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

---

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

Low molecular weight heparins from a variety of commercial sources were examined. These had been prepared by several methods including peroxidative cleavage, nitrous acid cleavage, chemical beta-elimination, enzymatic beta-elimination, and chromatographic fractionation. The molecular weight and polydispersity of these low molecular weight heparins showed greater differences than were observed for typical commercial heparin preparations. Considerable differences were also observed in the antithrombin III mediated anti factor Xa activity, the heparin cofactor II mediated antifactor IIa activity, and the USP activity of these low molecular weight heparins. An oligosaccharide-mapping technique (comparable to the peptide mapping of proteins) was applied to these low molecular weight heparins in an effort to understand the structural features responsible for their activity differences. Heparin lyase from Flavobacterium heparinum was first used to depolymerize the low molecular weight heparin into its constituent oligosaccharides. The oligosaccharides present in the resultant mixture were identified and quantitated by using standard oligosaccharides of defined structure on gradient polyacrylamide gel electrophoresis and strong anion exchange high pressure liquid chromatography. Six of the oligosaccharide products have been identified and represent nearly 90 wt % of heparin's mass. Even though all the low molecular weight heparins showed these six oligosaccharide components, their content in each varied greatly, accounting for 20 to over 90% of their mass. The antithrombin III mediated anti factor Xa activities of the low molecular weight heparins correlated only poorly to the concentration of a hexasaccharide containing a portion of heparin's antithrombin III binding site. The heparin cofactor II mediated antifactor IIa activity, however, could not be correlated to these six oligosaccharides of known structure nor to the molecular weight or charge density of these low molecular weight heparins. The low molecular weight heparins prepared by different methods each showed a new distinctive oligosaccharide in their maps. Their isolation and structural characterization, which included two-dimensional NMR and fast atom bombardment mass spectrometry, indicated that these unusual oligosaccharides result from end-sugar modification during chemical depolymerization. Both gel electrophoresis and high-pressure liquid chromatography mapping techniques showed a greater structural diversity between low molecular weight heparins than had previously been observed between similarly analyzed commercial heparins.
...
PMID:Oligosaccharide mapping of low molecular weight heparins: structure and activity differences. 216 May 37

Size homogeneous heparin oligosaccharides were prepared from nitrous acid depolymerized heparin by means of repeated gel filtration chromatography. These oligosaccharides were then further separated with respect to affinity for antithrombin by means of affinity chromatography. All the high-affinity oligosaccharides thus obtained had a strong ability to potentiate factor Xa inhibition while their ability to inhibit factor IIa abruptly dropped below a chain length of 20 monosaccharides. In a rabbit stasis model, high-affinity oligosaccharides below a chain length of 20 units also showed a continuous decrease in antithrombotic effect with increasing degree of depolymerization. However, there was no distinct drop paralleling the thrombin inhibiting capacity. Low-affinity oligosaccharides also exhibited a weak antithrombotic effect, although they did not always contribute to an increased anti-factor Xa activity ex vivo. This was the case whether or not they were administered alone or in combination with high-affinity oligosaccharides. Low-affinity oligosaccharides may therefore exert an antithrombotic effect per se with a mechanism of action that is independent of antithrombin III.
...
PMID:Antithrombotic effects of heparin oligosaccharides. 273 63

Studies were conducted to define the location of components and sequences in heparin with respect to their distance from the peptide linkage in the native proteoglycan. A purified heparin-oligopeptide was linked via its amino terminus to a matrix containing an azo bond and an activated carboxyl group. The polysaccharide chain was maximally degraded, either with heparinase or nitrous acid, and the soluble products were removed. The heparin-oligopeptide fragments that remained on the matrix were released by reductive cleavage of the azo linkage and characterized. The fragments, as well as heparin released without prior degradation, contained serine and glycine as the principal amino acids; the ratio of galactose to xylose was 2:1. The ratio of glucosamine to serine of 33:1 in the undegraded heparin was reduced to 6:1 and 1:1 in the heparinase-treated and nitrous acid-treated products, respectively. The undegraded sample and the fragments contained phosphate in equivalent amounts, demonstrating its presence in the heparin-protein linkage region. The heparin-oligopeptide preparation was also fractionated by gel filtration and high and low molecular weight fractions thus obtained were each linked to the insoluble matrix. The products that were subsequently released were subfractionated on a molecular weight-calibrated column of Sephadex G-200, and eluates were assayed for activity in promoting the neutralization of thrombin and factor Xa by antithrombin. The results revealed a sharp decrease in specific activity in heparin-oligopeptide fractions below Mr = 15,000 indicating that the anticoagulant-conferring segment is located at about 20 disaccharide units away from the peptide linkage region.
...
PMID:Location of specific oligosaccharides in heparin in terms of their distance from the protein linkage region in the native proteoglycan. 333 97

The influence of heparin on the inhibition of factor Xa has been studied under conditions where factor Xa is bound to collagen-thrombin-stimulated platelets to form the prothrombinase complex. Unfractionated heparin was found to cause a concentration-dependent acceleration of the inhibition of the platelet prothrombinase complex up to a maximum rate constant of 4.1 X 10(7) M-1 X min-1 at heparin concentrations of 0.2 microM and above. This is equivalent to a 4800-fold acceleration over the rate constant for the inhibition in the absence of heparin, and is 6.8-fold lower than the rate constant for the inhibition of uncomplexed factor Xa in the presence of saturating concentrations of heparin which was determined as 2.8 X 10(8) M-1 X min-1. The effects of three Mr fractions of heparin were also studied. These were a gel-filtered heparin of Mr 15000, a gel-filtered heparin of Mr 6000 and a heparin oligosaccharide (primarily 8-10 monosaccharide units) prepared by nitrous acid depolymerization, each with high affinity for antithrombin III. These fractions all accelerated the rate of the antithrombin III inhibition of the platelet prothrombinase complex, with maximum rate constants of 6.8 X 10(7), 1.4 X 10(7) and 9.8 X 10(6) M-1 X min-1, respectively. On comparison with the effect of these heparin fractions on the rate of inhibition of uncomplexed factor Xa a progressively increasing disparity between the rate of inhibition of uncomplexed and complexed factor Xa was observed, rising from 1.7-fold with the oligosaccharide to 6.8-fold with the unfractionated heparin. A possible mechanism for this differential activity between uncomplexed and complexed factor Xa with the various heparin fractions is discussed in terms of an involvement of heparin binding to factor Xa.
...
PMID:The acceleration of the inhibition of platelet prothrombinase complex by heparin. 395 24

We have isolated from nitrous acid cleavage products of heparin two major octasaccharide fragments which bind with high affinity to human antithrombin. Octasaccharide S, with the predominant structure iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid-----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, is sensitive to cleavage by Flavobacterium heparinase as well as platelet heparitinase and binds to antithrombin with a dissociation constant of (5-15) X 10(-8) M. Octasaccharide R, with the predominant structure iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, is resistant to degradation by both enzymes and binds antithrombin with a dissociation constant of (4-18) X 10(-7) M. The occurrence of a 15-17% replacement of N-sulfated glucosamine 3,6-di-O-sulfate with N-sulfated glucosamine 3-O-sulfate and a 10-12% replacement of iduronic acid with glucuronic acid in both octasaccharides indicates that these substitutions have little or no effect on the binding of the oligosaccharides to the protease inhibitor. When bound to antithrombin, both octasaccharides produce a 40% enhancement in the intrinsic fluorescence of the protease inhibitor and a rate of human factor Xa inhibition of 5 X 10(5) M-1 s-1 as monitored by stopped-flow fluorometry. This suggests that the conformation of antithrombin in the region of the factor Xa binding site is similar when the protease inhibitor is complexed with either octasaccharide.
...
PMID:Sequence variation in heparin octasaccharides with high affinity for antithrombin III. 652 37

Two types of LMW heparin were prepared by gel filtration of standard heparin (LMW fraction) and by degradation of heparin by nitrous acid (LMW fragment), respectively. The effects on factor Xa inhibition (XaI), APTT, platelet aggregation and AT III level of these preparations were studied after subcutaneous administration to humans and compared with those of standard heparin. At a dose of 5000 IU (XaI) the LMW fraction and LMW fragment induced peak plasma XaI activity of 0.32 IU/ml and 0.41 IU/ml respectively, compared to 0.07 IU/ml for heparin. Still 11.5 h after administration both LMW preparations gave higher activities than heparin ever induced. Following administration of 10,000 IU (XaI) of the LMW fragment the plasma peak XaI activity was 0.81 IU/ml. This prolonged the APTT from 36 sec to 46 sec only. The half-lives of the XaI activity in plasma were between 3 and 4 hours. No effect on platelet aggregation or AT-III level was demonstrated.
...
PMID:Anticoagulant effects of two types of low molecular weight heparin administered subcutaneously. 665 20

We have compared the hemorrhagic and antithrombotic effects of a low molecular weight (LMW) heparin fraction and standard heparin in rabbits. Similar LMW heparin fractions have antithrombotic effects when tested in animals, but their hemorrhagic effects relative to standard heparin have not been established. Standard porcine mucosal heparin (mol wt 15,000 daltons) was depolymerized by nitrous acid to a low molecular weight fraction (mol wt 4600 daltons). Using equal USP units, the standard and Dep LMW heparin were compared in vitro, ex vivo, and in vivo. In vitro, when diluted in rabbit plasma, the Dep LMW heparin at equivalent anti-Xa activity showed less prolongation of thrombin clotting times or activated partial thromboplastin times. Ex vivo, platelets from rabbits treated with the Dep LMW heparin showed less inhibition of collagen-induced aggregation. The relative hemorrhagic properties of the two heparins were compared in vivo in rabbits using a sensitive blood loss assay, and the antithrombotic properties were compared in a thrombin-induced venous stasis model. By using an optimal threshold heparin dose in each test system, it was possible to demonstrate that equal USP units of Dep LMW heparin caused less blood loss but showed greater antithrombotic activity than standard heparin.
...
PMID:The relationship between the hemorrhagic and antithrombotic properties of low molecular weight heparin in rabbits. 708 26

Tissue factor pathway inhibitor (TFPI) is a heparin-binding protein involved in the extrinsic blood coagulation system. In order to elucidate the minimal size of heparin chain required for the interaction with TFPI, we prepared a series of heparin-derived oligosaccharides with tailored chain length ranged from disaccharide to eicosasaccharide after the successive treatments of heparin, including partial N-desulphation, deaminative cleavage with nitrous acid and gel-filtration. Affinity chromatography study of each oligosaccharide fraction using TFPI as the ligand indicated that increasing the degree of polymerisation causes increased affinity, and that a remarkable change in the affinity occurs between the decamers and dodecamers. Measurement of factor Xa inhibitory activity of TFPI in the presence of each oligosaccharide fraction indicated that the fractions shorter than dodecamers only slightly enhanced the TFPI activity for factor Xa inhibition, while the fractions larger than octadecamers had an effect comparable to full-length heparin. These were compatible to the results from the kinetic analyses of the interaction between TFPI and heparin-derived oligosaccharide with an evanescent wave-based biosensor system, IAsys, using a TFPI C-terminal peptide as the ligand.
...
PMID:Effect of heparin chain length on the interaction with tissue factor pathway inhibitor (TFPI). 1206 17

Endogenous N-sulfated glycosaminoglycans (GAGs) comigrating with standard heparin and sensitive to nitrous acid treatment were isolated from plasma of healthy donors. The amount of these compounds was 7-10 microg/ml, and activated partial thromboplastin time, anti-Xa and anti-IIa activities were similar to those of standard heparin of high molecular mass. Analysis with gradient PAGE of the putative endogenous heparin showed a mean molecular mass of 12 kD. These N-sulfated GAGs could be isolated only after removal of binding peptides that impaired purification by ion-exchange chromatography. We used SDS-PAGE as a tool to separate peptides from endogenous GAGs. N-sulfated GAGs exited the gel before peptides when the electrophoresis was overrun. Endogenous GAGs could be recovered by ion-exchange chromatography of the SDS-PAGE buffer, 'free' from associating peptides. These results strongly support the hypothesis that endogenous heparin is associated in vitro with a variety of proteins and that this association could be responsible for modification of both heparin and protein activities.
...
PMID:Isolation of endogenous anticoagulant N-sulfated glycosaminoglycans in human plasma from healthy subjects. 1221 63

Heparin-like glycans with diverse disaccharide composition and high anticoagulant activity have been described in several families of marine mollusks. The present work focused on the structural characterization of a new heparan sulfate (HS)-like polymer isolated from the mollusk Nodipecten nodosus (Linnaeus, 1758) and on its anticoagulant and antithrombotic properties. Total glycans were extracted from the mollusk and fractionated by ethanol precipitation. The main component (>90%) was identified as HS-like glycosaminoglycan, representing approximately 4.6 mg g(-1) of dry tissue. The mollusk HS resists degradation with heparinase I but is cleaved by nitrous acid. Analysis of the mollusk glycan by one-dimensional (1)H, two-dimensional correlated spectroscopy, and heteronuclear single quantum coherence nuclear magnetic resonance revealed characteristic signals of glucuronic acid and glucosamine residues. Signals corresponding to anomeric protons of nonsulfated, 3- or 2-sulfated glucuronic acid as well as N-sulfated and/or 6-sulfated glucosamine were also observed. The mollusk HS has an anticoagulant activity of 36 IU mg(-1), 5-fold lower than porcine heparin (180 IU mg(-1)), as measured by the activated partial thromboplastin time assay. It also inhibits factor Xa (IC(50) = 0.835 microg ml(-1)) and thrombin (IC(50) = 9.3 microg ml(-1)) in the presence of antithrombin. In vivo assays demonstrated that at the dose of 1 mg kg(-1), the mollusk HS inhibited thrombus growth in photochemically injured arteries. No bleeding effect, factor XIIa-mediated kallikrein activity, or toxic effect on fibroblast cells was induced by the invertebrate HS at the antithrombotic dose.
...
PMID:Unique extracellular matrix heparan sulfate from the bivalve Nodipecten nodosus (Linnaeus, 1758) safely inhibits arterial thrombosis after photochemically induced endothelial lesion. 2005 99


1 2 Next >>

---