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)

The activation of prothrombin by factor Xa is strongly accelerated by negatively charged phospholipids plus calcium ions. In this paper we report that positively charged membranes can also stimulate prothrombin activation provided that the activation reaction is carried out in the absence of calcium ions. Membranes composed of a mixture of phosphatidylcholine (PC) and positively charged lipids like stearylamine, sphingosine, or hexadecyltrimethylammonium bromide caused a more than 1000-fold increase of the rate of prothrombin activation. Prothrombin activation by the factor Xa-factor Va complex was also considerably stimulated by such membranes. Stimulation of prothrombin activation by positively charged membranes was suppressed at high ionic strength. This suggests that electrostatic attraction of negatively charged proteins by positively charged membranes is the major driving force in the association of prothrombin and factor Xa with the lipid surface. Calcium ions strongly inhibited prothrombin activation on vesicles composed of PC and stearylamine (80/20 M/M), which indicates that the regions of prothrombin and/or factor Xa containing gamma-carboxyglutamic acid (gla) are important for the interaction of these proteins with positively charged membranes. The importance of the gla domain was confirmed by the observation that PC/stearylamine vesicles had much less effect on the reactions between proteins that lack gla residues [gla-domainless (des-1-45) prothrombin, prethrombin 1, prethrombin 2, or gla-domainless (des-1-44) factor Xa]. The efficiency of prothrombin and prothrombin derivatives to act as substrate decreased in the order prothrombin greater than des-1-45-prothrombin = prethrombin 1 greater than prethrombin 2, while prothrombin activation by gla-domainless (des-1-44) factor Xa was hardly stimulated by positively charged membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Calcium-independent activation of prothrombin on membranes with positively charged lipids. 323 22

Hexadimethrine bromide was evaluated as a heparin-neutralizing agent in a simple modification of the activated partial thromboplastin time test in canine plasma. Addition of various amounts of heparin in vitro to canine plasma indicated that heparin could be neutralized by adding 0.5 micrograms of hexadimethrine bromide 15 s before CaCl2 was added to the reaction mixture of the activated partial thromboplastin time test. In 8 dogs given (subcutaneous injection) 500 USP units of sodium heparin/kg, marked individual variations in clotting time prolongations were observed over the 12-hour period of study. The hexadimethrine bromide modification effectively neutralized the heparin-related clotting time prolongations to values that were not significantly different from base-line (preheparin) activated partial thromboplastin time values. The modification seems to be useful in confirming the presence of heparin and in monitoring heparin therapy in dogs.
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PMID:Use of hexadimethrine bromide as a heparin-neutralizing agent in canine plasma. 356 7

Known allyl 4,6-O-benzylidene-alpha-D-glucopyranoside was first converted into methyl (prop-1-enyl 2,3-di-O-benzyl-4-O-chloroacetyl-alpha-D-glucopyranosid)-uronate. Acid hydrolysis, followed by treatment with (bromomethylene)dimethyl-ammonium bromide, gave methyl (2,3-di-O-benzyl-4-O-chloroacetyl-alpha-D-glucopyranosyl bromide)uronate. Condensation of this bromide with 3-O-acetyl-1,6-anhydro-2-azido-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-bet a-D-glucopyranose. Acetolysis, followed by treatment with titanium tetrabromide, then gave 3,6-di-O-acetyl-2-azido-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-alp ha-D-glucopyranosyl bromide. Condensation of this bromide with benzyl 6-O-acetyl-3-O-benzyl-2-benzyloxy- carbonylamino-2-deoxy-4-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L- idopyranosyluronate)-alpha-D-glucopyranoside provided benzyl O-(methyl 2,3-di-O-benzyl-4-O-chloroacetyl-beta-D-glucopyranosyluronate)-(1- ---4)-O-(3,6-di-O-acetyl- -2-azido-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-ac etyl-3-O- acetyl-3-O-benzyl-2-benzyloxycarbonylamino-2-deoxy-alpha-D-gluc opyranoside. O-Dechloroacetylation followed by condensation with 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl bromide provided benzyl O-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-beta-D-glucopyranosyl)- (1----4)-O-(methyl 2,3-di-O-benzyl-beta-D-glucopyranosyluronate)-(1----4)- O-(3,6-di-O-acetyl-2-azido-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(m ethyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-ac etyl-3-O- benzyl-2-benzyloxycarbonylamino-2-deoxy-alpha-D-glucopyranoside in 70% yield. O-Deacetylation followed by re-esterification, O-sulfation, saponification, catalytic hydrogenolysis, and N-sulfation gave the decasodium salt of O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D- glucopyranosyl)-(1----4)-O-(beta-D-glucopyranosyluronic acid)-(1----4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-gl ucopyranosyl)-(1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic+ ++ acid)-(1----4)-2-deoxy-2-sulfamido-6-O-sulfo-D-glucopyranose. This synthetic pentasaccharide binds to antithrombin III with an association constant similar to that of high-affinity heparin and elicits a potent anti-factor Xa activity in plasma.
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PMID:Synthesis of heparin fragments. A chemical synthesis of the pentasaccharide O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl)-(1-4 )-O-(beta-D-glucopyranosyluronic acid)-(1-4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-glu copyranosyl)-(1-4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1-4)-2-deoxy-2-sulfamido-6-O-sulfo-D-glucopyranose decasodium salt, a heparin fragment having high affinity for antithrombin III. 370 27

The composition, surface properties, and capacity of lipid particles to bind activated factor X can be correlated both with the influence of various phosphatide combinations on activated factor X activity in vitro and on the intensity and duration of the thrombogenic stimulus as measured by a standard bioassay for thrombus formation. The measurable activity of activated factor X in vitro increased as a linear function of lipid concentration from 0 to 40 micro moles/liter. The effectiveness of the lipids examined was in the following decreasing order: phosphatidyl serine-phosphatidylcholine mixture, "cephalin", and phosphatidyl serine alone. An increase in the duration of hypercoagulability with increasing lipid concentration was also observed, but, with regard to the three lipid fractions tested, the in vivo system appeared to be more discriminatory than in vitro assays. Lipid mixtures containing phosphatidyl serine with either phosphatidylcholine, phosphatidyl ethanolamine, or cetyltrimethylammonium bromide markedly enhanced the in vitro activity of activated factor X. Phosphatidic acid-phosphatidylcholine mixtures had a similar but smaller effect, and phosphatidyl ethanolamine-phosphatidylcholine mixtures were inert. The duration of the hypercoagulability was similarly related to the composition of the phosphatide infused. In mixtures containing phosphatidyl serine, the surface charge density of the lipid particles and the binding of the activated factor X activity to lipid showed some correlations with the in vitro activated factor X assay and with the intensity and duration of the thrombogenic stimulus. These data suggest that the catalytic effect of phosphatides on prothrombin activation and their role in the retardation of in vivo compensatory mechanisms directed against circulating activated factor X, are dependent on the affinity of activated factor X for the lipid surface.
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PMID:Reactions of activated factor X-phosphatide mixtures in vitro and in vivo. 541 83

Human isolated monocytes possess low levels of procoagulant activity, which was stimulated 10-30 fold by brief (2 hr) exposure to 10 micrograms/ml endotoxin. This activity was expressed in normal or factor XII-deficient plasma, but lost in plasma deficient in factors X or VII, indicating that it was due to thromboplastin. The stimulation of monocyte thromboplastin by endotoxin was inhibited in a dose-dependent manner by two phospholipase A2 inhibitors, 4-bromophenacyl bromide and quinacrine, and by two lipoxygenase inhibitors, eicosatetraynoic acid and nordihydroguaiaretic acid. Two cyclooxygenase inhibitors, aspirin and indomethacin, prevented endotoxin-induced increases in thromboxane B2 production but had no effect on thromboplastin production. These results suggest that a component in the sequence of lipid deacylation, arachidonic acid release, and metabolism via lipoxygenase may mediate the stimulation of monocyte thromboplastin activity by endotoxin.
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PMID:Effects of inhibitors of arachidonic acid metabolism on thromboplastin activity in human monocytes. 642 35

Clotting tests based upon the activated partial thromboplastin time are disturbed by heparin. This brief communication shows that Factor XII clotting activity in plasma samples heparinized in vitro and in vivo can be measured accurately by means of an aPTT-test using congenitally deficient substrate plasma when the plasma dilution buffer contains hexadimethrine bromide (Polybrene), 15 mg/L. This method of neutralizing heparin obviates more complicated procedures such as heparin adsorption to anion exchange resins.
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PMID:Assay of Factor XII clotting activity in heparinized plasma. 662 10

Activation of bovine plasma prekallikrein was investigated with several proteinases. Highly purified bovine plasma prekallikrein was rapidly activated to kallikrein [EC 3.4.21.8] by bovine activated Hageman factor, trypsin [EC 3.4.21.4] and Pronase P (proteinases from Streptomyces griseus) and more gradually by papain [EC 3.4.22.2] and ficin [EC 3.4.22.3]. Activation of prekallikrein was also observed with bovine plasmin [EC 3.4.21.7], but not with bovine clotting factors Xa (Stuart factor) [EC 3.4.21.6] and IXa (Christmas factor) or thrombin [EC 3.4.21.5]. Urokinase [EC 3.4.99.26], Reptilase, collagenase [EC 3.4.24.3], elastase [EC 3.4.21.11], alpha-chymotrypsin [EC 3.4.21.1], Nagarse [EC 3.4.21.14], and stem bromelain [EC 3.4.22 4] did not convert prekallikrein to kallikrein. Plasma kallikrein activated to Hageman factor released kinin rapidly from bovine high molecular weight (HMW) kininogen. However, from bovine low molecular weight (LMW) kininogen, liberation of kinin was extremely slow. The kallikrein activity was inhibited by soybean trypsin inhibitor (SBTI), Trasylol, diisopropylfluorophosphate (DFP), and N-alpha-tosyl-L-lysine chloromethylketone (TLCK), but not by egg-white trypsin inhibitor (EWTI), lima bean trypsin inhibitor (LBTI), heparin or hexadimethrine bromide (Polybrene). The kallikrein formed an enzyme-inhibitor complex with SBTI and Trasylol, but not with LBTI. Prekallikrein did not react with SBTI. Prekallikrein consists of a single polypeptide chain of molecular weight about 90,000, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Activation of prekallikrein by Hageman factor was found to involve cleavage of the single peptide bond on the disulfide-bridged polypeptide chain, and no change of molecular weight was observed during the activation. The peptide bond cleaved in prekallikrein by the activation was an Arg-X peptide bond on a disulfide-bridged polypeptide chain.
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PMID:Studies on prekallikrein of bovine plasma. II. Activation of prekallikrein with proteinases and properties of kallikrein activated by bovine Hageman factor. 676 24

Heparin kinetics were determined in four normal subjects, each of whom received three different doses (25, 50, and 75 units/kg body weight) by intravenous injection. Multiple blood samples were collected after each dose and each plasma sample was assayed for heparin activity using three different assay methods. Two of these assays are based on coagulation tests, i.e., activated partial thromboplastin time and thrombin time, while the third is based on chemical neutralization of heparin using hexadimethrine bromide. Heparin kinetics showed pronounced dose-dependent changes, irrespective of the assay method used, which were characterized by increasing biologic half-life and decreasing total clearance (Cl) with increasing dose. No changes were noted in apparent volume of distribution (Vd). This data also showed that there were differences in kinetic parameters of heparin depending on the assay method. In general, values for total Cl and apparent Vd based on chemical neutralization were approximately 1.5 to 2.0 times these parameters based on coagulation tests. We conclude that the immediate mechanism of the dose-dependent heparin kinetics is decreasing total clearance with increasing dose and suggest that in vivo activation of the anticoagulant properties of heparin may explain the assay-dependent kinetics.
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PMID:Heparin kinetics determined by three assay methods. 705 98

The cloning, purification and characterization of full-length annexin V, expressed intracellularly in Saccharomyces cerevisiae is detailed. Following homogenization in a glass bead mill, clarification by ultracentrifugation and fractional ammonium sulfate precipitation, the 319 amino acid protein was purified by column chromatography on phenyl-Sepharose and heparin-Sepharose. Annexin V elutes on reverse phase C4 silica as a single peak with greater than 97% homogeneity and is further characterized by a molecular mass of 34 kDa from electrophoresis under reducing conditions on SDS gels. Dynamic light scattering experiments reveal annexin V exists as a monomer in solution. Amino terminal Edman degradation afforded no sequence, therefore the carbamidomethylated protein was chemically cleaved with cyanogen bromide. Separation of the resulting peptide fragments on reverse phase HPLC followed by N-terminal sequencing and electrospray mass spectrometry supported the correct sequence as well as the existence of an acetyl blocking group on the N-terminus. The protein exhibits an isoelectric point of 4.73 by column chromatofocusing. Secondary structure predictions from CD spectroscopy indicate that the molecule is correctly folded. In anticoagulant assays, the purified protein exhibits dose-response effects in activated partial thromboplastin time (APTT) prolongation and doubles the clotting time of control human plasma at 70 micrograms ml-1. More specifically, in a factor Xa inhibition assay in which the activation of factor X via the tissue factor-factor VIIa complex is monitored by the cleavage of a factor Xa chromogenic substrate, recombinant annexin V exhibits a 50% inhibitory concentration (IC50) in the low nanomolar range.
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PMID:Isolation and characterization of recombinant annexin V expressed in Saccharomyces cerevisiae. 776 33

A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.
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PMID:Isolation and characterization of an intracellular serine proteinase inhibitor from a monkey kidney epithelial cell line. 840 7


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