Natural sulfated polysaccharides, such as galactans [1–5], arabinans , and fucoidans [1, 4, 7–11], present in seaweeds, as well as sulfated fucans of invertebrates [1–3, 7, 12], are regarded as the perspective basis to develop drugs for different applications. Among these polysaccharides, the fucoidans raise the largest interest, as it can be concluded from the statistical records for corresponding papers summarized in Fig. 1 (comp. with ref. ). Great attention to fucoidans can be connected with their facile availability from brown seaweeds, lack of toxicity, great biocompatibility, as well as with broad structural variability, which stimulates their structural analysis to assess the relation between structure and biological activity.
Influence of these biopolymers on the hemostatic system depends on the ability of polymeric sulfates to interact with positively charged groups in proteins responsible for hemostasis, leading to the formation of stabilized complexes. The anticoagulant properties of sulfated polysaccharides are mainly connected with thrombin inhibition mediated by antithrombin III (ATIII) and/or heparin cofactor II (HCII), with different efficiencies depending on the structural features of carbohydrates. Other mechanisms, such as direct inhibition of thrombin, are also possible.
A number of studies revealed certain structure-activity relationships for these macromolecules [2, 5, 8, 10, 12–16]. The main structural features of sulfated polysaccharides, which should be taken into account regarding their anticoagulant properties, include the monosaccharide composition, the degree and pattern of sulfation, molecular weight, and types of glycosidic bonds. The level of sulfation exceeding one sulfate per monosaccharide residue was shown to be important for high anticoagulant activity of fucans and galactans [17, 18]. High molecular weight fucans demonstrated a greater anticoagulant effect than structurally similar polysaccharides having lower molecular weight [17, 19].
The pattern of O-sulfation together with the structure of the backbone and branches, but not only total negative charge of sulfates, has a substantial impact on the activity of the biopolymers under discussion (see, for example, ref. ). Thus, in a series of polysaccharides isolated from invertebrates it has been shown that 2-O-sulfated (1 → 3)-linked α-l-galactan, but not an α-l-fucan, with a similar sulfation pattern and molecular size, is a potent thrombin inhibitor mediated by ATIII or HCII . In the case of HCII-mediated inhibition, the major structural requirement for the activity is the presence of selectively 4-O-sulfated fucose units . In addition, the linear (1 → 3)-linked α-l-fucans, enriched in 2,4-di-O-sulfated units, were shown to have an amplifying effect on the ATIII-mediated anticoagulant activity [2, 7].
Fucoidans from brown algae possess a significantly more complicated structure than fucans from invertebrates due to the presence of numerous branches, non-fucose monosaccharide constituents, and acetates . It was found that, besides ATIII- and HCII-mediated thrombin inhibition activities, which are typical of fucans, the algal fucoidans could also be direct inhibitors of thrombin. For the first time, this behavior was shown for fucoidans from Fucus vesiculosus and Laminaria brasiliensis, which are built up of alternating (1 → 3)- and (1 → 4)-linked α-l-fucose residues sulfated at C-2 and/or C-4, and bearing fucose branches . The similar mechanism of action was shown for the fucoidans from the brown seaweeds Saccharina latissima and F. distichus, but the polysaccharides from Cladosiphon okamuranus and Analipus japonicus were inactive . Notably, a linear arabinan consisting of (1 → 3)-linked β-l-arabinose residues sulfated at C-2 and/or C-4 was also found to be the potent direct thrombin inhibitor .
Here we summarize the results of the study on anticoagulant activity of several fucoidans from brown seaweeds, their chemically modified derivatives, and synthetic oligosaccharides, related to the fucoidans. The studied compounds differed in the monosaccharide composition, types of glycosidic bonds, pattern and degree of sulfation, and molecular weight.
Stuctural diversity of brown algal fucoidans
As a rule, fucoidans present in different algal species vary not only in sulfation pattern, but also in the structures of their carbohydrate moieties. Moreover, even a crude fucoidan obtained from the single algal species may be a mixture of polysaccharides having different chemical structures. For example, it was shown by detailed chemical analysis that a mixture of sulfated polysaccharides extracted from the brown alga S. latissima contained at least four structurally different components . Fractions enriched in the main components of this mixture, namely, in a sulfated fucan and a sulfated fucoglucuronomannan, respectively, were obtained using anion-exchange chromatography on DEAE-Sephacel. Recently it was shown that sulfated fucan, but not sulfated fucoglucuronomannan is of primary importance for the biological activity of a total fucoidan from S. latissima . Similar approach was used for isolation of the highly sulfated fucoidan fractions from a number of other brown seaweed species, namely, Chordaria flagelliformis (CF) , Cladosiphon okamuranus (CO) , Punctaria plantaginea (PP) , Fucus evanescens (FE) , Fucus distichus (FD) , Sargassum polycystum (SP)  (Table 1). The main structural features of these polymers are shown in Fig. 2, and their monosaccharide and sulfate content is presented in Table 1. Elucidation of these structures was described elsewhere [20–26].
Several chemical modifications of the natural polysaccharides have been performed. Thus, the per-O-sulfated derivatives SL-S and CO-S have been prepared from the polymers SL and CO, respectively, by treatment with sulfating reagents (Scheme 1, Table 1) .
The polysaccharide CF was subjected to another chemical transformation. Reduction of carboxyl groups in its structure gave rise to the polymer CF-R bearing glucosyl branches instead of glucuronyl ones (Scheme 2, Table 1) . Smith degradation of the branched xylofucan sulfate PP led to the linear sulfated fucan PPX devoid of xylose residues (Scheme 3, Table 1) .
The studied polysaccharides were different in monosaccharide content, degree of sulfation, types of glycosidic bonds, molecular weight. The homogeneity of the fucoidan fractions was evidenced by the results of electrophoresis of the samples in agarose gel (Fig. 3).
Synthesis of the oligosaccharides related to fucoidans
Besides the polymeric compounds the low molecular weight fucosides have been studied. The linear and branched oligosaccharides OS1–OS5 (Fig. 4) have been synthesized [27–29]. The tetrasaccharide OS1 and the octasaccharide OS3 built up of (1 → 3)-linked α-l-fucopyranosyl residues could be considered as the backbone fragments of the highly sulfated polysaccharides SL-S and CO-S. The octasaccharide OS2 has the same chain length as OS3, but it bears sulfate groups only at O-2. The compound OS4 built up of the alternating (1 → 3)- and (1 → 4)-linked α-l-fucopyranosyl residues is the isomer of the octasaccharide OS3. The tetrasaccharide OS5 is related to the branched fragment of the polysaccharide SL-S. Key synthetic steps are shown below (Scheme 4) and are applicable also for the preparation of much larger oligosaccharides including the 16-fucoside .
For the synthesis of the compounds OS1–OS5 the efficient method of the α-l-fucosylation has been developed [27, 30–32]. The stereoselectivity of the reaction is determined by the presence of acyl groups at O-3 and/or O-4 of a fucosyl donor, which stabilize the glycosyl cation in a manner favorable for the α-attack of a glycosyl acceptor. Notably, not only monosaccharide, but also di- and tetrasaccharide glycosyl donors bearing acyl groups at O-3 and/or O-4 were successfully used for the preparation of the α-linked fucosides. This led to develop efficient blockwise strategies for large carbohydrate chains assembling.
The fragment of the convergent synthesis of the octasaccharide OS4 is shown on Scheme 4. The presence of an allyl aglycon and an acetyl group at O-3″′ in a structure of the tetrasaccharide 1 permitted its selective transformation either to the glycosyl acceptor 2 or to the glycosyl donor 3. Thus, acidic O-deacetylation of 1 gave the tetrasaccharide 2 in a yield of 82 %. Deallylation of 1 followed by trichloroacetimidation afforded the glycosyl donor 3 as a 1:1 mixture of α- and β-isomers in a total yield of 78 %. Coupling of the tetrasaccharides 2 and 3 proceeded stereospecifically with the formation of the α-linked octafucoside 4 in a yield of 76 %. Deprotection of 4 followed by per-O-sulfation  gave the compound OS4.
Influence of different structural features of fucoidans on blood coagulation
Poly- and oligosaccharides described above were assessed on anticoagulant activity in different in vitro experiments. General clotting assays were performed as described previously  with the use of normal plasma, which was incubated with the samples. Commercially available low-molecular-weight heparin Clexane® (enoxaparin) was chosen as a reference, because this polysaccharide is intensively used in medical practice as heparinoid anticoagulant with low risk of side effects [34, 35].
The influence of the samples on the intrinsic pathway of coagulation was evaluated in the activated partial thromboplastin time (APTT) assays. The dose-depended changes in the APTT value are shown on Fig. 5, and the values 2APTT (the concentration of a sample, at which double increasing of control value of APTT is observed) are presented in Table 2.
The biological effect was shown to depend on structural features of the tested sample. Among the parent fucoidans (Fig. 5a), the samples SL and FD demonstrated high level of activity, even exceeding that for Clexane®. The values 2APTT for SL and FD were ∼1.1 μg/mL and ∼2.8 μg/mL, respectively, while this value for Clexane® was ∼3.3 μg/mL. Slightly lower effects were detected for CF and SP (2APTT were ∼3.5 μg/mL and ∼4.9 μg/mL, respectively). Moderate activity was shown for FE (∼25.1 μg/mL), while the polysaccharides CO and PP were inactive even at a concentration of 100 μg/mL.
Chemical sulfation of the fucoidans CO and SL changed their properties. Thus, per-O-sulfation of the inactive fucoidan CO gave the product CO-S (DS is 2.0, Table 1) with pronounced anticoagulant effect. This sample prolonged blood coagulation by 2 times at a concentration of ∼2.5 μg/mL. An opposite situation was observed in the case of the samples SL and SL-S. Additional introduction of sulfates into a structure of SL led to a slight decrease in the anticoagulant effect, however, the sample SL-S (2APTT ∼1.9 μg/mL) was slightly more active than CO-S (Fig. 5b).
Reduction of caboxyl groups in a structure of CF gave the polysaccharide CF-R, which was less active than the parent fucoidan (2APTT were 3.5 μg/mL and 4.9 μg/mL for CF and CF-R, respectively). Dexylosylation of the sample PP did not influence on the anticoagulant properties of the polysaccharide. The preparation PPX was inactive similarly to PP.
Among the synthetic oligosaccharides only the per-O-sulfated octasaccharides OS3 and OS4 deserved attention because of moderate anticoagulant effect (2APTT were 5.0 μg/mL and 7.5 μg/mL for OS3 and OS4, respectively). Notably, OS3 built up of (1 → 3)-linked α-l-fucopyranosyl residues was more active, than its isomer OS4 consisted of alternating (1 → 3)- and (1 → 4)-linked α-l-fucosyl units. Neither the per-O-sulfated tetrasaccharides OS1 and OS5, nor the selectively 2-O-sulfated octasaccharide OS2 demonstrated anticoagulant properties (data not shown).
The influence of the samples on thrombin-induced clot formation was also investigated. The values of 2TT (the concentration of a sample, at which double increasing of control value of TT was observed) are presented in Table 2. High level of activity was determined for SL, CF, SL-S, FD, CO-S (2TT 2.2–4.75 μg/mL), which was comparable with the effect of Clexane® (2TT ∼2.2 μg/mL). Moderate effect was observed for the octasaccharides OS3 and OS4, as well as for the polysaccharide CF-R (2TT 16.1–18.2 μg/mL). The fucoidan SP showed low anticoagulant activity, while CO, PP, FE and PPX were inactive in this test.
To investigate further the mechanism of anticoagulant action of the fucoidans and their derivatives, the experiments with purified proteins have been performed. These studies were based on the assay of amidolytic activity of thrombin (IIa) or factor Xa using chromogenic substrates, as described previously . The ability of the samples to inhibit thrombin and factor Xa was assessed in the presence and in the absence of antithrombin III (ATIII). The results are shown on Fig. 6 and in Table 3.
Effectiveness of binding to thrombin in the presence of ATIII was high for the samples SL, CF, FD, SL-S, and CO-S (IC50 0.4–0.9 μg/mL), which was similar to Clexane® activity (IC50 0.6 μg/mL). These polysaccharides are enriched in 2,4-di-O-sulfated fucosyl residues, which was previously shown to be essential for the anticoagulant activity [2, 7]. Branched polysaccharides from this series, namely SL, SL-S, CO-S and CF, bear negatively charged group (sulfate or carboxyl) at a branch fragment. It is remarkable, that the preparation CF-R with non-charged glucosyl units as branches demonstrated low anti-IIa activity (IC50 41.3 μg/mL). The polysaccharide SP containing sulfate groups at O-4 showed moderate effect (IC50 6.5 μg/mL), while the 2-O-sulfated fucoidan FE with the same degree of sulfation (1.0, Table 1) was inactive in this test.
Opposed to the polymeric compounds with the same backbone, the octasaccharides OS3 and OS4 showed no anti-IIa activity in the presence of ATIII. It could be connected with insufficient chain length, because it was established earlier [36–38] that at least 16 monosaccharide units in the chain of the heparinoid structure are required for the formation of a ternary complex with thrombin and ATIII.
The fucoidans CO, PP, and PPX with low degree of sulfation (0.4–0.7, Table 1) were inactive in all tested concentrations. On the contrary, three of the tested samples, namely SL, SL-S, and CO-S, were found to bind with thrombin in the absence of ATIII (Table 3). However, higher concentrations of the polysaccharides were required for this test. Thus, the samples SL, SL-S, and CO-S showed 50 % inhibition at concentrations of ∼45.9, ∼55.9, and ∼58.9 μg/mL, respectively.
The polysaccharides SL, SL-S, and CO-S efficiently bind to factor Xa only in the presence of ATIII (the values of IC50 were ∼1.1, ∼1.9, and ∼2.0 μg/mL, respectively), but their activity was significantly lower than that for heparinoid Clexane® (the value of IC50 was ∼0.059 μg/mL). The linear fucoidan FD consisted of alternating (1 → 3)- and (1 → 4)-linked α-l-fucosyl units was less active than branched polysaccharides SL, SL-S, and CO-S built up of (1 → 3)-linked α-l-fucopyranosyl residues. Surprisingly, the synthetic per-O-sulfated octasaccharide OS3 demonstrated moderate anti-Xa activity in the presence of ATIII (IC50 were ∼13.0 μg/mL). Neither fucoidans, nor heparinoids bind to factor Xa in the absence of ATIII (data not shown).
The trends found in amidolytic experiments (see Table 3 and Fig. 6 showing anti-IIa and anti-Xa activities) for the studied samples correlated well with the results obtained in clotting assays. Thus, the polysaccharides SL, FD, CF, SL-S, and CO-S enriched in 2,4-di-O-sulfated (1 → 3)-linked α-l-fucose units demonstrated high effects in clotting assays and also possessed significant anti-IIa activity. These results are in a good correlation with the data obtained previously for linear highly sulfated fucans from invertebrates [2, 7]. Moreover, it is noticeable that inhibition of thrombin by the polysaccharides SL, SL-S, and CO-S was performed both in the presence and in the absence of ATIII, which coincides well with the published data for other branched fucoidans from brown seaweeds [12, 13]. Additionally, the samples SL, SL-S, and CO-S demonstrated significant anti-Xa activity. The synthetic per-O-sulfated octasaccharide OS3, which was structurally related to the polysaccharides SL, SL-S, and CO-S, possessed a moderate effect on clot formation connected mainly with its moderate anti-Xa activity.
Fucoidans isolated from different brown seaweed species vary in monosaccharide content, types of glycoside bonds, degree and pattern of sulfation, presence of branches, and molecular weight. The effect of fucoidans on blood coagulation was shown to depend on their structural features. Thus, the polysaccharides SL, CF, FD, SL-S, and CO-S enriched in 2,4-di-O-sulfated (1 → 3)-linked α-l-fucose units demonstrated high effect in clotting assays. The polysaccharides CO, PP, PPX with the (1 → 3)-linked α-l-fucose backbone with degree of sulfation 0.4–0.7 did not influence on blood coagulation, and this behaviour could be explained by the absence of a sufficient amount of sulfate groups in their structure. The trends found in clotting assays correlated well with the results obtained in experiments with individual proteins. High anti-IIa activity was shown for the samples SL, CF, FD, SL-S, and CO-S in the presence of ATIII, while CO, PP, PPX were inactive. It is noticeable that the branched polysaccharides SL, SL-S, CO-S were also shown to be direct thrombin inhibitors and, hence, they differ from heparinoids and linear fucans from invertebrates. Additionally SL, SL-S, and CO-S demonstrated significant anti-Xa activity. The synthetic per-O-sulfated octasaccharide OS3, which was structurally related to the polysaccharides SL, SL-S, and CO-S, possessed a moderate effect on clot formation connected mainly with its moderate anti-Xa activity. This result indicated that longer and probably branched fucoidan fragments are required for the efficient inhibition of blood coagulation.
This work was supported in part by the Russian Foundation for Basic Research (grants 12-04-01749, 12-03-92703, 13-03-91170, KOMFI-grant 13-04-40315-K and its parts 13-04-40313-H, 13-04-40314-H, and 13-04-40315-H).
Russian Foundation for Basic Research, (Grant/Award Number: 12-04-01749, 12-03-92703, 13-04-40313-H, 13-03-91170, KOMFI-grant 13-04-40315-K, 13-04-40314-H, 13-04-40315-H).
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