Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dermatan sulfate increases the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 1000-fold by providing a catalytic template to which both the inhibitor and the protease bind. Dermatan sulfate is a linear polymer of D-glucuronic acid (GlcA) or L-iduronic acid (IdoA) alternating with N-acetyl-D-galactosamine (GalNAc) residues. Heterogeneity in dermatan sulfate results from varying degrees of O-sulfation and from the presence of the two types of uronic acid residues. To characterize the HCII-binding site in dermatan sulfate, we isolated the smallest fragment of dermatan sulfate that bound to HCII with high affinity. Dermatan sulfate was partially N-deacetylated by hydrazinolysis, cleaved with nitrous acid at pH 4, and reduced with [3H]NaBH4. The resulting fragments, containing an even number of monosaccharide units with the reducing terminal GalNAc converted to [3H]2,5-anhydro-D-talitol (ATalR), were size-fractionated and then chromatographed on an HCII-Sepharose column. The smallest HCII-binding fragments were hexasaccharides, of which approximately 6% bound. Based on ion-exchange chromatography, the bound material appeared to comprise a heterogeneous mixture of molecules possessing four, five, or six sulfate groups per hexasaccharide. Subsequently, hexasaccharides with the highest affinity for HCII were isolated by overloading the HCII-Sepharose column. The high-affinity hexasaccharides were fractionated by strong anion-exchange chromatography, and one major peak representing approximately 2% of the starting hexasaccharides was isolated. The high-affinity hexasaccharide was cleaved to disaccharides that were analyzed by anion-exchange chromatography, paper electrophoresis, and paper chromatography. A single disulfated disaccharide, IdoA(2-SO4)----ATalR(4-SO4) was observed, indicating that the hexasaccharide has the following structure: IdoA(2-SO4)----GalNAc(4-SO4)----IdoA(2-SO4)---- GalNAc(4-SO4)----IdoA(2-SO4)----ATalR(4-SO4). Since IdoA(2-SO4)----GalNAc(4-SO4) comprises only approximately 5% of the disaccharides present in intact dermatan sulfate, clustering of these disaccharides must occur during biosynthesis to form the high-affinity binding site for HCII.
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PMID:Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity. 221

Polysaccharide was isolated from human spleen mastocytoma by proteolytic digestion, precipitation with cetylpyridinium chloride, digestion with chondroitinase ABC, and ion-exchange chromatography on DEAE-cellulose. The final product (0.7 mg per g of starting material, MW 8000) behaved like standard heparin on ion-exchange chromatography and on electrophoresis, and contained D-glucuronic acid, L-iduronic acid, D-glucosamine and sulfate in the proportions expected for heparin. Affinity chromatography on antithrombin-Sepharose separated a distinct high-affinity fraction (4-5% of the total material). Structural analysis of this fraction showed that about 10% of the D-glucosamine residues were N-acetylated, the remainder N-sulfated. The anticoagulant activity of the isolated heparin was 71 B.P. units per mg (whole-blood system), or 30 units per mg (anti-thrombin and chromogenic substrate). 205 and 10-15 units per mg (chromogenic assay) were found for high and low affinity fractions, respectively. These results demonstrate conclusively the occurrence of heparin in a human tissue.
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PMID:Isolation and characterization of heparin from human mastocytoma tissue. 678 Oct 95

A natural lacquer polysaccharide with complex branches was separated into two fractions, LPH (MW 16.9x10(4)) and LPL (MW 6.85x10(4)). Results of 13C NMR and FT-IR indicated they had the same structure. The treatment of LPL with sodium periodate led to a partial cut-off of side chains with 4-O-methyl-D-glucuronic acid in the terminal. These polysaccharides were sulfated in the presence of Py*SO3/DMSO. Depending on the reaction conditions, the products showed a different degree of sulfation (DS) ranging from 0.57 to 1.57 and different molecular weights ranging from 1.71x10(4) to 3.49x10(4). FT-IR analysis showed the equatorial primary OH at O-6 and the axial secondary OH at O-4 were sulfated. Activated partial thromboplastin time (APTT), prothrombin time and thrombin time (TT) assays showed the sulfated polysaccharides could prolong APTT and TT, but not TP. These activities strongly depended on the DS, the molecular weights (MW) and the branching structure of polysaccharides. DS of above 0.8 was essential for anticoagulant activity. The anticoagulant activity increased with the DS and the molecular weights. The molecular weights played a more important role. The branching structure of polysaccharides increased the activities. In our studies, the sulfated polysaccharides with the DS of 1.15 and the highest MW of 3.49x10(4) had the best blood anticoagulant activities.
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PMID:Chemical modification, characterization and structure-anticoagulant activity relationships of Chinese lacquer polysaccharides. 1255 27

Fucosylated chondroitin sulfate is a glycosaminoglycan from sea cucumber, made up of alternating beta-D-glucuronic acid and N-acetyl-beta-D-galactosamine units, like mammalian chondroitin sulfate. But the beta-D-glucuronic acid residues have branches of sulfated fucose, which confer high anticoagulant and antithrombotic properties on this compound. We have now compared the anticoagulant, bleeding and antithrombotic effects of this fucosylated chondroitin sulfate and its carboxyl-reduced derivative. Both compounds have similar anticoagulant action, mostly due to acceleration of thrombin inhibition in the presence of heparin cofactor II. The native glycosaminoglycan shows a correlation among anticoagulant, bleeding and antithrombotic effects. Inhibition of thrombosis in an arterio-venous shunt occurs at low doses, which are almost ineffective in modifying the anticoagulant activity of plasma. In a venous experimental model, on the contrary, antithrombotic activity requires high doses and occurs concomitantly with an increase in the anticoagulant activity of plasma. The action of fucosylated chondroitin sulfate on thrombosis is apparently unrelated to its effect on platelet aggregation. The carboxyl-reduced derivative of fucosylated chondroitin sulfate prevented thrombosis in the arterio-venous shunt, but not in the venous experimental model. This derivative did not increase bleeding, in spite of its potent anticoagulant activity. Therefore, our results reveal a dissociation of the anticoagulant, bleeding and antithrombotic effects of the glycosaminoglycan. Furthermore, we demonstrate that a polysaccharide may be a potent inhibitor of one type of thrombotic episode, but inactive on another. We propose that the different effects of fucosylated chondroitin sulfate and its carboxyl-reduced derivative on venous thrombosis may be related to adherence of the glycosaminoglycan to the vessel wall.
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PMID:Venous and arterial thrombosis in rat models: dissociation of the antithrombotic effects of glycosaminoglycans. 1516 43

Dermatan sulfate (DS) accelerates the inhibition of thrombin by heparin cofactor II (HCII). A hexasaccharide consisting of three l-iduronic acid 2-O-sulfate (IdoA2SO3)-->N-acetyl-D-galactosamine 4-O-sulfate (GalNAc4SO3) subunits was previously isolated from porcine skin DS and shown to bind HCII with high affinity. DS from porcine intestinal mucosa has a much lower content of this disaccharide but activates HCII with potency similar to that of porcine skin DS. Therefore, we sought to characterize oligosaccharides from porcine mucosal DS that interact with HCII. DS was partially depolymerized with chondroitinase ABC, and oligosaccharides containing 2-12 monosaccharide units were isolated. The oligosaccharides were then fractionated by anion-exchange and affinity chromatography on HCII-Sepharose, and the disaccharide compositions of selected fractions were determined. We found that the smallest oligosaccharides able to bind HCII were hexasaccharides. Oligosaccharides 6-12 units long that lacked uronic acid (UA)2SO3 but contained one or two GalNAc4,6SO3 residues bound, and binding was proportional to both oligosaccharide size and number of GalNAc4,6SO3 residues. Intact DS and bound dodecasaccharides contained predominantly IdoA but little D-glucuronic acid. Decasaccharides and dodecasaccharides containing one or two GalNAc4,6SO3 residues stimulated thrombin inhibition by HCII and prolonged the clotting time of normal but not HCII-depleted human plasma. These data support the hypothesis that modification of IdoA-->GalNAc4SO3 subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate molecules with HCII-binding sites and anticoagulant activity.
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PMID:N-Acetylgalactosamine 4,6-O-sulfate residues mediate binding and activation of heparin cofactor II by porcine mucosal dermatan sulfate. 1662 94

Previously, we reported that the sulphated polysaccharides (SPS)-CF, a water-soluble polysaccharide isolated and purified from Korean green alga Maesaengi (Capsosiphon fulvescens, Chlorophyta), is a glucuronogalactomannan based mainly on the monosaccharide composition determined by high-performance liquid chromatography (HPLC) analysis after 1-phenyl-3-methyl-5-pyrazolone (PMP) labelling of sugars in the acid (trifluoroacetic acid (TFA)) hydrolyzates of SPS-CF, which showed mannose (55.4 mol %), galactose (25.3 mol %) and glucuronic acid (16.3 mol %) as major sugars (Na et al., Int Immunopharmacol 10:364-370, 2010). However, the results of the present study re-performed for monosaccharide composition of this polysaccharide using, in addition to HPLC of PMP-labelled sugars, other separation methods, i.e. high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), gas chromatography with flame ionising detection (GC-FID) and thin-layer chromatography (TLC), clearly demonstrated that the most prominent neutral monosaccharides of SPS-CF are xylose (38.6-49.4 mol %) and rhamnose (39.6-45 mol %), while mannose and galactose are present at a much lesser extent or in negligible amount. These extensive monosaccharide analyses, correlation nuclear magnetic resonance (NMR), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) measurements confirmed the sulphated glucuronorhamnoxylan (ulvan) type of SPS-CF polysaccharide, whose backbone is composed of alternating sequence of 4-linked L-rhamnose-3-sulphate and D-xylose residues (ulvobiose U3s) carrying monomeric D-glucuronic acid or D-glucuronic acid-3-sulphate on O-2 of some L-rhamnose-3-sulphate units as the side chains. The SPS-CF exhibited significant in vitro anti-coagulant activity by which the activated partial thromboplastin time (aPTT) and thrombin time (TT) were significantly prolonged. The results of this study demonstrated that the ulvan SPS-CF isolated from Korean Maesaengi C. fulvescens can be considered a potential anti-coagulant agent.
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PMID:Structural Features and Anti-coagulant Activity of the Sulphated Polysaccharide SPS-CF from a Green Alga Capsosiphon fulvescens. 2633 23