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)

Bovine high molecular weight kininogen (bHMWK) partially corrects the activated plasma thromboplastin time (aPTT) of Fitzgerald trait plasma which is congenitally deficient in HMWK. The relationship between the structure and activity of HMWK was clarified by studying the effects of different fragments of bHMWK on the aPTT of Fitzgerald-trait plasma. The peptides studied were lys-bradykinin-free HMWK, bradykinin-fragment 1-2-free HMWK, heavy chain, fragment 1-2-light chain, and light chain. All fragments were tested in equimolar concentrations. Bradykinin-fragment 1-2-free HMWK, heavy chain, and light chain have little or no correcting activity upon Fitzgerald-trait plasma aPTr. Fragment 1-2 light chain has the same correcting activity as intact bHMWK, while that of lys-bradykinin-free HMWK appears to be higher. Both fragment 1-2 and fragment 2 inhibit the clotting time of normal human plasma. When compared on a molar basis, fragment 2 is a more active inhibitor than fragment 1-2. When the effects of bovine plasma kallikrein upon bHMWK and hHMWK were studied, it was found that it released kinins from both kininogens. However, while the correcting activity of bHMWK was completely destroyed after 60 min of incubation, that of hHMWK was fully retained. These data suggest that: (a) the active part of bHMWK is comprised of the fragment 1-2 light chain portion; (b) fragment 1-2 or fragment 2 is the binding site to negatively charged surfaces, while the light chain interacts with other components of the surface-mediated reactions; and (c) bovine plasma kallikrein releases kinins, but probably does not cause the release of fragment 1-2 from human HMWK.
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PMID:Relation between structure and correcting activity of bovine high molecular weight kininogen upon the clotting time of Fitzgerald-trait plasma. 42 63

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

The amino-acid sequence of the heavy chain of bovine blood coagulation factor X1 (Stuart factor) isolated before and after activation has been determined. Sequence analysis was performed on fragments obtained by cleavage with cyanogen bromide and by tryptic digestion. Comparison of the complete sequence with those of other hepatic and pancreatic serine proteases demonstrates homology of the heavy chain of activated factor X1 (factor X1a) with the B chain of bovine thrombin as well as with bovine trypsin, chymotrypsins A and B, and porcine elastase. The activation peptide cleaved near the amino terminus by a protease from Russell's viper venom differs in both size and sequence from those of other serine proteases. With three exceptions, all of the residues which are important in the catalytic functions of trypsin and chymotrypsin occur in corresponding loci in the heavy chain of factor Xa. These finding suggest that the three-dimensional structure of the heavy chain is similar to that of the pancreatic serine proteases and that these enzymes have evolved from a common ancestral gene.
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PMID:Bovine factor X1 (Stuart factor): amino-acid sequence of heavey chain. 105 93

The crude venom of many elapid snakes appeared to contain proteins that activated blood coagulation factor V. The factor V activator present in the venom of Naja naja oxiana was purified to homogeneity by chromatography on a mono-S column. The activator was a single chain protein with an apparent mol. wt of 48,000, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by gel permeation chromatography on Sephacryl S200. Activation of bovine factor V by the purified venom activator was accompanied by proteolytic cleavage of factor V and resulted in the formation of two major polypeptide chains with mol. wts of about 90,000 and 77,000. The final product obtained was compared with thrombin-activated factor V for its ability to function as cofactor in factor Xa-catalysed prothrombin activation in the presence of negatively charged phospholipid vesicles (5 mole% phosphatidylserine/95 mole% phosphatidylcholine). The Km for prothrombin obtained at a saturating amount of venom-activated factor Va was nine-fold higher than with thrombin-activated factor V (0.83 microM vs 0.09 microM, respectively) whereas both factor Va molecules stimulated the Vmax of thrombin formation some 6000-fold. Both forms of factor Va promoted the binding factor Xa to negatively charged phospholipid vesicles. However, the apparent Kd for factor Xa was less favorable in the presence of venom-activated factor V (0.67 x 10(-9) M) than in the presence of thrombin-activated factor V (0.043 x 10(-9) M). Thrombin cleaved a peptide bond in the 77,000 mol. wt polypeptide chain of venom-activated factor V, which resulted in the formation of a normal factor Va light chain. This peptide bond cleavage was, however, not associated with a change of cofactor activity. Venom treatment of thrombin-activated factor V, on the other hand, did remove a small fragment (mol. wt approximately 4000) from the heavy chain of factor Va (94,000), yielding a molecule with reduced cofactor activity. The diminished cofactor activity of venom-activated factor V is, therefore, likely due to the fact that a small peptide fragment, involved in the interaction with prothrombin and factor Xa, is missing from the heavy chain of venom-activated factor V.
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PMID:Activation of bovine factor V by an activator purified from the venom of Naja naja oxiana. 144 Jun 44

Factor X, a vitamin K-dependent protein, is the plasma zymogen for the active serine protease factor Xa. Factor Xa is the proteolytic enzyme for prothrombinase, the multi-protein membrane complex that catalyses the cleavage of prothrombin to thrombin. A panel of 10 monoclonal antibodies (identified by their corresponding clone numbers: 1, 2, 3, 5, 7, 26, 27, 54, 73, and 79) to factor X were produced by immunizing mice with purified factor X. All of the antibodies bound both human factor X and factor Xa in a solid-phase ELISA and binding of the antibodies was not affected by removal of Ca2+ with EDTA. In immunoblot analysis, antibody alpha HFX-54 bound to the light chain and antibodies alpha HFX-1, -5, -7, and -26 bound to the heavy chain of reduced factor X. Antibodies alpha BFX-2b, alpha HFX-27, -54, and -73 prolonged both the factor X-dependent clotting time and activated partial thromboplastin time (APTT) of normal plasma while antibody alpha HFX-1 only prolonged the APTT. None of the antibodies significantly inhibited factor X activation by purified Russell's viper venom factor X activator. In prothrombin activation assays using purified factor Xa, factor Va, prothrombin, Ca2+ and phospholipid vesicles, seven of the antibodies (alpha HFX-1, -3, -26, -27, -54, -73 and alpha BFX2b) showed some inhibition of thrombin generation ranging from 18 to 60% of the control. The decrease in factor X plasma clotting activity was most likely due to inhibition of factor Xa activity in prothrombinase, although some antibody-dependent inhibition of factor X activation may contribute to the observed inhibition of plasma clotting. Prothrombinase activity on platelets was inhibited in an identical manner by the monoclonal antibodies. When prothrombin was activated in the absence of factor Va, only antibody alpha BFX-2b inhibited activation. Calcium-independent determinants on both the heavy chain (determinants 1 and 26) and light chain (determinant 54) of factor X may play a role in prothrombin activation by prothrombinase. Other epitopes (antibodies alpha HFX-3, -27, -73) appeared to be influenced by association of factor Xa with factor Va. Topographic regions on factor X important for factor X activation and factor Xa function may be identified by the use of these monoclonal antibodies.
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PMID:Neutralization of factor X activity by factor X-specific monoclonal antibodies. 145 Mar 23

A panel of monoclonal antibodies against human prekallikrein was raised in mice and characterized with respect to the major antigenic epitopes. Of 18 antibodies, nine were directed against the light chain portion performing the proteolytic function of activated kallikrein, and nine recognized the heavy chain mediating the binding of prekallikrein to high molecular weight (H-)kininogen. Among the anti-heavy chain antibodies, one (PK6) interfered with the procoagulant activity of prekallikrein, and prolonged in a concentration-dependent manner the activated partial thromboplastin time of reconstituted prekallikrein-deficient plasma (Fletcher type). Antibody PK6 was subtyped IgG1,k and had an apparent Kass of 6.8 +/- 0.44.10(8) M-1 for prekallikrein. Functional analyses revealed that PK6 does not interfere with prekallikrein activation by activated Hageman factor (beta-F XIIa), and has no effect on the kininogenase function of activated kallikrein. Monoclonal antibody PK6 but none of the other anti-heavy chain antibodies completely prevented complex formation of prekallikrein with H-kininogen, and readily dissociated preformed complexes of prekallikrein and H-kininogen. Likewise, Fab' and F(ab')2 fragments of PK6 blocked H-kininogen binding to prekallikrein. A synthetic peptide of 31 amino acid residues encompassing the entire prekallikrein binding region of H-kininogen effectively competed with PK6 for prekallikrein binding indicating that the target epitope of PK6 is juxtaposed to, if not incorporated in the H-kininogen-binding site of prekallikrein. Extensive cross-reactivity of PK6 with another H-kininogen-binding protein of human plasma, i.e. factor XI, suggested that the structure of the target epitope of PK6 is well conserved among prekallikrein and factor XI, as would be expected for the kininogen-binding site shared by the two proteins. It is anticipated that monoclonal antibody PK6 will be an important tool for the precise mapping of the hitherto unknown kininogen-binding site of prekallikrein.
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PMID:High molecular weight kininogen-binding site of prekallikrein probed by monoclonal antibodies. 169 51

A 29-year-old women, who had been treated by hemodialysis for 5 years because of chronic renal failure, developed bleeding tendency in March 1989. Laboratory data showed prolonged activated partial thromboplastin time which was not corrected by addition of normal plasma; factor VIII activity was less than 1% and factor VIII inhibitor 70 Bethesda units/ml. The inhibitor was eluted in the second peak which corresponded to IgG when the plasma was subjected to Sephacryl S 200 column. The further purified IgG fraction by passing through protein A column showed a factor VIII inhibitor activity of 52 Bethesda units/ml. The factor VIII inhibitor epitopes were examined by western blotting technique using factor VIII purified by monoclonal antibody as the antigen. The factor VIII preparation used was composed of a doublet of light chain (Mr 80,000) and three heavy chains (Mr 160,000-200,000) when examined by immunoblotting using anti-factor VIII light and heavy chains monoclonal antibodies after SDS-PAGE. Factor VIII inhibitor that arose in a hemophilia A patient recognized the light chain, and the inhibitor in this case reacted to the heavy chain of factor VIII.
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PMID:[Characterization of the factor VIII inhibitor in a patient with chronic renal failure]. 170 30

We have found that rat plasma corrected the non-activated PT of human normal or factor-X deficient plasma, and the factor Xa-like activity being constantly detected in every 1 ml of blood collected via the cannulated carotid artery of rats. The present study was undertaken to characterize the factor Xa-like activity in rat plasma by preparing rat factor X and a monoclonal antibody against it. Factor X was purified from a BaCl2 eluate of rat plasma by chromatographies on columns of DEAE-Sepharose CL-6B and Sulfate Cellulofine or on a column of Affi-Gel 10 conjugated with a monoclonal antibody against rat factor X. Factor Xa-like activity in rat plasma was eliminated by the treatment of rat plasma with a monoclonal antibody which recognized the heavy chain portions of rat factors X and Xa. A kinetical study demonstrated that rat factor Xa was strongly inhibited by rat antithrombin III, with a Ki of 2.2 x 10(-11) M, in the presence of heparin. However, in the absence of heparin, the second order rate constant for the inhibition of rat factor Xa by rat antithrombin III was 2.6 x 10(4) M-1.min-1, which was one forty-third that for the inhibition of human factor Xa by human antithrombin III. Furthermore, rat factor Xa was resistant to the inhibition by rat alpha-1-antitrypsin and alpha-2-macroglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of rat factors X and Xa: demonstration of factor Xa in rat plasma. 171 49

The time-course of factor VIII (FVIII) proteolysis during whole blood coagulation was monitored using polypeptide-specific enzyme-linked immunosorbent assays (ELISAs) developed with monoclonal antibodies to FVIII. Such assays of whole blood from normal subjects revealed that, within 30 min after coagulation had started, levels of the amino-terminal region of FVIII light chain and in the middle region of FVIII heavy chain had decreased to baseline in parallel with factor VIII procoagulant activity, but that levels of the 70 kDa thrombin digest decreased more slowly, with 75 U/dl of FVIII antigen (FVIII:Ag) remaining even after 2 h. Similar analysis of the blood of a patient with congenital hypoprothrombinemia indicated a lag phase of 20 min before proteolysis started. No significant change was observed until 1 h after calcium chloride was added to prothrombin-depleted plasma. On the other hand, in the presence of tissue thromboplastin, levels of FVIII:Ag in all ELISAs decreased rapidly. These results indicate a requirement for thrombin generation in the proteolysis of FVIII during the process of whole blood clotting.
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PMID:Changes in polypeptide-specific factor VIII antigen levels during whole blood clotting. 172 92

Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. To identify the regions on the surface that mediate anticoagulant activity, 26 synthetic peptides were prepared representing 90% of the human protein C heavy chain primary structure and tested for their ability to inhibit APC anticoagulant activity. Peptide-(390-404) specifically inhibited APC activity in activated partial thromboplastin time and Xa-1-stage coagulation assays in normal, in protein S-depleted and Factor VIII-deficient plasma with 50% inhibition at 5 microM peptide. Polyclonal antibodies raised against this peptide and immunoaffinity-purified on a protein C-Sepharose column inhibited APC anticoagulant activity in activated partial thromboplastin time and Xa-1-stage assays in normal, protein S-depleted, and Factor VIII-deficient plasma with half-maximal inhibition at 30 nM anti-(390-404) antibody. Neither the peptide-(390-404) nor the anti-(390-404) antibodies inhibited APC amidolytic activity or the reaction of APC with recombinant [Arg358] alpha 1-antitrypsin. Furthermore, in a purified system, peptide-(390-404) inhibited APC-catalyzed inactivation of Factor Va in the presence as well as in the absence of phospholipids with 50% inhibition at 4 microM peptide. These data suggest that the region containing residues 390-404 in APC is essential for anticoagulant activity and is available to interact with antibodies or with other proteins such as the macromolecular substrates Factors Va or VIIIa.
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PMID:Identification of a sequence of human activated protein C (residues 390-404) essential for its anticoagulant activity. 176 51


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