Structure and Anticoagulant Activity of Sulfated Fucans
COMPARISON BETWEEN THE REGULAR, REPETITIVE, AND LINEAR FUCANS FROM ECHINODERMS WITH THE MORE HETEROGENEOUS AND BRANCHED POLYMERS FROM BROWN ALGAE*

Mariana S. PereiraDagger , Barbara Mulloy§, and Paulo A. S. MourãoDagger

From the Dagger  Laboratório de Tecido Conjuntivo, Hospital Universitário and Departamento de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ 21941-590, Brazil and § National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Sulfated fucans are among the most widely studied of all the sulfated polysaccharides of non-mammalian origin that exhibit biological activities in mammalian systems. Examples of these polysaccharides extracted from echinoderms have simple structures, composed of oligosaccharide repeating units within which the residues differ by specific patterns of sulfation among different species. In contrast the algal fucans may have some regular repeating structure but are clearly more heterogeneous when compared with the echinoderm fucans. The structures of the sulfated fucans from brown algae also vary from species to species. We compared the anticoagulant activity of the regular and repetitive fucans from echinoderms with that of the more heterogeneous fucans from three species of brown algae. Our results indicate that different structural features determine not only the anticoagulant potency of the sulfated fucans but also the mechanism by which they exert this activity. Thus, the branched fucans from brown algae are direct inhibitors of thrombin, whereas the linear fucans from echinoderms require the presence of antithrombin or heparin cofactor II for inhibition of thrombin, as reported for mammalian glycosaminoglycans. The linear sulfated fucans from echinoderms have an anticoagulant action resembling that of mammalian dermatan sulfate and a modest action through antithrombin. A single difference of one sulfate ester per tetrasaccharide repeating unit modifies the anticoagulant activity of the polysaccharide markedly. Possibly the spatial arrangements of sulfate esters in the repeating tetrasaccharide unit of the echinoderm fucan mimics the site in dermatan sulfate with high affinity for heparin cofactor II.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Since the description of high amounts of sulfated fucans in marine brown algae over 50 years ago (1), these polysaccharides have been widely tested for biological activities in different mammalian systems. Algal-sulfated fucans have anticoagulant activity as measured in several different assays (2-5) and have also venous antithrombotic activity (6). These polysaccharides are inhibitors of native (7) and recombinant (8) human immunodeficiency virus reverse transcriptase activity in vitro. Furthermore, because of their interference with molecular mechanisms of cell-to-cell recognition, algal-sulfated fucans are potent blockers of a wide range of biological processes. Thus, algal-sulfated fucans are inhibitors of cell invasion by retroviruses such as human immunodeficiency virus, herpes, cytomegalovirus, and African swine fever virus (9-11). Algal-sulfated fucans inhibit invasion of erythrocytes by Plasmodium falciparum merozoites and cytoadherence of parasitized erythrocytes to endothelial cells (12). In addition, algal-sulfated fucans can act as anti-angiogenic agents (13), can block selectin-mediated cell-cell binding (14), are inhibitors of sperm binding to oviductal epithelium (15), can block sperm-egg binding in diverse species (16-18), have antiproliferative and antitumoral properties (19), and are inhibitors of vascular smooth muscle cell (20, 21) and fibroblast proliferation (22).

The mechanisms by which the algal fucans exert their anticoagulant action remain controversial. Mechanisms related to both antithrombin and heparin cofactor II-mediated activity have been described for algal-sulfated fucans from different species (2, 4, 6, 23). Direct anticoagulant activity by an antithrombin-independent pathway has also been reported for some preparations of algal fucans (24). In addition, some fucans have anti-factor Xa activity (2), whereas others do not (4). The structures of sulfated fucans vary from species to species (25) and must give rise to variation in the detailed mechanisms of anticoagulant action.

The structural components of sulfated fucan necessary for all these biological activities have not been determined. Most of the difficulties for these studies arise from the fact that algal-sulfated fucans are heterogeneous polysaccharides, which give complex NMR spectra with broad signals hampering resolution (25, 26). Recently, modified methylation analysis of these polysaccharides have allowed more definitive conclusions about some structural features of algal fucans (25, 26).

We have also isolated and characterized the structure of several sulfated fucans from echinoderms (25, 27). In contrast with the algal fucans, these echinoderm polysaccharides have simple structures, composed of oligosaccharide repeating units within which the residues differ by specific patterns of sulfation (Fig. 1). The specific pattern of sulfation and the position of the glycosidic linkage varies among different species. Thus, the regular repeating sequences of residues in the sulfated fucan from the sea urchin Lytechinus variegatus are as follows: [3-alpha -L-Fucp-2(OSO3)-1right-arrow 3-alpha -L-Fucp-4(OSO3)-1right-arrow 3-alpha -L-Fucp-2,4(OSO3)-1right-arrow3-alpha -L-Fucp-2(OSO3)-1]n (25), whereas the sequences of residues in the sulfated fucan from the sea urchin Arbacia lixula are as follows: [4-alpha -L-Fucp-2(OSO3)-1right-arrow4-alpha -L-Fucp-2(OSO3)-1right-arrow4-alpha -L-Fucp-1right-arrow4-alpha -L-Fucp-1] n (27). Finally, the sea cucumber Ludwigothurea grisea contains a sulfated fucan with the following sequences of residues: [3-alpha -L-Fucp-2,4(OSO3)-1right-arrow3-alpha -L-Fucp-1right-arrow3-alpha -L-Fucp-2(OSO3)-1right-arrow3-alpha -L-Fucp-2(OSO3)-1]n (27, 28).


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Fig. 1.   Structures of the sulfated fucans isolated from echinoderms. Sulfated fucans from echinoderms are linear polymers, composed of tetrasaccharide repeating units in which the four residues differ only by specific patterns of sulfation at the O-2 and O-4. See Refs. 25 and 27 for further details.

One way to determine the relationship between structure and biological activity of sulfated polysaccharides is to compare the activities in various assays where the nature of the polysaccharide backbone and the extent and position of sulfation have been fully characterized. In this line of work the new echinoderm-sulfated fucans constitute a valuable tool. In the present study we report the anticoagulant activity of the regular and repetitive fucans from echinoderms. In addition, their activity was compared with sulfated fucans from three species of brown algae. Algal fucans were extensively purified, and their major structural features were partially characterized by a combination of methylation and NMR analysis. Our results indicate that different structural features determine not only the anticoagulant potency of the sulfated fucans but also the mechanism by which they exert this activity.

    EXPERIMENTAL PROCEDURES

Sulfated Fucans from Echinoderms

Sulfated fucans were extracted from the body wall of the sea cucumber L. grisea and from the egg jelly coat of the sea urchins L. variegatus and A. lixula and were purified by anion exchange and gel filtration chromatographies, as described (25, 27).

Sulfated Fucans from Brown Algae

Extraction-- A commercial crude preparation of sulfated polysaccharides, extracted from Fucus vesiculosus, was obtained from Sigma. The brown algae Laminaria brasiliensis and Ascophyllum nodosum were collected from the sea, immersed immediately in acetone, and kept for 24 h at 4 °C. The sulfated polysaccharides were extracted from the dried tissue (~2 g) by papain and partially purified by cetylpyridinium chloride and ethanol precipitation, as described (29). About 120 mg (dry weight) of crude extract was obtained after these procedures.

Purification-- The crude polysaccharide (200 mg) was applied to a DEAE-cellulose column (9 × 2 cm) equilibrated in 50 mM sodium acetate (pH 5.0) and washed with 200 ml of the same buffer containing 0.2 M NaCl. Thereafter, the column was eluted by a linear gradient prepared by mixing 150 ml of 50 mM sodium acetate (pH 5.0) containing 0.2 M NaCl with 150 ml of 1.2 M NaCl in the same buffer. The flow rate of the column was 12 ml/h. Fractions of 3 ml were collected and checked by the phenol-H2SO4 (30) and carbazole reactions (31) and by metachromasia using 1,9-dimethylmethylene blue (32). In some experiments, the sulfated fucan (100 mg) partially purified on the DEAE-cellulose column was re-chromatographed on a new DEAE-cellulose column, under the same experimental conditions described above.

The DEAE-cellulose-purified fucan (~20 mg) was applied to a Mono Q column-FPLC1 (HR 5/5) (Amersham Pharmacia Biotech) equilibrated in 20 mM Tris-HCl (pH 8.0). The column was developed with a linear gradient of 0-2.0 M NaCl in the same buffer. The flow rate of the column was 0.45 ml/min, and fractions of 0.5 ml were collected and assayed by metachromasia (32) and by the phenol-H2SO4 (30) and carbazole (31) reactions.

Agarose Gel Electrophoresis-- Sulfated fucans were analyzed by agarose gel electrophoresis as described (27, 29). Purified sulfated fucan (~15 µg) was applied to a 0.5% agarose gel and run for 1 h at 110 V in 0.05 M 1,3-diaminopropane/acetate (pH 9.0). The sulfated fucans in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution. After 12 h, the gel was dried and stained with 0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5, v/v).

Polyacrylamide Gel Electrophoresis-- The molecular masses of the sulfated fucans were estimated by polyacrylamide gel electrophoresis. Sulfated fucan samples (~10 µg) were applied to a 6% 1-mm-thick polyacrylamide gel slab in 0.02 M sodium barbital (pH 8.6). After electrophoresis (100 V for 30 min), the gel was stained with 0.1% toluidine blue in 1% acetic acid and then washed for about 4 h in 1% acetic acid. The molecular mass markers were the same as those used previously (33).

Chemical Analyses-- Total fucose was measured by the method of Dische and Shettles (34) and by the method of Dubois et al. (30). After acid hydrolysis of the polysaccharides (5.0 M trifluoroacetic acid for 5 h at 100 °C), sulfate was measured by the BaCl2/gelatin method (35). The percentages of hexoses and 6-deoxyhexoses were estimated by paper chromatography in 1-butanol/pyridine/water (3:2:1, v/v) for 48 h and by gas-liquid chromatography of derived alditol acetates (36). Optical rotations were measured with a digital polarimeter (Perkin-Elmer model 243-B).

Desulfation and Methylation of the Fucans-- Desulfation of the sulfated fucans was performed by solvolysis in dimethyl sulfoxide, as described previously for desulfation of other types of polysaccharides (29, 37). The native and desulfated fucans (~5 mg) were subjected to three rounds of methylation as described (38) with the modifications suggested by Patankar et al. (26). The methylated fucans were hydrolyzed with 6 M trifluoroacetic acid for 5 h at 100 °C and reduced with borohydride, and the alditols were acetylated with acetic anhydride/pyridine (1:1, v/v) (36). The alditol acetates of the methylated sugars were dissolved in chloroform and analyzed in a gas chromatography/mass spectrometry unit.

NMR Spectroscopy-- 1H spectra were recorded at 500 MHz and 13C spectra at 125 MHz using a Varian Unity 500 spectrometer. The sulfated fucan sample (~10 mg) was converted to the sodium salt by passage through a column 10 × 1 cm of Dowex 50-X8 Na+ form, and all samples were dissolved in approximately 0.7 ml of 99.8% D2O. The spectra were recorded at 60 °C with suppression of the HOD signal by presaturation. 13C spectra were recorded with full proton decoupling. Two-dimensional double-quantum filtered COSY, TOCSY, and NOESY experiments were performed using pulse sequences supplied by Varian. TOCSY spectra were run with a spin-lock field of about 20 kHz and a mixing time of 100 ms; NOESY spectra were run with a mixing time of 100 ms. All chemical shifts were relative to internal or external trimethylsilylpropionic acid.

Anticoagulant Action Measured by Activated Partial Thromboplastin Time-- Activated partial thrombloplastin time clotting assays were carried out by the method of Anderson et al. (39). Normal human platelet-poor plasma (90 µl) was mixed with 10 µl of a solution of sulfated polysaccharide (0-5 µg) and incubated for 1 min at 37 °C. Then, 100 µl of celite + rabbit phospholipid reagent (Reagent Celite, Biolab, Mérieux) was added to the mixture and incubated for 2 min at 37 °C. Thereafter, 100 µl of 0.25 M CaCl2 was added and the clotting time recorded on a KC4A coagulometer (Heinrich Amelung, Germany). The activity was expressed as international units/mg using a parallel standard curve based on the 4th International Heparin Standard (193 international units/mg).

Effect of Sulfated Polysaccharides on the Inactivation of Thrombin or Factor Xa by Antithrombin or Heparin Cofactor II-- These experiments were based on the assay of amydolytic activity of thrombin or factor Xa using chromogenic substrate, as described previously (40, 41).

Inhibition of Thrombin by Antithrombin in the Presence of Sulfated Polysaccharides-- Ten µl of the sulfated polysaccharide solution (0-50 µg) was mixed with 5 µl of 1 unit/ml purified human antithrombin (National Institute for Biological Standards and Control reference reagent) and 85 µl of 15 mM Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl and 1 mg/ml polyethylene glycol (TS/PEG buffer). Then, 15 µl of 10 units/ml purified human thrombin (National Institute for Biological Standards and Control reference reagent) was added to initiate the reaction. After a 60-s incubation at 25 °C, 500 µl of 0.24 mM chromogenic substrate S-2238 from Chromogenix AB (Mondal, Sweden) was added, and the absorbance at 405 nm was recorded continuously for 120 s. The rate of change of absorbance was proportional to the thrombin activity remaining in the incubation.

Inhibition of Thrombin by Heparin Cofactor II in the Presence of Sulfated Polysaccharides-- The assay was essentially as described above except that 5 µl of 1.5 µM heparin cofactor II from Diagnostica Stago (Asniére, France) instead of antithrombin was added to the incubation mixtures.

Inhibition of Factor Xa by Antithrombin in the Presence of Sulfated Polysaccharides-- This assay was performed as described for the inhibition of thrombin by antithrombin except that 4 units/ml of factor Xa (Diagnostic Reagents Ltd, Thame, Oxon, UK) instead of thrombin was added to the incubation mixtures. In addition, the chromogenic substrate employed in this experiment was S-2765 (Chromogenix AB, Mondal, Sweden).

In the short incubation periods used to measure activity versus concentration of the sulfated polysaccharide, no inhibition occurred when thrombin or factor Xa was incubated with antithrombin or heparin cofactor II in the absence of sulfated polysaccharide.

    RESULTS

Purification and Structural Analysis of the Sulfated Fucans from Three Species of Brown Algae

Purification of the Algal-sulfated Fucans-- The sulfated fucans from three species of brown algae were purified by a combination of ion exchange chromatographies on DEAE-cellulose and on Mono Q-FPLC (Figs. 2-4). These exhaustive fractionation procedures are necessary to achieve purified algal fucans for the anticoagulant assays.


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Fig. 2.   Purification of the sulfated fucan from the brown alga L. brasiliensis by DEAE-cellulose (A and B) and Mono Q-FPLC (C) chromatograhies. A, the crude polysaccharides from the brown alga L. brasiliensis (200 mg) were purified by DEAE-cellulose chromatography as described under "Experimental Procedures." B, the partially purified sulfated fucan (100 mg) was then re-chromatographed on a new DEAE-cellulose column, under the same experimental conditions. C, finally, the DEAE-cellulose-purified fucan (~20 mg) was applied to a Mono Q-FPLC column and purified as described under "Experimental Procedures." Fractions were checked by the phenol-H2SO4 (open circle ) and carbazole (black-triangle) reactions, for metachromasia () and NaCl concentration (- - -). The fractions indicated by horizontal bars were pooled, dialyzed against distilled water, and lyophilized. The vertical arrows in C indicate the elution of standard chondroitin 6-sulfate from shark cartilage (CS) and heparin from porcine intestinal mucosa on the Mono Q-FPLC column.

Anion exchange chromatography on a DEAE-cellulose column separated the acidic polysaccharides from brown algae into two major peaks (Figs. 2A, 3A, and 4A). The peak eluted at the beginning of the salt gradient, containing hexuronic acid (closed triangles in the panels), corresponds to a heterogeneous sulfated polysaccharide. Its electrophoretic mobility on agarose gel and chemical composition (not shown) indicate that it has a complex sugar composition and an electrophoretic mobility different from that of the sulfated fucans. In the case of the F. vesiculosus this heterogeneous sulfated polysaccharide does not occur as a very clear and distinct peak. However, a detailed analysis of the various fractions from the DEAE-cellulose column indicate the heterogeneous sulfated polysaccharide is at the left shoulder of the major peak (Fig. 3A and Ref. 25).


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Fig. 3.   Purification of the sulfated fucan from the brown alga F. vesiculosus by DEAE-cellulose (B) and Mono Q-FPLC (B) chromatographies. A, the crude polysaccharides from the brown alga F. vesiculosus (200 mg) were purified by DEAE-cellulose. B, the DEAE-cellulose-purified fucan (~20 mg) was re-purified by a Mono Q-FPLC column. Fractions were checked by the phenol-H2SO4 (open circle ) and carbazole (black-triangle) reactions, for metachromasia () and NaCl concentration (- - -). Further details are described under "Experimental Procedures" and in the legend of Fig. 2.

The sulfated fucans were eluted at high salt concentration. The purity of these fucans was confirmed by re-chromatography on another DEAE-cellulose column (Fig. 2B) and/or on Mono Q-FPLC (Figs. 2C, 3B, and 4, B and C). These final sulfated fucans preparations are devoid of hexuronic acid contaminant (closed triangles in the panels), have strong metachromatic property (closed circles), and high fucose content (open circles), as revealed by the Dubois et al. (30) reaction.

Sulfated fucan from A. nodosum showed a very wide peak, and thus it was further separated into two subfractions denominated F1 and F2 (Fig. 4A). On Mono Q-FPLC these two subfractions show slight differences in the salt concentrations necessary for elution from the column (Fig. 4, B and C).


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Fig. 4.   Purification of the sulfated fucan from the brown alga A. nodosum by DEAE-cellulose (A) and Mono Q-FPLC (B and C) chromatographies. Fractions were checked by the phenol-H2SO4 (open circle ) and carbazole (black-triangle) reactions, for metachromasia () and NaCl concentration (- - -). The sulfated fucan from A. nodosum showed a very wide peak on the DEAE-cellulose column, and thus it was separated into two subfractions denominated F1 and F2, as indicated by the horizontal bars in A. The DEAE cellulose-purified subfractions F1 (B) and F2 (C) were further purified by Mono Q-FPLC column. Further details are described under "Experimental Procedures" and in the legend of Fig. 2.

In contrast with F. vesiculosus and A. nodosum, the sulfated fucan from L. brasiliensis showed a very narrow peak on Mono Q-FPLC (compare Fig. 2C with Figs. 3B and 4, B and C) perhaps denoting a more homogeneous polysaccharide in terms of molecular mass and/or charge density.

Electrophoretic Mobility, Molecular Mass, and Chemical Composition of the Sulfated Fucans-- Agarose gel electrophoresis in 1,3-diaminopropane/acetate followed by toluidine blue staining showed variation in mobilities among the various sulfated fucans (Fig. 5A). The electrophoretic migration of sulfated polysaccharides in 1,3-diaminopropane/acetate depends on the structure of the polysaccharide, which forms a complex with the diamino buffer (42). Thus the different electrophoretic mobility of the various sulfated fucans is a first indication of distinctive structures of these polysaccharides. Polyacrylamide gel electrophoresis (Fig. 5B) indicates the sulfated fucans, except that from F. vesiculosus, stay at the origin of the gel, denoting high molecular weight.


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Fig. 5.   Agarose (A) and polyacrylamide (B) gel electrophoresis of the sulfated fucans from brown algae and echinoderms. A, purified sulfated fucans (~15 µg of each) were applied to a 0.5% agarose gel and run for 1 h at 110 V in 0.05 M 1,3-diaminopropane/acetate (pH 9.0). The sulfated fucans in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution. After 12 h, the gel was dried and stained with 0.1% toluidine blue in acetic acid/ethanol/water (0.1:5:5,v/v). B, sulfated fucans (~10 µg of each) were applied to a 6% 1-mm-thick polyacrylamide gel slab in 0.02 M sodium barbital (pH 8.6) and run for 30 min at 100 V. After electrophoresis the sulfated fucans were stained with 0.1% toluidine blue in 1% acetic acid and then washed for about 4 h in 1% acetic acid. The molecular mass markers were low molecular weight dextran sulfate (Dex 8) (8 kDa), chondroitin 4-sulfate from whale cartilage (C-4-S) (15 kDa), dermatan sulfate from pig skin (DS) (20 kDa), chondroitin 6-sulfate from shark cartilage (C-6-S) (60 kDa), and high molecular weight dextran sulfate (Dex 500) (~500 kDa).

Chemical analysis of the sulfated fucans from different species of brown algae (Table I) reveals a high content of sulfate ester. Galactose (~12% of total sugar residues) was also detected in the sulfated fucan from L. brasiliensis, whereas galactose and mannose (10% of each) were found in subfraction F1 from A. nodosum. The high negative charge density of these polysaccharides was confirmed by the sulfate/total sugar molar ratios. In fact, the sulfated fucans were eluted from the Mono Q-FPLC column at higher NaCl concentrations than mammalian glycosaminoglycans (Figs. 2C, 3B, and 4, B and C), and a correlation was observed between the sulfate content and the NaCl concentrations necessary to elute the sulfated fucans from the Mono Q-FPLC column (Table I).

                              
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Table I
Chemical composition, specific optical rotation, and concentration of NaCl necessary for elution from the Mono Q-FPLC column of sulfated fucans from different species of brown algae

Methylation Analysis-- When the sulfated fucans from different species of brown algae were submitted to three rounds of methylation, under the same experimental conditions, the proportions of methylated derivatives obtained varied significantly among the species (Table II and Fig. 6). Thus, the proportions of methyl derivatives are similar for F. vesiculosus and A. nodosum, but both species are markedly distinctive from L. brasiliensis. Sulfated fucan from L. brasiliensis yields larger proportions of di-O-methyl derivatives from fucose than fucans from F. vesiculosus and A. nodosum (Fig. 6), indicating the presence of unsulfated fucoses in the linear part of the polysaccharide.

                              
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Table II
Methylated fucose (Fuc) derivatives obtained from native and partially desulfated fucans from three species of brown algae


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Fig. 6.   Proportions of methylated fucose derivatives obtained from native and partially desulfated fucans from three species of brown algae. The native and desulfated fucans (~5 mg) were subjected to three rounds of methylation as described under "Experimental Procedures." The methylated fucans were hydrolyzed with 6 M trifluoroacetic acid for 5 h at 100 °C and reduced with borohydride, and the alditols were acetylated with acetic anhydride/pyridine (1:1, v/v). The alditol acetates of the methylated sugars were analyzed in a gas chromatography/mass spectrometry unit.

Desulfation of the fucan from L. brasiliensis results in a decrease of unmethylated fucose and a concomitant increase of tri-O-methyl and di-O-methyl derivatives (Fig. 6). Possibly, unmethylated fucose yields monomethyl derivatives after desulfation which in turn produces di-O-methyl and tri-O-methyl derivatives. The overall result is that the proportion of monomethyl derivatives does not change after desulfation of the fucan. The presence of 2,4-di-O-methylfucose and 2,3-di-O-methylfucose after methylation of the partially desulfated fucan from L. brasiliensis indicates the presence of 3- and 4-linked units, respectively, whereas 2,3,4-tri-O-methylfucose shows this fucan is a branched polymer with fucosyl units at nonreducing ends (Table II).2 The presumable increase in the proportions of 2- and 4-methylfucose after desulfation, as a consequence of decreased proportions of unmethylated fucose, suggests the presence of sulfate esters at O-2 and O-4.

NMR Spectroscopy-- The 1H spectra at 500 MHz of the fucans are shown in Fig. 7. Consistent with the methylation analysis results (Table II and Fig. 6), there are strong similarities between the 1H spectra of the sulfated fucans from F. vesiculosus and A. nodosum (Fig. 7, B and C), whereas the fucan from L. brasiliensis gives a distinct spectrum (Fig. 7A). Two-dimensional TOCSY and NOESY techniques were used to give a partial assignment of the spectra of all three compounds, as summarized in Table III.


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Fig. 7.   1H NMR spectra at 500 MHz of the sulfated fucans from the brown algae L. brasiliensis (A), F. vesiculosus (B), and A. nodosum (C). The spectra were recorded at 60 °C for samples in D2O solution. Chemical shifts are relative to internal or external trimethylsilylpropionic acid at 0 ppm. The HOD signal has been suppressed by presaturation. Expansions of the 5.9 to 4.5 ppm region are shown in the panels. Peaks curtailed with diagonals are from contaminants. Assignments marked in the spectra are as summarized in Table III.

                              
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Table III
Proton chemical shifts (ppm) for residues of alpha -L-fucose in the sulfated fucans from brown algae
The 1H spectrum was recorded at 500 MHz, 60 °C in 99.8% D2O. Chemical shifts are referenced to internal trimethylsilylpropionic acid. Values in boldface indicate positions bearing sulfate ester.

In the one-dimensional spectrum of the sulfated fucan from L. brasiliensis, six distinguishable anomeric resonances with chemical shifts between 5.0 and 5.6 ppm give rise to partial spin systems consistent with alpha -L-fucopyranose residues (A-F in Fig. 7 and Table III). This indicates an underlying regular repeating structure, although other broader signals are present, and the structure of the fucan is clearly heterogeneous. By analogy with earlier work on fucans of marine echinoderms (25, 27, 28), the residues giving rise to systems A and B are shown to be 2-O- and 4-O-sulfated (in accordance with methylation results) and therefore either terminal or 3-linked. Residues C and D are 3-O-sulfated, and residues E and F show no sulfation at C-2 or C-3.

The integrals of the six H-1 resonances in the 1H spectrum of the sulfated fucan from L. brasiliensis are reported in Table III. These integrals are intensity from poorly resolved signals and thus require a careful interpretation. Nevertheless, they are consistent with a 1:1:1:1:1:1 ratio of the six residues. If we assume that a six-residue repeating structure is present, then 33.3% of the residues (E and F) are totally unsulfated, 33.3% are monosulfated (C and D), and 33.3% are disulfated (A and B). These results are consistent with the methylation analysis reported in Table II and Fig. 6.

A NOESY spectrum of the fucan from L. brasiliensis (Fig. 8) shows clear cross-peaks corroborating these assignments. In addition, a cross-peak is visible between two anomeric protons, H1C and H1D. An inter-residue cross-peak is also seen between H1D and H2C. This is strong evidence for a 1right-arrow2 linkage D1-C2. No other linkage information is available from the NOESY spectrum. Both the one- and two-dimensional spectra of the fucan from L. brasiliensis show signals from non-fucose residues, further complicating the spectra.


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Fig. 8.   NOESY spectrum of the sulfated fucan from the brown algae L. brasiliensis. The NOESY spectrum of the fucan from L. brasiliensis shows intra-residue cross-peaks from H-1 of residues A-F to other ring protons, confirming assignments made by correlation spectroscopy. Only two inter-residue cross-peaks are marked, between H-1 of residues C and D, and between H-1 of residue D and H-2 of residue C. All these cross-peaks are good evidence for the existence of a partially ordered structure in this heterogeneous polysaccharide.

Proton spectra of the fucans from F. vesiculosus and A. nodosum differ in detail but show strong overall similarities. Two-dimensional assignment techniques pick out only two partial spin systems from alpha -L-fucopyranose residues, A and B in Fig. 7, B and C, and Table III. Of these two residues, A is both 2-O- and 3-O-sulfated, and B is 2-O-sulfated. Neither residue is 4-O-sulfated, and indeed the strong signal from H3A in the spectrum (Fig. 7, B and C) is the only strong signal between 4.7 and 5.0 ppm, where the H-4 signal of 4-O-sulfated residues is found, implying a low level of 4-O-sulfation compared with the fucan from L. brasiliensis.

Residues A and B, the only structures making an interpretable contribution to the 1H NMR spectra of the sulfated fucans from F. vesiculosus and A. nodosum, only comprise ~50% of the intensity in the anomeric region. From Table III, residue A is disulfated and residue B is monosulfated. For these fucans therefore, ~50% of the structure has 1.5 sulfates per fucose residue, but the remainder cannot be determined by NMR.

13C NMR spectra of the three fucans are shown in Fig. 9. Although no detailed assignment is available for these spectra, it is again clear that the fucans from F. vesiculosus and A. nodosum are similar, whereas the fucan from L. brasiliensis is distinct from either.


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Fig. 9.   13C NMR spectra at 125 MHz of the sulfated fucans from the brown algae L. brasiliensis (A), F. vesiculosus (B), and A. nodosum (C). The spectra were recorded at 60 °C for samples in D2O solution. Chemical shifts are relative to internal or external trimethylsilylpropionic acid at 0 ppm. Anomeric (C1) and fucose C6 signals are marked. Although the spectrum from a limited sample of the fucan from A. nodosum (C) is not strong, it can be seen to resemble the spectrum of the F. vesiculosus (B) fucan. The spectrum of the fucan from L. brasiliensis (A) is more complex than the other two. Peaks marked with × are from contaminants.

Anticoagulant Action of Sulfated Fucans from Brown Algae and Echinoderms

Anticoagulant Potencies of the Various Sulfated Fucans-- Anticoagulant activities of the purified sulfated fucans from brown algae were measured in several in vitro assays and were compared with the same activities of sulfated fucans from marine echinoderms, which have regular, repetitive, and well defined structures (Fig. 1).

The activated partial thrombloplastin time assays summarized in Table IV indicate that crude sulfated polysaccharides from brown algae have anticoagulant action (d, g, and i in Table IV), and purification of the sulfated fucans results in increased anticoagulant potencies (e, h, j, and k in Table IV). Comparison among sulfated fucans from the three species of brown algae show similar activities for fucans from F. vesiculosus and A. nodosum (h, j, and k in Table IV), whereas L. brasiliensis expressed much stronger activity (e in Table IV). Thus, sulfated fucans with similar structural features, as those from F. vesiculosus and A. nodosum, have similar anticoagulant potency, distinguished from the sulfated fucan of L. brasiliensis. Desulfation totally abolishes the anticoagulant activity (f in Table IV).

                              
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Table IV
Anticoagulant potencies of sulfated fucans from echinoderms and brown algae and of standard vertebrate glycosaminoglycans

Among the echinoderms only the sulfated fucan from L. variegatus has a significant anticoagulant action (a in Table IV). Since the only structural difference between the tetrasaccharide repeating unit of the sulfated fucans from L. variegatus and L. grisea is the presence of a 4-O-sulfated fucose unit in L. variegatus (Fig. 1), this single modification on the tetrasaccharide repeating unit is responsible for a 4-fold difference in anticoagulant potency.

Effect of Sulfated Fucans on Thrombin Inhibition in the Absence and in the Presence of Antithrombin or Heparin Cofactor II-- The sulfated fucan from the brown alga L. brasiliensis inhibits thrombin amidolytic activity in the presence of antithrombin, but higher concentrations are required to obtain the same effect with sulfated fucans from the brown algae F. vesiculosus and A. nodosum or from the sea urchin L. variegatus and even higher concentrations with the sulfated fucan from the sea cucumber L. grisea (Table V). In particular, a comparison between the two sulfated fucans from echinoderms indicates that the removal of a single 4-O-sulfate ester from the tetrasaccharide repeating unit (Fig. 1) results in an approximately 4-fold increase in the IC50 for thrombin inhibition (compare a and b in Table V). These sulfated fucans were compared with mammalian glycosaminoglycans. Unfractionated heparin and low molecular weight heparin inhibit thrombin at lower doses than the sulfated fucans, whereas mammalian dermatan sulfate has no effect on this assay, as expected.

                              
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Table V
IC50 of sulfated fucans from echinoderms and brown algae, and of standards vertebrate glycosaminoglycans, for thrombin or factor Xa inhibition in the presence of antithrombin or heparin cofactor II

The effects of the sulfated fucans are essentially the same if factor Xa instead of thrombin is the target protein for antithrombin inactivation. However, marked differences are observed in the concentrations for thrombin and factor Xa inhibition in the presence of antithrombin (Table V). For example, the IC50 for sulfated fucan from L. brasiliensis inhibition of thrombin and factor Xa are ~17 and ~75 times greater when compared with IC50 for unfractionated heparin inhibition, respectively. These differences are even greater for the sulfated fucans from F. vesiculosus and L. variegatus.

Finally, sulfated fucans also inactivate thrombin in the presence of heparin cofactor II. In this case, the inhibitory effect of the sulfated fucans from brown algae (in particular those from L. brasiliensis and F. vesiculosus) occurs in the range of concentration below that required for mammalian dermatan sulfate (Table V). Again, a single difference in the 4-O-sulfation of the tetrasaccharide unit of the sulfated fucans from L. variegatus and L. grisea results in ~5-fold difference in the IC50 for thrombin inhibition in the presence of heparin cofactor II.

We investigated whether the various sulfated fucans could have different effects on thrombin inhibition (Fig. 10). In contrast with mammalian glycosaminoglycans, the sulfated fucans from brown algae are direct thrombin inhibitors in the absence of antithrombin or heparin cofactor II (open circles in Fig. 10). Total inhibition is not achieved in dose-effect curves, and about 20% of thrombin activity remains. The residual thrombin activity is abolished if antithrombin or heparin cofactor II is added to the incubation mixtures. Direct inhibition by sulfated fucans is not dependent on the molecular mass of the polysaccharide since the species L. brasiliensis and F. vesiculosus markedly differ in molecular mass (Fig. 5B), but both are direct inhibitors of thrombin. Sulfated fucan from the sea urchin L. variegatus has a negligible inhibitory effect on thrombin in the absence of antithrombin or heparin cofactor II, but the addition of either of these two plasma proteins to the incubation mixtures results in a marked thrombin inhibition (Fig. 10C), as reported for mammalian glycosaminoglycans (Fig. 10D).


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Fig. 10.   Effect of sulfated fucans from brown algae (A and B) and from echinoderm (C) and of heparin (D) in thrombin inactivation in the absence and in the presence of antithrombin or heparin cofactor II. Thrombin (0.15 units) was incubated in the absence (open circle ) and in the presence of antithrombin () or heparin cofactor II (black-square) with increasing concentrations of sulfated fucans (A-C) or unfractionated heparin (D). After 60 s, the remaining thrombin activity was determined with the chromogenic substrate S-2238 (A404 nm/min).


    DISCUSSION

Sulfated Fucans from the Brown Algae F. vesiculosus and A. nodosum Have Similar Structure Features but Are Markedly Different from L. brasiliensis

Methylation Analysis of the Sulfated Fucans from Brown Algae Reveals Highly Complex Structures-- We employed methylation analysis as one approach to probe the complexity of sulfated fucans from brown algae. However, some limitations of this method must be considered. Methylation of sulfated polysaccharides does not always yield reliable proportions of methylated alditols (26, 37, 43, 44). This may be a consequence of steric hindrance due to the sulfate esters, which does not allow complete methylation of these polymers. The more drastic conditions necessary to remove sulfate esters may also destroy some of the methylated fucose derivatives (26). Alternative methods involving methylation of the desulfated polysaccharide are not always possible since it is difficult to obtain totally desulfated fucans. The fucose-linked sulfate esters appear to be more resistant than other sulfated polysaccharides to solvolysis in dimethyl sulfoxide (29). Nevertheless methylation analysis may offer valuable information concerning the position of the glycosidic linkage and the site of sulfation (29, 37, 45-47).

Methylation of the sulfated fucans from F. vesiculosus and A. nodosum yielded mainly unmethylated fucose. This is not merely a product of an incomplete reaction since its proportions remain unchanged after an additional methylation (not shown).3 Its presence may be an artifact of incomplete methylation caused by steric hindrance by the sulfate esters. Analysis of the methyl derivatives obtained from the partially desulfated F. vesiculosus polysaccharide (Table II) reveals a highly complex structure. All possible methyl derivatives from fucose are present in this mixture of methylated alditols including the very unusual 2,3,5-tri-O-methyl fucose, indicative of fucofuranosyl units at non-reducing terminals. Again, the presence of 2,4-di-O-methylfucose and 2,3-di-O-methylfucose after methylation of the desulfated fucan of F. vesiculosus indicates the presence of 3-linked and 4-linked fucose units, respectively, but in different proportions when compared with the fucan from L. brasiliensis.

Subfractions F1 and F2 prepared from A. nodosum yielded approximately similar proportions of methyl derivatives, indicating that there are no marked structural differences between them.

Overall, methylation analysis of the sulfated fucans from brown algae indicates these polymers have complex and heterogeneous structures. Nevertheless, some structural features can be deduced from these analyses. These fucans are branched polysaccharides, with different proportions of 3- and 4-linked residues and of sulfation at O-2 and/or O-4. In addition, a distinct variation occurs among the structures of sulfated fucans from different species of brown algae. F. vesiculosus and A. nodosum have similar structural features but are markedly different from L. brasiliensis.

NMR Analysis Suggests Some Regular Repeating Structure in the Algal-sulfated Fucans-- Because of the complexity and heterogeneity of the fucans, interpretation of the 1H and 13C spectra yields only a partial analysis of the structures of the three sulfated fucans from brown algae. The one-dimensional 1H and 13C spectra show distinguishable anomeric resonances (Figs. 7 and 9 and Table III). This indicates some regular repeating structures in these polysaccharides, although other broad signals are present, and the sulfated fucans are clearly heterogeneous.

In the case of the sulfated fucans from F. vesiculosus and A. nodosum, it may be speculated that residues A and B form the backbone of both polysaccharides, with the remaining residues forming heterogeneous branches and giving rise to the remaining broad, unresolved intensity in the NMR spectrum and the wide variety of derivatives in the methylation analysis of the fucan from F. vesiculosus.

From this limited NMR study two main differences arise between the fucan from L. brasiliensis and those from F. vesiculosus and A. nodosum. One is the presence of 1right-arrow2 linkages in the L. brasiliensis fucan, and the other is the presence of a higher level of 4-O-sulfation in the fucan from this alga compared with the other two.

Additional Structural Complexity in the Sulfated Fucans from F. vesiculosus and A. nodosum

If the unmethylated, monomethylated, and dimethylated fucose residues obtained after the methylation reactions are internal units, they must contain 2, 1, and no sulfate esters, respectively. In this case we expect that the sulfated fucan from L. brasiliensis would contain ~94 sulfate esters per 100 fucose residues (54 from the unmethylated and 40 from the monomethylated residues, see Table II). This result is in good agreement with the sulfate/fucose molar ratio reported in Table I and with the integrals for the di-, mono-, and unsulfated fucose residues estimated by 1H NMR spectra (Table III).

Similar rationale for the fucans from F. vesiculosus and A. nodosum, based on the methylation analysis (Table II), suggested ~137 and ~150 sulfate esters per 100 fucose residues, respectively, which are above the experimental values obtained in Table I. Undermethylation cannot occur in significant proportion to explain these differences, as estimated by infrared spectra of the polysaccharide obtained after successive and exhaustive methylation steps. The 1H NMR analysis cannot help to reconcile these results, since only about 50% of the structures make interpretable contribution to the 1H spectra.

One possible explanation for the controversial results observed for the sulfated fucans from F. vesiculosus and A. nodosum is the presence of the unusual fucofuranosyl units. These extremely labile residues may be partially lost during the methylation reaction.

We believe the structure of these extremely complex polysaccharides needs future investigation, possibly involving the preparation of small oligosaccharides by specific degradation procedures.

Is There a Correlation between Structure and Anticoagulant Action of the Sulfated Fucans?

Sulfated Fucans from Brown Algae with Similar Structural Features Have Similar Anticoagulant Potencies-- Sulfated fucans from brown algae are complex, and heterogeneous polysaccharides and the controversy surrounding their anticoagulant actions (summarized under the Introduction) may be related to variations in the structure of sulfated fucans from different species of brown algae. The complete structure of the sulfated fucans from brown algae was not obtained (especially in the case of F. vesiculosus and A. nodosum), but some important structural features were established. Even with this limitation a clear correlation can be traced between structure and anticoagulant action of these polysaccharides. We have found that sulfated fucans from the brown algae F. vesiculosus and A. nodosum, which have similar structural features (Figs. 6, 7, and 9 and Tables II and III), have the same anticoagulant potency (Table IV), unlike the sulfated fucan from another brown algae, L. brasiliensis, which has a different structure. The exact nature of the structural features that confer a higher anticoagulant activity on the sulfated fucan from L. brasiliensis is not clear, but it is notable that the sulfate content of this fucan is lower than that of the fucan from F. vesiculosus (Table I), so the increased biological activity is unlikely to be merely a charge density effect. Both the NMR and methylation analysis of the fucan from L. brasiliensis indicate that the polysaccharide contains highly sulfated fucose branches, which may confer anticoagulant activity, as has been reported for a fucosylated chondroitin sulfate from sea cucumber (33). The F. vesiculosus and A. nodosum fucans have less 4-O-sulfate substitution, which may indicate that branch residues are less highly sulfated.

Branched Sulfated Fucans from Brown Algae, but Not the Linear Fucans from Echinoderms, Are Direct Inhibitors of Thrombin-- Mammalian glycosaminoglycans have anticoagulant action mediated mainly by plasma protease inhibitors. Thus, heparin inhibits thrombin, factor Xa, and other coagulation enzymes in the presence of antithrombin (48, 49). Dermatan sulfate and heparin have an additional inhibitory effect on coagulation through heparin cofactor II (50). The exact mechanism of anticoagulant action of sulfated fucans remains controversial (see Introduction).

In order to trace a parallel between the anticoagulant actions of mammalian glycosaminoglycans and that of sulfated fucans, we compared the influence of these sulfated polysaccharides on thrombin and factor Xa inactivation in the absence and in the presence of antithrombin or heparin cofactor II. Although the highly branched sulfated fucans from brown algae directly inhibited thrombin (Fig. 10, A and B), the linear fucans from echinoderm require the presence of antithrombin or heparin cofactor II for inhibition of thrombin (Fig. 10C), similar to mammalian glycosaminoglycans (Fig. 10D). A direct inhibition of thrombin amidolytic activity by sulfated polysaccharides has been reported previously, for the mildly anticoagulant pentosan polysulfate (51).

The Linear and Repetitive Sulfated Fucan from the Sea Urchin L. variegatus Has an Anticoagulant Action Resembling That of Mammalian Dermatan Sulfate-- The linear sulfated fucan from L. variegatus has an anticoagulant action that resembles in some respects that of mammalian dermatan sulfate, despite the marked structural differences between these two polysaccharides. Both compounds are devoid of direct inhibitory effect on thrombin (Fig. 10) and have a similar IC50 for thrombin inhibition in the presence of heparin cofactor II (Table V). Mammalian dermatan sulfate does not inhibit thrombin or factor Xa in the presence of antithrombin, whereas the sulfated fucan from L. variegatus has a modest effect in this system (Table V).

Comparison between two sulfated fucans from echinoderms showed that a single difference of one sulfate ester per tetrasaccharide repeating unit modifies the anticoagulant activity of the polysaccharide markedly. It is reasonable to suppose that the extra sulfate group of the L. variegatus polysaccharide makes it possible for the polysaccharide to present a spatial arrangement of sulfate esters which mimics the site in dermatan sulfate with high affinity for heparin cofactor II. The structural requirements for interaction between dermatan sulfate and heparin cofactor II are specific: a sequence in which 4-O-sulfated galactosamine residues alternate with 2-O-sulfated iduronate residues is necessary (52). In agreement with this structural stringency, dermatan sulfate of ascidian origin with a high proportion of 2-O-sulfated iduronate residues, but with the galactosamine residues 6-O-sulfated rather than 4-O-sulfated, had no measurable anticoagulant activity (53).

Taken together, these results indicate that structural analysis of sulfated polysaccharides from algae and echinoderms and their test on specific biological assays are useful tools to investigate molecular mechanisms of anticoagulant activity in mammals, besides their obvious pratical implications.

    ACKNOWLEDGEMENTS

We are grateful to Adriana A. Eira and Fabio S. Araujo for technical assistance, to Dr. Catherine Boisson-Vidal (Université Paris Nord) for the sample of brown alga A. nodosum, and to Dr. George A. Reis for assistance in the preparation of this manuscript.

    FOOTNOTES

* This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: FNDCT, PADCT, and PRONEX), Financiadora de Estudos e Projetos (FINEP), and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Fax: 55.21.270.8647; E-mail: mourão{at}server.bioqmed.ufrj.br.

2 Following desulfation the increase in these trimethylated fucoses could also result from hydrolysis of the polysaccharide. Thus, a linear oligosaccharide containing 8 fucose units would yield ~12% of 2,3,4-tri-O-methylfucose. In order to exclude this possibility, the native and desulfated fucans from F. vesiculosus were analyzed on a Superose 6 FPLC (HR 10/30) column. Although desulfation reduces the average molecular mass of the polysaccharide from ~30 to ~10 kDa, this decrease is far from sufficient to explain the marked increase in the proportions of the trimethylated fucoses reported in Table II.

3 The sulfated fucans obtained from four successive methylation reactions were analyzed by infrared spectra. The OH stretch band was diminished, with concomitant increase in intensity of the CH stretch bands at 3,000 to 2,800 cm-1, with no loss in the bands corresponding to sulfate. From the third to the fourth methylation steps, there was no additional modification of the infrared spectra, indicating the reactions reach almost completeness.

    ABBREVIATIONS

The abbreviation used is: FPLC, fast protein liquid chromatography.

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