A comparative study of the anticoagulant activities of eleven fucoidans

Abstract

Seven fucoidans that differed only with respect to the average molecular weight and four fucoidans that differed with respect to both the molar ratio of fucose to galactose and the average molecular weight were obtained. The anticoagulant activities of these fucoidans were determined in order to understand the effects of the average molecular weight and the content of galactose. Fucoidans Y5–Y11, with the average molecular weight in the range of 50.1–8.4 kDa, showed decreasing activity at three concentrations and dose-dependent activity in the APTT and TT assays. Y1–Y4, which differed with respect to the average molecular weight and the molar ratio of fucose to galactose, exhibited complicated results in the APTT and TT assays. It was shown that not only the average molecular weight but also the molar ratio of fucose to galactose affected the anticoagulant activity of fucoidans.

Introduction

Fucoidans, a family of sulfated heteropolysaccharides, are found in brown algae and marine invertebrates. The amount of fucose present is dependent on the origin of the algae, the time of harvest and the species of algae. Owing to the complexity of fucoidan, it is still difficult to elucidate the relationship between the structure of this compound and its activities. However, there is abundant information on the structure and the activities. Patankar, Oehninger, Barnett, Williams, and Clark (1993) revised the structure of fucoidan extracted from Fucus vesiculosus. The structure of this fucoidan was a branched 1 → 3 linked α-l-fucan with sulfation at position O-4, and every two or three fucose residues had a branched fucose at the 2 or 3 position. Chevolot, Mulloy, Ratiskol, Foucault, and Colliec-Jouault (2001) observed that fucoidans from Ascophyllum nodosum and Fucus vesiculosus consisted of the repeating disaccharide unit [→3)-α-l-Fuc(2SO₃⁻)-(1 → 4)-α-l-Fuc(2,3-diSO₃⁻)-(1]. A structure of alternating 3-linked α-l-fucopyranose 2,4-disulfate and 4-linked α-l-fucopyranose 2-sulfate units was also identified from Fucus distichus (Bilan et al., 2004). Chandía and Matsuhiro (2008) obtained fucoidan from Lessonia vadosa (Phaeophyta) with a backbone of 1 → 3 linkages with sulfate groups primarily at position O-4 with infrequent sulfation at position O-2. The fucoidan from Saccharina latissima was made up of α-(1 → 3) linked fucopyranose residues, which were sulfated at C-4 and/or at C-2 and branched at C-2 with singly sulfated α-l-fucopyranose residues (Bilan et al., 2010).

Fucoidans have diverse biological activities, such as anticancer activity (Cho et al., 2011, Ermakova et al., 2011), immunomodulating activity (Caipang, Lazado, Berg, Brinchmann, & Kiron, 2010), antiviral activity (Karmakar, Pujol, Damonte, Ghosh, & Ray, 2010), antiangiogenic activity (Koyanagi, Tanigawa, Nakagawa, Soeda, & Shimeno, 2003), antitumor activity (Sokolova et al., 2011, Synytsya et al., 2010) and antioxidant activity (Wang, Zhang, Zhang, Song, & Li, 2010). The anticoagulant activity of these compounds is by far the most widely studied (Croci et al., 2011, Pomin, 2004). This activity depends on the structural features, the sulfate position, the composition of the monosaccharides and the molecular weight (Barros et al., 2011). It has been reported that the structural features influence not only the anticoagulant activity of fucoidan but also the mechanism of this activity (Pereira, Mulloy, & Mourão, 1999). Chandía and Matsuhiro found that 4-O-sulfate 1 → 3 linked fucan exerted anticoagulant activity, whereas 2-O-sulfate 1 → 3 linked fucan had a deleterious effect. In addition, 2,4-disulfate 1 → 3 linked fucan had high anticoagulant activity (Chandía & Matsuhiro, 2008). It was confirmed that the difference in the anticoagulant activities of sulfated polysaccharides might depend on the different patterns of sulfation of the fucose branch of the chondroitin sulfate, especially 2,4-O-disulfation (Chen et al., 2011). Cumashi et al. (2007) examined the anticoagulant activity of nine polysaccharides from Laminaria saccharina (Saccharina latissima), L. digitata, Fucus evanescens, F. serratus, F. distichus, F. spiralis, Ascophyllum nodosum, and Cladosiphon okamuranus. They found that only the polysaccharide from C. okamuranus, which had substantial levels of 2-O-α-d-glucuronpyranosyl branches in the linear (1 → 3)-linked α-l-fucopyranoside chain, did not display anticoagulant activity as determined by the activated partial thromboplastin time (APTT). All of the other polysaccharides exhibited different anticoagulant activities. The effect of fucoidan on thrombin inactivation mediated by heparin cofactor II was reduced dramatically by a slight decrease in the molecular size of the sulfated fucan. A total of 178 monosaccharide units were necessary for the interaction with heparin cofactor II, and 408 monosaccharide units were necessary for complete thrombin inhibition (Pomin, 2004).

According to a previous study (Wang, Zhang, Zhang, Zhang, & Niu, 2010), the backbone of fucoidan LF2 extracted from the brown seaweed Laminaria japonica (Saccharina japonica), consisting of 1 → 3 linkages (75%) and 1 → 4 linkages (25%). The branched points are at C-4, with β-d-galactopyranose residues (35%), or at C-2, with 4-sulfate fucopyranose residues (65%). Sulfation is sometimes present at position C-4 or C-2 with fucopyranose residues and C-3 and/or C-4 with galactopyranose residues.

In this study, we prepared seven polysaccharides (Y5–Y11) that differed only with respect to the average molecular weight and four polysaccharides (Y1–Y4) that differed with respect to both the molar ratio of fucose to galactose and the average molecular weight. We used these polysaccharides to further study the relationship between structure and anticoagulant activity. Based on the results of a previous study (Cumashi et al., 2007), it was hypothesized that the branching units of the 2-O-α-d-glucuronpyranosyl residues had a negative effect on anticoagulant activity. In this study, the relationship between the molar ratio of fucose to galactose and the anticoagulant activity was elucidated for the first time. According to our results, it is necessary to consider the content of galactose when evaluating the quality of commercially available crude fucoidans for use as anticoagulant agents. Although there have been many reports on the relationship between molecular weight and anticoagulant activity, this study was the first to determine the anticoagulant activities of samples that differed only with respect to the average molecular weight. We found that both the average molecular weight and the molar ratio of fucose to galactose influenced the activity of fucoidans in the APTT and TT assays. The effect was not as obvious in the PT assay.