence was Asp-Ala-Ala-Asp-Leu-Asp-Pro-; two other secondary sequences Ser-Ile-Thr-Trp-Lys-Pro- and Glu-Asn-Pro-Phe-Asp-Leu were found. The penultimate amino acid on the carbonyl side of the hydrolysed amide bond plays a critical role for the recognition of the cleavage site of cysteine proteinases. These data indicate that the penultimate amino acid for the primary cleavage site of factor X by CP is proline-20 and for the secondary sites, proline-13 and proline-28. This is in contrast to arginine-52 that determines the specificity of the cleavage by normal serine proteinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The site of activation of factor X by cancer procoagulant. 179 60

Factor IXLong Beach has a single amino acid substitution at 397 (Ile to Thr) in the catalytic domain which results in severe hemophilia B. Recent investigations have shown that the substitution of threonine for isoleucine at 397 may affect a part of the macromolecular substrate binding site. Because threonine has a hydroxyl group in its side chain, it is possible that this hydroxyl group makes new hydrogen bonds and disturbs the substrate binding site. We used three techniques: molecular biology, which includes site-directed mutagenesis and recombinant protein expression in tissue culture; computer-aided kinetic data analysis; and molecular modeling to study this mutation site. We have produced two mutant factor IX molecules that have isoleucine 397 replaced by valine or threonine. Factor IXwild type and the two mutants (factor IXVal and factor IXThr) were expressed in human kidney cells and purified using a conformation-specific monoclonal antibody column. After the activation by factor XIa, these three molecules were able to bind p-aminobenzamidine and increase its fluorescence intensity in a similar manner. Factor IXVal and factor IXwild type had indistinguishable activities in an activated partial thromboplastin time (aPTT) assay and similar kinetic parameters with factor X as a substrate. Factor IXThr had only 5% clotting activity compared with normal factor IX, a slightly lower Km and significantly reduced kcat, using factor X as a substrate. We developed energy-refined (AMBER v.3.1) computer models of the three factor IX molecules based on previous work. Three factor IXa models (Ile, Val, or Thr at 397) with a fragment of the factor X activation site were used to predict the effect of the mutation at 397 and evaluate the significance of the new hydrogen bond thought to form between the side chain hydroxyl group of threonine 397 and the carbonyl oxygen of tryptophan 385. This new hydrogen bond would affect the position of an amide proton of adjacent glycine 386 which has been proposed to make a hydrogen bond with a backbone carbonyl oxygen of the P3 residue of factor X. In addition to the new hydrogen bond, there is significant movement in the side chain of tryptophan 385 between the factor IXawild type-factor X model and the factor IXaThr-factor X model that could interfere with substrate binding. This movement could be caused by the change in the molecular volume, the orientation of the side chain at 397, and the new hydrogen bond.
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PMID:Expression and characterization of human factor IX. Factor IXthr-397 and factor IXval-397. 190 72

Antithrombin III Hamilton is a structural variant of antithrombin III (AT-III) with normal heparin affinity but impaired serine protease inhibitory activity. The molecular defect of AT-III-Hamilton is a substitution of threonine for alanine at amino acid residue 382. Recently it has been shown that both plasma-derived and cell-free-derived AT-III-Hamilton polypeptides act as substrates rather than inhibitors of thrombin and factor Xa. In the present study, the cell-free expression phagemid vector pGEM-3Zf(+)-AT-III1-432 was mutated at amino acid residue 382 of AT-III to generate 7 cell-free-derived variants. All these cell-free-derived AT-III variants were able to bind heparin as effectively as cell-free-derived normal AT-III. In terms of alpha-thrombin inhibitory activity each variant reacted differently. Variants could be grouped into 3 categories with respect to thrombin-AT-III complex formation: (1) near normal activity (glycine, isoleucine, leucine, valine); (2) low activity (threonine, glutamine); (3) no detectable activity (lysine). These data suggest that mutations at position 382 of AT-III may have a variable effect on protease inhibitory activity, depending on either the stability of the P12-P9 region of the exposed loop of AT-III, or the inability of the amino acid residue at position 382 to interact with a conserved hydrophobic pocket consisting of phenylalanine (at positions 77, 221 and 422) and isoleucine (position 412) residues.
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PMID:Site-directed mutagenesis of alanine-382 of human antithrombin III. 201 20

Antithrombin-III-Hamilton has been shown to be a structural variant of antithrombin-III (AT-III) with normal heparin affinity but impaired protease inhibitory activity. The molecular defect of AT-III-Hamilton is the substitution of Thr for Ala at amino acid residue 382. The plasma of affected individuals contains approximately equal quantities of normal AT-III and AT-III-Hamilton. When AT-III was isolated from the plasma of the propositus by heparin-Sepharose chromatography, it had identical mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to normal plasma-derived AT-III, under both reducing and nonreducing conditions. However, the AT-III-Hamilton species, separated from the propositus' normal AT-III by a combination of heparin-Sepharose and thrombin-Sepharose chromatography, had increased mobility on reductive SDS-PAGE compared with AT-III from the propositus isolated by heparin-Sepharose chromatography alone. Under nonreducing conditions this AT-III-Hamilton species had decreased mobility compared with AT-III from the propositus (or normal AT-III) isolated only by heparin-Sepharose chromatography. When incubated with either human alpha-thrombin or human factor Xa, this AT-III-Hamilton species was unreactive. Approximately 50% of the AT-III from the propositus isolated by heparin-Sepharose chromatography, when incubated with either human alpha-thrombin or factor Xa, did not form complex but was cleaved, presumably at the reactive center Arg393-Ser394. To further substantiate the biological behavior of this variant, AT-III-Hamilton polypeptides were synthesized in a cell-free system. This recombinantly produced AT-III-Hamilton, when incubated with either human alpha-thrombin or factor Xa, was cleaved by both these proteases, but did not show any complex formation. The results indicate that AT-III-Hamilton does not form a stable covalent inhibitory complex with these serine proteases but can be cleaved at the reactive center. Thus, the inhibition of serine proteases by their natural inhibitors (the serpins) involves at least two separate, but interrelated events; hydrolysis at the reactive center followed by complex formation. AT-III-Hamilton is capable of only the first of these events.
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PMID:Antithrombin-III-Hamilton, Ala 382 to Thr: an antithrombin-III variant that acts as a substrate but not an inhibitor of alpha-thrombin and factor Xa. 202 79

A group of leupeptin analogues was found in Streptomyces griseus strain 254, isolated from a soil sample from Fujian Province, China. The inhibitors excreted in the culture filtrate were purified by adsorption on macroporous resin, followed by sequential ion exchange chromatography on DEAE-52 cellulose, CM-32 cellulose and affinity chromatography with immobilized trypsin. The preparation thus obtained was further purified by preparative HPLC. Several major components were found and characterized, which possessed different inhibitory properties toward trypsin. Based upon amino acid and mass spectrophotometric analysis, these peptides were placed in four major structural categories, viz., R-Val-Val-argininal, R-Leu-Leu-argininal, R-Ile-Ile-argininal and R-Thr-Thr-argininal, this latter component representing a newly identified leupeptin analogue. The structural variability of the R-group was partly responsible for the multiplicity of the peaks obtained with HPLC. All peptides displayed varying degrees of inhibitory activity toward proteases involved in blood coagulation and fibrinolysis, including plasmin, factor Xa, activated protein C and thrombin. Among these peptide inhibitors, the molecule containing threonine showed the strongest inhibitory activity.
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PMID:The inhibition of the enzymic activity of blood coagulation and fibrinolytic serine proteases by a new leupeptin-like inhibitor, and its structural analogues, isolated from Streptomyces griseus. 250 6

Factor IX Chicago-2 and prothrombin Madrid were purified from patients with hemophilia B and congenital dysprothrombinemia, respectively. Each protein displays defects in zymogen activation secondary to the failure to cleave one of the sessile bonds whose cleavage is necessary for full coagulant activity. These proteins were isolated by immunoaffinity chromatography using conformation-specific antibodies directed at either factor IX or prothrombin. Factor IX Chicago-2 is cleaved abnormally by factor XIa, yielding a pattern consistent with the failure to cleave the sessile bond between Arg 145 and Ala 146. Prothrombin Madrid is cleaved abnormally by factor Xa, yielding a pattern consistent with the failure to cleave the sessile bond between Arg 271 and Thr 272. Peptide mapping was performed on reduced and alkylated factor IX, factor IX Chicago-2, prothrombin, and prothrombin Madrid, and the hydrolysates were separated by high-performance liquid chromatography. The mutant peptide in factor IX Chicago-2 was identified by automated Edman degradation as residues 143 through 188 of factor IX, and had a histidine substituted for arginine at residue 145. The mutant peptide identified in prothrombin Madrid corresponds to residues 267 through 285 of prothrombin and has the substitution of cysteine for arginine at residue 271. These mutations, each occurring at arginines, are identical to those in factor IX Chapel Hill and prothrombin Barcelona. These results suggest that a limited repertoire of point mutations, many affecting arginine residues, may be responsible for hereditary defects of the vitamin K-dependent proteins in patients with normal antigen levels.
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PMID:Molecular defects of factor IX Chicago-2 (Arg 145----His) and prothrombin Madrid (Arg 271----cys): arginine mutations that preclude zymogen activation. 275 9


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