Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human alpha-thrombin, the thromboplastin activation product of prothrombin with high clotting and esterase activity, was produced from Cohn Fraction III paste. The procedure started with 0.4 to 3.2 kg of frozen paste and was completed in 2 or 3 days. Some 23 g of thrombin were recorded for 65 quantitated preparations made from 11 lots of Fraction III paste. These preparations were obtained at protein concentrations of 3.9 +/- 1.3 mg/ml with a yield of 340 +/- 110 mg/kg of paste, which represented 48 +/- 14% of the clotting potential extracted as prothrombin. They had specific clotting activities of 2.8 +/- 0.4 U.S. (NIH) units/microng of protein and titrated to 88 +/- 8% active with p-nitrophenyl-p'-guanidinobenzoate (NPGB). Those (N - 29) examined by labeling with [14C]diisopropyl phosphorofluoridate (iPr2P-F) and electrophoresing in sodium dodecyl sulfate (SDS)-polyacrylamide gels were found to contain only (N = 4) or predominantly alpha-thrombin (97 +/- 3%) and corresponding amounts of ists degradation product, beta-thrombin (2.6 +/- 3.1%). No plasmin(ogen), prothrombin complex factors (II, VII, IX, IXalpha, X, Xalpha), or prothrombin fragments were detected in representative preparations. As produced in 0.75 M NaCl, pH approximately 6, thrombin was stable for approximately 1 week at 4 degrees and for greater than 1 year at less than or equal to 50 degrees; freeze-dried thrombin stored at 4 degrees for greater than 1 year displayed stable clotting activity and no vial to vial variation, permitting its use for reference purposes. Human thrombin generated by Taipan snake venom activation was compared with that produced by rapid thromboplastin activation: after treatment with [14C]iPr2P-F, greater than 95% of the label in both thrombins migrated at the same rate during electrophoresis in SDS; identical pairs of NH2-terminal residues were released in three consecutive Edman degradation cycles.
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PMID:Human thrombins. Production, evaluation, and properties of alpha-thrombin. 1 8

Improved methods are described to obtain bovine prothrombin, Factor IX, Protein C, and autoprothrombin III (Factor X, Auto-III) in purified form. The prothrombin had a specific activity of 4,340 Iowa units/mg. Theoretically, a preparation of clean thrombin should have a specific activity of 8,200 U/mg, because 47.08% of the protein in prothrombin is lost when thrombin forms. Such thrombin preparations have been obtained (Arch. Biochem. Biophys. 121, 372 (1967)). The prothrombin concentration of bovine plasma is near 60 mg/liter. Protein C, first isolated by Stenflo (J. Biol. Chem. 251, 355 (1976)), was found to be the precursor of autoprothrombin II-A (Auto-II-A), discovered earlier (Thromb. Diath. Haemorrh. 5, 218 (1960)). Protein C (Factor XIV) was converted to Auto-II-A (Factor XIVa) by thrombin. Digesting purified Auto-III with purified thrombin removed a small glycopeptide from the COOH-terminal end of the heavy chain to yield Auto-IIIm. Auto-III thrombin leads to Auto-IIIm + peptide. Auto-IIIm was not converted to the active enzyme with thromboplastin, and furthermore, inhibited the activation of purified native Auto-III with thromboplastin. Auto-IIIm was also not converted to the active enzymes when the procoagulants consisted of purified Factor VIII, purified Factor IXa, platelet factor 3 and calcium ions. The "activation peptide" released by RVV-X from the NH2-terminal end of the heavy chain and the active enzyme (Auto-Cm) were purified. Auto-III was also activated with purified RVV-X. The same "actid of Auto-Cm. Purified Factor IX developed anticoagulant activity when reacted with an optimum concentration of purified thrombin. A suitable reagent for the assay of Factor IX was prepared by removing prothrombin complex from anticoagulated bovine plasma and restoring the prothrombin and Auto-III concentration with use of the respective purified proenzymes.
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PMID:Improved procedures for the purification of selected vitamin K-dependent proteins. 78 72

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

We describe a method for the purification of recombinant proteins based upon the selective interaction of the choline-binding domain of the pneumococcal murein hydrolase and tertiary amines. Proteins of interest, fused to the binding domain by a peptide linker, containing the cleaving sequence recognized by blood coagulation factor Xa, can either be assayed for biological activities in vitro and in vivo or have the binding moiety removed to yield a totally unmodified form, suitable for clinical and functional studies. The method can also be applied to the production of low molecular mass peptides. The principle of the technique is illustrated with acidic fibroblast growth factor and with a neuropeptide-like fragment of ten amino acids contained within its sequence.
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PMID:Single-step purification on DEAE-sephacel of recombinant polypeptides produced in Escherichia coli. 137 76

A method to increase the amount and improve the bioactivity of heparin (HEP) immobilized on a polymer surface was developed. The surface of polyurethane-urea (PU) coated glass beads was first modified with diisocyanates, followed by surface grafting of polyfunctional polymers (PFP), including: poly(vinyl alcohol), poly(ethyleneimine), and poly(allylamine). The functional groups of the surface grafted PFP (-OH, -NH, or -NH2) were modified with diisocyanates (TDI) to amplify the surface concentration of isocyanate groups, alpha, omega-diamino-terminated polyethylene oxide (PEO; molecular weight, 4,000 daltons) was then coupled to the surface grafted PFP, and the free amino groups were derivatized with TDI. Finally, HEP was coupled to the amplified surface through free -NCO groups of PU-PFP-PEO. The surfaces were quantified during each step of the procedures for -NCO groups and HEP. All grafted surfaces showed a four to eightfold increase in -NCO content and a twofold increase in immobilized HEP content compared with HEP immobilized directly onto the PU surface. The HEP bioactivity tests (including activated partial thromboplastin time, thrombin times, and factor Xa) demonstrated an increased bioactivity of HEP when immobilized through PFP-PEO compared with PFP and PU alone.
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PMID:Heparin immobilization by surface amplification. 145 39

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

Factor IX is a vitamin K-dependent zymogen of a serine protease. The NH2-terminal half of the molecule consists of a Ca(2+)-binding gamma-carboxyglutamic acid (Gla)-containing module and two modules homologous to the epidermal growth factor (EGF) precursor. To elucidate the role of these non-catalytic modules of factor IXa beta in factor X activation, we have isolated and characterized fragments of bovine factor IX, containing one or both of the EGF-like modules as well as these modules linked to the Gla module. The fragments were used as inhibitors of factor IXa beta-mediated factor X activation in a plasma clotting system and in systems with purified components of the Xase complex. Fragments consisting of either the two EGF-like modules of factor IX linked together or the NH2-terminal EGF-like module alone were found to inhibit factor Xa generation both in the presence and absence of the cofactor, factor VIIIa. Moreover, a fragment consisting of the corresponding modules of factor X had a similar effect. We therefore propose that factor IXa beta and factor X interact directly through their EGF-like modules on or in the vicinity of a phospholipid surface. We have also found that the isolated Gla module of factor IX inhibits the formation of factor Xa both in the presence and absence of phospholipid but not in the absence of factor VIIIa. Our results are compatible with a model of the Xase complex, in which both the serine protease part and the Gla module of factor IXa beta interact with factor VIIIa.
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PMID:Effects of gamma-carboxyglutamic acid and epidermal growth factor-like modules of factor IX on factor X activation. Studies using proteolytic fragments of bovine factor IX. 173 81

A new case of heterozygous dysfibrinogenemia characterized by an amino acid replacement in the NH2-terminal region of the fibrin alpha-chain was found in a 27-year-old woman with a bleeding problem. Her one-stage prothrombin time and activated partial thromboplastin time were slightly prolonged, and the purified fibrinogen from this patient had a markedly prolonged thrombin or reptilase time. Release of fibrinopeptides A and B was normal, but the polymerization of fibrin monomers was impaired. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fibrinogen under the reduced condition showed no abnormalities in the apparent molecular weights of its three chains. Reverse-phase high performance liquid chromatography (HPLC) of the lysylendopeptidase-cleaved purified A alpha-chains showed a decrease in one peptide compared with the normal amount and the appearance of an abnormal peptide peak. These peptides were treated with thrombin and further separated on HPLC. Amino acid sequence analysis of the abnormal peptide indicated that A alpha proline-18, the second residue from the NH2-terminus of the fibrin alpha-chain, was replaced by leucine. The synthetic peptide Gly-Pro-Arg-Pro inhibited both thrombin- and reptilase-induced fibrin aggregation, but Gly-Leu-Arg-Pro showed little or no inhibition under the same conditions. The discovery of this abnormal fibrinogen supports the findings that A alpha proline-18 is important as part of the polymerization site in the NH2-terminus of the fibrin alpha-chain. The propositus' mother had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Kyoto II.
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PMID:Fibrinogen Kyoto II, a new congenitally abnormal molecule, characterized by the replacement of A alpha proline-18 by leucine. 207 49

Previous studies have shown that extrinsic pathway inhibitor (EPI) is an effective inhibitor of factor Xa alone or factor VIIa-tissue factor complex in the presence of factor Xa. Since tissue factor exposure is implicated in thrombogenesis, we hypothesized that EPI may be valuable in the treatment of some thromboembolic episodes. Furthermore, EPI may be an important factor in bleeding complications in hemophiliacs. In the present study, human EPI was expressed in baby hamster kidney cells using a mammalian expression vector. Transfected cells expressed 1-2 micrograms/ml of recombinant EPI (rEPI) which was purified to homogeneity by heparin-Sepharose chromatography, ion-exchange chromatography, and reverse phase high performance liquid chromatography. Purified rEPI exhibited a specific activity of 30,000 units/mg and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 42,000. In addition, the NH2-terminal sequence of rEPI was identical to that of HepG2 EPI and HeLa EPI. The ability of rEPI to inhibit factor X activation by a complex of factor VIIa-tissue factor was then examined in the presence and absence of plasma concentrations of human factors VIII and IX. Using relipidated human brain tissue factor apoprotein, rEPI inhibited the factor VIIa-mediated activation of factor X half-maximally at 2.5 and 1 nM in the presence and absence of factors VIII and IX, respectively. Using monolayers of a human bladder carcinoma cell line (J82) as the source of tissue factor, the activation of factor X by cell-bound factor VIIa was inhibited half-maximally by 5 nM rEPI in the presence of factors VIII and IX. The proteolytic activity of J82 cell-bound factor Xa toward prothrombin was inhibited half-maximally at approximately 5 nM rEPI, while the amidolytic activity of factor Xa in solution was inhibited by rEPI with a Ki of 130 pM. Recombinant EPI also inhibited the amidolytic activity of factor VIIa half-maximally at 10 nM rEPI in the presence of relipidated tissue factor apoprotein and calcium. These results indicate that, in the presence of plasma concentrations of factors VIII and IX, at least 10 times the plasma concentration of EPI is required to reduce factor VIIa-dependent factor X activation one order of magnitude in vitro. In the absence of functional factor VIII and IX, rEPI at plasma levels was a potent inhibitor of factor VIIa-mediated factor X activation, and this activity presumably accounts for the inability of hemophiliacs to initiate hemostasis via the extrinsic pathway.
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PMID:Recombinant human extrinsic pathway inhibitor. Production, isolation, and characterization of its inhibitory activity on tissue factor-initiated coagulation reactions. 221 93

Coagulation factor Va is a cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. The phospholipid-binding domain of bovine factor Va has been reported to be located on the light chain of the molecule and more precisely on a fragment of Mr = 30,000 which is obtained after digestion of factor Va light chain by factor Xa. This proteolytic fragment is located in the NH2-terminal part of factor Va light chain (residues 1564-1765). In order to further characterize the lipid-binding domain of bovine factor Va, isolated bovine light chain was preincubated with synthetic phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine) and digested with trypsin, chymotrypsin, and elastase. Two peptide regions protected from proteolytic cleavage were identified and characterized from each proteolytic digestion. A comparison of the NH2-terminal sequence and amino acid composition of the two tryptic peptides with the deduced sequence of human factor V indicates a match with residues 1657-1791 of the light chain of human factor V for one peptide and residues 1546-1656 for the other peptide. When chymotrypsin or elastase were used for digestion, the NH2-terminal sequence of one peptide showed a match with residues 1667-1797 of the light chain, while the other peptide presented an NH2-terminal sequence identical with the previously described for the bovine factor Va light chain. When these peptides were assayed for direct binding to phospholipid vesicles, only the tryptic and the chymotryptic peptides covering the middle region of the A3 domain of the bovine factor Va light chain demonstrated an ability to interact with phospholipid vesicles. Thus, knowing that the factor Xa cleavage site on the factor Va light chain is located between residues 1765 and 1766 of the light chain this lipid-binding region of the bovine factor Va is further localized to amino acid residues 1667-1765.
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PMID:Identification and characterization of a phospholipid-binding site of bovine factor Va. 225 16


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