Takashi Nishino

Isolation, purification, and characterization of fucose-containing sulfated polysaccharides from the brown seaweed ecklonia kurome and their blood-anticoagulant activities

A sulfated polysaccharide fraction, obtained from the hot-water extract of the brown seaweed, Ecklonia kurome by removing laminaran and the major part of alginic acid, gave sulfated polysaccharides (B-I, B-II, C-I, and C-II) by both anion-exchange chromatography on a column of Ecteola-cellulose and by fractional precipitation with ethanol containing 0.3% calcium acetate, and then by gel-filtration chromatography on a Sepharose 4B column. B-I and B-II are composed of fucose, galactose, mannose, xylose, glucuronic acid, and ester sulfate in the approximate molar ratios of 1.00:0.36:0.48:1.08:1.85:2.35 and 1.00:0.81:0.18:0.45:0.61:2.00, respectively. C-I and C-II are composed of fucose, galactose, glucuronic acid, and ester sulfate in approximate molar ratios of 1.00:0.03:0.03:1.61 and 1.00:0.19:0.07:1.48, respectively. Blood-anticoagulant activities with respect to activated partial thromboplastin time (APTT) were approximately 24, 19, 81, and 85% of that of heparin for B-I, B-II, C-I, and C-II, respectively. All the polysaccharides showed slight antithrombin activity. No antifactor Xa activity was observed for any of the polysaccharides.

Isolation and partial characterization of a noval amino sugar-containing fucan sulfate from commercial Fucus vesiculosus fucoidan☆

Commercial crude fucoidan (Sigma) from the brown seaweed Fucus vesiculosus was fractionated into its polysaccharide components by gel filtration and anion-exchange chromatography to clarify the structure-anticoagulant activity relationship. The products comprised a wide spectrum of fucans ranging from typical fucoidans (major components) containing mainly fucose, sulfate, and no uronic acid to low sulfate-containing heteropolysaccharide-like fucans (minor components) being composed of neutral sugars other than fucose and a high content of uronic acid(s). The polysaccharide components also had a wide range of molecular weight. The typical fucoidans showed a potent anticoagulant activity, whereas the other fucans had no or only slight activity. One of the fractions found as a minor component, was a novel polysaccharide containing an appreciable amount (11.5%) of glucosamine and a small amount (5.2%) of protein in addition to fucose and sulfate, and having a low apparent molecular weight of 6800. This is the first report that a proteoglycan-like, amino sugar-containing fucan sulfate, composed of fucose, galactose, glucose, mannose, xylose, uronic acid, glucosamine, and sulfate in the molar ratio of 1.00:0.04:0.01:0.48:0.24:0.18:0.56:1.90, could be obtained from brown seaweed. However, this polysaccharide showed no anticoagulant activity.

Anticoagulant and antithrombin activities of oversulfated fucans

Three species of oversulfated fucans having different sulfate contents (the ratio of sulfate/total sugar residues, 1.38-1.98) were prepared by chemical sulfation of a fucan sulfate (sulfate/sugar ratio, 1.28) isolated from the brown seaweed Ecklonia kurome. The anticoagulant activities of the oversulfated fucans were compared with that of a parent fucan with respect to activated partial thromboplastin time (APTT) and thrombin time (TT) in plasma. The respective activities (for APTT and TT) of the oversulfated fucans increased to 110-119% and 108-140% of the original values with increase in their sulfate content. The anticoagulant activity with respect to APTT (173 units/mg) of an oversulfated fucan (sulfate/sugar ratio, 1.98) was higher than that (167 units/mg) of heparin used as a standard. The heparin cofactor II-mediated antithrombin activity of the oversulfated fucans also increased significantly with increase in sulfate content. The maximum activity was higher than those of the parent fucan and heparin. However, the increment of the anticoagulant and the antithrombin effects gradually decreased with increase in the sulfate content of the fucans. These results indicate that the effects of the fucan sulfate are dependent on its sulfate content until a plateau is reached.

STRUCTURAL CHARACTERIZATION OF A NEW ANTICOAGULANT FUCAN SULFATE FROM THE BROWN SEAWEED ECKLONIA KUROME

Methylation analysis of a fucose-containing, sulfated polysaccharide (C-II), which was isolated from the brown seaweed Ecklonia kurome and has a potent anticoagulant activity, showed the presence of 3-O- and 3,4-O-disubstituted fucopyranosyl residues in addition to small proportions of nonreducing, terminal fucofuranosyl and fucopyranosyl groups, and 2,3-di-O- and 2,3,4-tri-O-substituted fucopyranosyl and galactopyranosyl residues with various glycosidic linkages. Methanolysis of C-II gave several neutral oligosaccharide fractions in small proportions and two high-molecular-weight acidic fractions in large proportions. Methylation analysis of the low-sulfated acidic fraction showed that the proportion of 3-O-linked fucosyl residues increases and that of 3,4-O-disubstituted decreased as compared to C-II. Methylation and g.l.c.-m.s. analysis of the neutral oligosaccharide fractions showed the presence of Fuc-(1----3)-Fuc and a fucosyl trisaccharide, in addition to small proportions of Gal-(1----4)-Fuc, Fuc-(1----2)-Fuc, Fuc-(1----4)-Fuc, Fuc-(1----2)-Gal, and Fuc----Gal----Fuc. Methylated C-II was also desulfated by methanolysis, followed by remethylation with (2H3)methyl iodide, and most of (2H3)methyl groups were linked to O-4 of the 3-O-linked fucosyl residues. These results suggested a highly branched, new type of fucan sulfate containing a backbone of (1----3)-linked L-fucosyl residues having sulfate groups mainly attached to C-4.

The influence of sulfate content and molecular weight of a fucan sulfate from the brown seaweed Ecklonia kurome on its antithrombin activity

The antithrombin effects of the sub-fractionated fucans with different molecular weights and sulfate contents, which were prepared from a fucan sulfate isolated from the brown seaweed Ecklonia kurome, were examined for their abilities to inhibit thrombin-fibrinogen reaction and amidolytic activity of thrombin, and to bind to fibrinogen. The inhibitory effects of the fucans on both fibrinogen clotting by thrombin and amidolysis of the protein in the presence of heparin cofactor II were improved with increase in their molecular weights and reduced with decrease in their sulfate contents. The binding abilities of the fucans with almost the same sulfate content to fibrinogen were unchanged independently of their molecular weights, although the ability diminished with decrease in the sulphate content. These results suggest that heparin cofactor II-mediated antithrombin activity of the fucan sulfate is dependent on both its sulfate content and molecular weight, and also that the inhibitory effect of the polysaccharide on fibrinogen clotting by thrombin may be attributable to the steric hindrance by its binding to fibrinogen.

Antithrombin activity of a fucan sulfate from the brown seaweed Ecklonia kurome.

The mechanism of antithrombin action of a fucan sulfate (C-II), which was isolated from the brown seaweed Ecklonia kurome, was examined by clotting method using a thrombin-fibrinogen system and by amidolytic method using a chromogenic substrate in the presence and the absence of antithrombin III (AT III) or heparin cofactor II (HC II). C-II significantly inhibited the clotting of fibrinogen by thrombin even in the absence of the protease inhibitors, and the amidolytic activity of the protein only in the presence of HC II. C-II was not adsorbed on an AT III-agarose column and its anticoagulant activity in AT III-depleted plasma was the same as that in normal one. Examination of interaction of C-II with fibrinogen by gel filtration chromatography demonstrated that C-II bound to the protein. These results indicated that the antithrombin activity of C-II was mediated by HC II and not by AT III, and that the polysaccharide bound to fibrinogen, thereby blocking thrombin action, and also that its direct thrombin inhibition was very weak.

Isolation and preliminary characterization of fucose-containing sulfated polysaccharides with blood-anticoagulant activity from the brown seaweed Hizikia fusiforme

We reported that a crude, sulfated polysaccharide (SPS) fraction from Hizikia fusiforme showed considerable anticoagulant activity. In order to identify the biologically active substances, we attempted to isolate each of the polysaccharide components from the SPS fraction. As a result, two purified, fucan-like sulfated polysaccharides having anticoagulant activity were obtained. We describe herein the isolation of the purified sulfated polysaccharides, their physical and chemical properties, and their blood-anticoagulant activities

Inhibition of the Generation of Thrombin and Factor Xa by a Fucoidan from the Brown Seaweed Ecklonia kurome

The effects of a fucoidan (C-II), which was purified from the brown seaweed Ecklonia kurome, on the generation of thrombin and factor Xa have been investigated by measuring the amidolytic activities by using the respective specific chromogenic substrates in both plasma and purified systems. C-II inhibited significantly the generation of thrombin in both the intrinsic and the extrinsic pathways, although the intrinsic inhibitory effect by C-II was more remarkable than the extrinsic one. On the other hand, C-II was a good inhibitor of the factor Xa generation in the intrinsic pathway, while it was a poor one in the extrinsic pathway. In the purified systems C-II also inhibited the formation of prothrombin-activating complex (i.e., prothrombinase), but not its activity. The concentration of C-II required for 50% inhibition of thrombin generation was about one-tenth to one-seventh of that of the activity of the generated thrombin in plasma. These results indicate that C-II has an inhibitory effect on the generation of thrombin by blocking the formation of prothrombinase and by preventing the generation of intrinsic factor Xa in addition to its antithrombin activity, and also that the generation-inhibitory effect is more remarkable than C-IIs enhancement effect on the antithrombin activity by heparin cofactor II in plasma.

Effects of a Fucoidan on the Activation of Plasminogen by u-PA and t-PA

The effect of an anticoagulant fucoidan (C-I-H) from the brown seaweed Ecklonia kurome on the fibrinolytic system was studied in vitro using S-2251 as a substrate of plasmin. C-I-H enhanced the activation of Glu- and Lys-plasminogen by high molecular weight urokinase-type plasminogen activator (HMW u-PA) very effectively, but the activation by low molecular weight u-PA was hardly enhanced with C-I-H. C-I-H also potentiated moderately the activation by single- and two-chain tissue-type plasminogen activators (sct- and tct-PA). These effects of C-I-H were higher than those of heparin used. But C-I-H had no effect on the amidolytic activity of plasmin to S-2251. These results indicate that C-I-H promotes the generation of plasmin in the plasminogen activation by HMW u-PA and t-PA, but not the activity of generated plasmin. Kinetic analyses suggest that C-I-H enhances the HMW u-PA-mediated plasminogen activation by increasing the affinity of the activator for Glu- and Lys-plasminogen and by increasing the molecular activity of the activator. On the other hand, C-I-H had no effect on the affinity of tct-PA for both plasminogens. The catalytic efficiencies of HMW u-PA and tct-PA for the activation of both plasminogens were increased with C-I-H about 8- and 2-fold, respectively. The present results suggest that C-I-H has the fibrinolytic activity by stimulating the plasminogen activation by HMW u-PA and t-PA. The mechanism of the enhancement effect of C-I-H on the activation is presumed to be that C-I-H binds to plasminogen, thereby inducing a structural change of plasminogen susceptible to the action of plasminogen activators.

An anticoagulant fucoidan from the brown seaweed Ecklonia kurome

The structure of a alpha-L-fucose-rich, sulphated polysaccharide (C-I) with a potent anticoagulant activity, which was isolated from the brown seaweed Ecklonia kurome, has been studied. Methylation analysis showed that C-I consisted mainly of 3-linked and 3,4-disubstituted fucopyranosyl residues in addition to non-reducing terminal fucofuranosyl and fucopyranosyl residues, 2,3-di- and 2,3,4-tri-substituted fucopyranosyl residues and galactopyranosyl residues with various glycosidic linkages. Methanolysis of C-I gave neutral di-, tri-, tetra- and highly polymerized-oligosaccharide fractions. GC-MS and methylation analysis indicated that di- and trisaccharide fractions consisted mainly of Fuc-(1----3)-Fuc and Fuc-(1----3)-Fuc-(1----3)-Fuc, respectively, in addition to small amounts of Fuc-(1----4)-Fuc, Fuc-(1----4)-Gal and Fuc-(1----3)-[Fuc-(1----2)-]Fuc. When methylated C-I was subjected to methanolysis for desulphation followed by remethylation with deuterated methyl iodide, most of deuteriomethyl groups substituted to position 4 of 3-linked Fuc.