Is there a correlation between structure and anticoagulant action of sulfated galactans and sulfated fucans?

Mariana S. Pereira, Fábio R. Melo and Paulo A.S. Mourão1

Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho 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

Received on January 27, 2002; revised on June 4, 2002; accepted on June 13, 2002


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We attempted to identify the specific structural features in sulfated galactans and sulfated fucans that confer anticoagulant activity. For this study we employed a variety of invertebrate polysaccharides with simple structures composed of well-defined units of oligosaccharides. Our results indicate that a 2-O-sulfated, 3-linked {alpha}-L-galactan, but not a {alpha}-L-fucan with a similar molecular size, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaced thrombin. The occurrence of 2,4-di-O-sulfated units is an amplifying motif for 3-linked {alpha}-fucan-enhanced thrombin inhibition by antithrombin. If we replace antithrombin by heparin cofactor II, then the major structural requirement for the activity becomes single 4-O-sulfated fucose units. The presence of 2-O-sulfated fucose residues always had a deleterious effect on anticoagulant activity. Overall, our results indicate that the structural requirements for interaction of sulfated galactans and sulfated fucans with coagulation cofactors and their target proteases are stereospecific and not merely a consequence of their charge density and sulfate content.

Key words: anticoagulant activity/heparin /sulfated fucan/sulfated galactan/sulfated polysaccharide


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Antithrombotic agents have been extensively used as an adjunct therapy in cardiovascular diseases, and heparin is now the initial choice (Kakkar and Hedges, 1989Go). Its anticoagulant (and possibly the antithrombotic) activity is mediated by specific plasma cofactors, named antithrombin and heparin cofactor II (Béguin et al., 1988Go). A specific local structure in the molecule of heparin, composed of a pentasaccharide sequence with a specific pattern of sugar composition and of sulfation pattern, is required to induce a conformational activation of the antithrombin (Thunberg et al., 1982Go; Lindahl et al., 1983Go). Heparin has an additional anticoagulant mechanism, resulting from bridging the protease and the antithrombin molecules (Streusand et al., 1995Go). This effect is determined by the size and bulk heparin structure. Dermatan sulfate is another mammalian glycosaminoglycan with anticoagulant activity, but in this case mediated exclusively by heparin cofactor II. A specific sequence of [4-{alpha}-L-IdUA-2(SO4)-1->3-ß-D-GalNAc-4(SO4)-1]n, where n >= 3 is required for the binding of dermatan sulfate to the plasma cofactor (Maimone and Tollefsen, 1990Go).

Heparin has several side effects, such as development of thrombocytopenia (Warkentin, 1999Go; Visentin, 1999Go), hemorrhagic effect (Kelton and Hirsh, 1980Go; Kakkar et al., 1986Go), ineffectiveness in congenital or acquired antithrombin deficiencies, incapacity to inhibit thrombin bound to fibrin (Liaw et al., 2001Go), and so on. In addition, heparin is mostly extracted from pig intestine or bovine lung, where it occurs in low concentrations. Furthermore, the incidence of prion-related diseases in mammals and the increasing requirement of anticoagulant therapy indicate that we may need to look for alternative sources of anticoagulant and antithrombotic compounds.

One abundant source of new anticoagulant polysaccharides is marine algae. They contain a variety of sulfated fucans (Church et al., 1989Go; Nishino et al., 1991Go; Colliec-Jouault et al., 1991Go; Colliec et al., 1994Go; Pereira et al., 1999Go) and sulfated galactans (Potin et al., 1992Go; Sem et al., 1994Go; Farias et al., 2000Go) with anticoagulant activity. These compounds are among the most abundant and widely studied of all the sulfated polysaccharides from nonmammalian origin. Several attempts to identify in these algal polysaccharides’ specific structural features necessary for their anticoagulant activity have been limited by the fact that algal fucans and galactans have complex, heterogeneous structures (Pereira et al., 1999Go; Farias et al., 2000Go). Their regular repeating sequences are not easily deduced; even high-field nuclear magnetic resonance (NMR) is at the limit of its resolution, and complete description of their structure is not available at present (Mulloy et al., 1994Go; Pereira et al., 1999Go; Farias et al., 2000Go). Obviously, identification of specific structural requirements in the algal polysaccharides necessary for interaction with coagulation cofactors is an essential step for a more rational approach to develop new anticoagulant and antithrombotic drugs.

Recently, we isolated and characterized several sulfated {alpha}-L-fucans and sulfated {alpha}-L-galactans from invertebrates (mostly from the egg jelly of sea urchins). In contrast to the algal fucans and galactans, these invertebrate polysaccharides have simple, linear structures, composed of well-defined repeating units of oligosaccharides (Santos et al., 1992Go; Alves et al., 1997Go, 1998; Vilela-Silva et al., 1999Go, 2002). The physiological role of these invertebrate polysaccharides is far distant from blood coagulation. They are either components of the extracellular matrix (Albano and Mourão, 1986Go; Santos et al., 1992Go) or involved in gamete interaction during fertilization (Alves et al., 1997Go, 1998; Vilela-Silva et al., 1999Go, 2002). Nevertheless, some of these polysaccharides have potent in vitro anticoagulant activity.

We undertook a systematic analysis of the anticoagulant activity of these invertebrate polysaccharides and took advantage of their wide diversity of regular and repetitive structures to elucidate structure–anticoagulant action relationship. Our aim was to identify in these compounds specific structural features necessary for activation of plasma serine-protease inhibitors.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Summary of the polysaccharide structures
Sulfated fucans and sulfated galactans from invertebrates have simple, linear structures, composed of well-defined units of oligosaccharides (Santos et al., 1992Go; Alves et al., 1997Go, 1998; Vilela-Silva et al., 1999Go, 2002). The specific pattern of sulfation and the position of the glycosidic linkage vary among the polysaccharides from different species of sea urchins. Arbacia lixula has a 4-linked sulfated {alpha}-L-fucan with two consecutive 2-O-sulfated residues followed by two unsulfated units (Figure 1J). Lytechinus variegatus and Strongylocentrotus pallidus have 3-linked sulfated {alpha}-L-fucans with tetrasaccharide repeating units, which differ by specific patterns of sulfation (Figures 1H and 1I, respectively). Strongylocentrotus purpuratus has two structures, found in different individuals: a monosaccharide with variable sulfation at one position (sulfated {alpha}-L-fucan I) and a trisaccharide repeat sequence (sulfated {alpha}-L-fucan II) (Figures 1F and 1G, respectively). Echinometra lucunter, Strongylocentrotus franciscanus and Strongylocentrotus droebachiensis (Figures 1A, 1D, and 1E, respectively) have polysaccharides with a single 2-O-sulfated monosaccharide unit, which differ either on the position of their glycosidic linkage or in their constituent monosaccharide. S. droebachiensis and S. franciscanus contain 4-linked and 3-linked {alpha}-L-fucopyranose, respectively, and E. lucunter has 3-linked {alpha}-L-galactopyranose. Herdmania monus contains a sulfated galactan composed of 3-O-sulfated, 4-linked {alpha}-L-galactopyranose (Figure 1B), whereas the galactan from Styela plicata has a similar backbone structure but with nonsulfated L-galactose as branched units linked to position O-2 to the central core (Figure 1C).




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Fig. 1. Structures of sulfated {alpha}-L-fucans and sulfated {alpha}-L-galactans from invertebrates. The figure shows 10 fully characterized structures of sulfated polysaccharides from the egg jelly of sea urchins or from the tunic of ascidians. The specific pattern of sulfation, the position of the glycosidic linkage, and the constituent monosaccharide vary among sulfated polysaccharides from different species. (See text and Santos et al., 1992Go; Alves et al., 1997Go, 1998; Vilela-Silva et al., 1999Go, 2002 for details.)

 
2-O-sulfate, 3-linked {alpha}-L-galactan is a distinguished anticoagulant polysaccharide
Initially we compared the anticoagulant activity of sulfated fucans and sulfated galactans enriched in 2-O- or 3-O-sulfate esters. The activated partial thromboplastin time (APTT) assays, summarized in Table I, indicate that the 2-O-sulfated 3-linked {alpha}-L-galactan from E. lucunter has a distinguished potent anticoagulant activity. Sulfation in a different position as well as the position of the glycosidic linkage (as in the galactan from H. monus) dramatically reduces the anticoagulant activity. Replacement of 2-O-sulfated {alpha}-L-galactose by 2-O-sulfated {alpha}-L-fucose in a polymer with either the same or a different position of the glycosidic linkage (as in the fucans from S. franciscanus and S. droebachiensis, respectively) does not restore the activity.


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Table I. Sulfate content, average mocecular mass, APTT, and IC50 of the 2-O- or 3-O-sulfated galactans and fucans for thrombin or factor Xa inhibition in the presence of antithrombin or heparin cofactor II
 
The sulfated galactan from E. lucunter enhances thrombin or factor Xa inhibition by antithrombin or heparin cofactor II with similar sigmoid curves as those observed for heparin or dermatan sulfate (Figure 2). Although higher concentrations were required to achieve the same effect as heparin, the IC50 for thrombin or factor Xa inhibition can be easily determined, as reported in Table I. For other invertebrate polysaccharides, we observed a dramatic shift to the right in relation to their effects on thrombin and factor Xa inhibition. In most cases, total inhibition was not achieved in the range of concentrations used in our experiments. For the sulfated galactan from H. monus we observe a slight decrease of the inhibitory effect at higher concentrations after achieve a ~60% inhibition at ~50 µg/ml (Figure 2A).



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Fig. 2. Dependence on the concentration of 2-O- or 3-O-sulfated {alpha}-L-galactans or {alpha}-L-fucans for inactivation of thrombin (A and B) or factor Xa (C) by antithrombin (A and C) or heparin cofactor II (B). Antithrombin (50 nM) or heparin cofactor II (68 nM) were incubated with thrombin (15 nM) or factor Xa (15 nM) in the presence of various concentrations of the sulfated {alpha}-L-galactans from E. lucunter (closed circles) and H. monus (open squares), sulfated {alpha}-L-fucans from A. lixula (closed squares) and S. franciscanus (open triangles), heparin (open circles), and dermatan sulfate (closed triangles). After 60 s, the remaining thrombin or factor Xa activity was determined with a chromogenic substrate (A405nm/min).

 
The experiments based on amidolytic activity (Figure 2, summarized in the IC50 values of Table I) permit more refined comparisons among the activities of the invertebrate polysaccharides. For example, insertion of two consecutive unsulfated residues between two 2-O-sulfated units (as in the fucan from A. lixula) increases the potency of the polysaccharide to enhance thrombin inhibition by either antithrombin or heparin cofactor II when compared with a totally 2-O-sulfated fucan (as in S. franciscanus) (Figure 2A, B). The difference between the activities of these two fucans is not very pronounced when thrombin is replaced by factor Xa (Figure 2C). Insertion of nonsulfated L-galactose residues as branched units linked to a 3-O-sulfated, 4-linked galactan core (as in the galactan from S. plicata) does not restore the anticoagulant effect of the polysaccharide (Table I).

The various sulfated polysaccharides from invertebrates have similar molecular masses, always > 50 kDa, as determined by polyacrylamide gel electrophoresis (data not shown). We undertook a more refined comparison between the molecular masses of the sulfated galactan and sulfated fucan from E. lucunter and S. franciscanus, respectively, using gel filtration on Superose 6-FPLC. As shown in Figure 3, these two polysaccharides did not diverge in their elution pattern from the column. Two subfractions of the E. lucunter galactan, eluted at different positions from the gel filtration chromatography, did not differ significantly in their anticoagulant activities. These results indicated that the differences in anticoagulant activity observed between the two invertebrate polysaccharides could not be ascribed to variation in the size of their chains.



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Fig. 3. Gel filtration chromatography of the sulfated {alpha}-galactan from E. lucunter (A) and the sulfated {alpha}-fucan from S. franciscanus (B) on a Superose-6 column. About 5 mg of each polysaccharide was applied to a Superose-6 (HR 10/30) column linked to a FPLC system. The column was eluted with 0.2 M NH4HCO3, at a flow rate of 0.5 ml/min and fractions of 0.5 ml were collected and assayed by metachromasia using 1,9-dimethylmethylene blue (Farndale et al., 1986Go). The fractions indicated by horizontal bars in (A) were pooled, dialyzed against distilled water, and lyophilized. The values above the horizontal bars on (A) are anticoagulant activities of these fractions determined by APTT assay (as in Table I) but expressed in the figure as IU/mg hexose.

 
Further information about the structure–anticoagulant activity relationship of the sulfated polysaccharides comes from literature data on a sulfated galactan from the red algae Botryocladia occidentalis (Farias et al., 2000Go). This compound has the repeating sequence [-4-{alpha}-D-Galp-1->3-ß-D-Galp-1->], but in contrast with the invertebrate polysaccharides, the algal galactan contains a variable sulfation pattern. However, it was possible to identify clearly that one-third of the total {alpha}-units are 2,3-di-O-sulfated and another third are 2-O-sulfated. The potent anticoagulant activity of this algal galactan was ascribed to the presence of 2,3-di-O-sulfated {alpha}-units (Farias et al., 2000Go).

Overall, the 2-O-sulfated 3-linked galactan from E. lucunter is an anticoagulant polysaccharide due to enhanced inhibition of thrombin and factor Xa by antithrombin and/or heparin cofactor II. Comparison with several closely related sulfated polysaccharides from marine invertebrates, mostly with the same charge density, indicates the structural requirements for interaction with coagulation cofactors are stereospecific and has no relation with the charge density of the polysaccharide. The presence of 2,3-di-O-sulfated {alpha}-galactose units has an amplifying effect on the anticoagulant activity of an algal galactan.

Insertion of 2,4-di-O-sulfated units into 3-linked {alpha}-fucans has an amplifying effect on the anticoagulant activity
On a further approach to trace structure/anticoagulant activity relationship of the invertebrate polysaccharides we employed closely related 3-linked sulfated {alpha}-L-fucans, which diverge exclusively on their patterns of 2-O- and 4-O-sulfation. The APTT assays indicate that sulfated fucan I from S. purpuratus, composed of ~80% 2,4-di-O-sulfated units (Figure 1F), has a distinguished potent anticoagulant activity (Table II). Sulfated fucan II from S. purpuratus and the sulfated fucan from L. variegatus, both also with 2,4-di-O-sulfated units but at a lower proportion (33% and 25% of the total residues, respectively; see Figures 1G and 1H), have a significant decrease in their anticoagulant activities.


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Table II. APTT and IC50 of the 2-O- and/or 4-O-sulfated 3-linked fucans for thrombin or factor Xa inhibition in the presence of antithrombin or heparin cofactor II
 
Differences among the actions of these 3-linked {alpha}-L-fucans are more evident on the experiments based on amidolytic activity (Figure 4). Sulfated fucan I from S. purpuratus enhances thrombin inhibition by antithrombin with a sigmoid curve like that of heparin, although higher concentrations of sulfated fucan are necessary to obtain the same effect (Figure 4A, Table II). The experiment of Figure 4A allows further conclusions based on comparison among activities of the various sulfated {alpha}-L-fucans. As the proportion of 2,4-di-O-sulfated fucose units decreases from sulfated fucan I to sulfated fucan II from S. purpuratus and to sulfated fucan from L. variegatus, up to virtual absence of this type of residue in the fucans from S. pallidus and S. franciscanus (see Figure 1), the inhibition curves of thrombin in the presence of antithrombin are shifted to the right and, in some cases, lost the typical sigmoid format (Figure 4A). Total inhibition was not achieved in the range of concentration tested, except for sulfated fucan I from S. purpuratus. For sulfated fucan II from S. purpuratus and the fucan from S. pallidus ~80% and ~30% thrombin inhibition is obtained at ~50 µg/ml, respectively, and increasing concentrations result in a decrease of the inhibitory effect.



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Fig. 4. Dependence on the concentration of 2-O- and/or 4-O-sulfated 3-linked {alpha}-L-fucans for inactivation of thrombin (A and B) or factor Xa (C) by antithrombin (A and C) or heparin cofactor II (B). Amidolytic activity was measured as described in the legend of Figure 2 in the presence of various concentrations of sulfated fucan I (closed squares) and sulfated fucan II (open squares) from S. purpuratus, sulfated fucans from L. variegatus (closed triangles) and S. pallidus (open triangles), heparin (open circles), and dermatan sulfate (closed circles). For a comparative purpose we added the values obtained with the sulfated fucan from S. franciscanus (broken line), based on the experiments of Figure 2.

 
Our interpretation of the experiments in Figure 4A is that 2,4-di-O-sulfated fucose unit is the "amplifying motif" because sulfated fucan enhances thrombin inhibition by antithrombin. The presence of exclusively 2-O-sulfated units has a deleterious effect, as demonstrated by the marked decrease in the activity of sulfated fucan from L. variegatus compared with that of sulfated fucan II from S. purpuratus. Both contain the amplifying motif but differ because the intercalate units are single 4-O-sulfated residues in sulfated fucan II from S. purpuratus and in L. variegatus 50% are exclusively 2-O-sulfated residues (Figures 1G and 1H). In addition, the sulfated fucan from S. franciscanus, composed exclusively of 2-O-sulfated fucose units, is almost devoid of activity, whereas sulfated fucan from S. pallidus, those 2-O-sulfated residues are intercalated by 4-O-sulfated units, shows a modest but detectable inhibitory effect on thrombin.

When the target protease is factor Xa instead of thrombin, a sigmoid curve was obtained with the sulfated fucan from S. pallidus, and total inhibition is achieved (compare curves in Figures 4A and 4C). Therefore, differences in the effect of the various sulfated {alpha}-L-fucans cannot be ascribed exclusively to variation in their affinities for antithrombin. It results from a more complex and still unclear effect of the sulfated {alpha}-L-fucans on the complex formed between the plasma cofactor and its target protease.

If we replace antithrombin by heparin cofactor II the difference between the 3-linked sulfated {alpha}-fucans from S. purpuratus and S. pallidus is not very pronounced (Figure 4B). Therefore, the presence of fucose units sulfated at both 2-O and 4-O positions was not essential for the fucan-enhanced thrombin inhibition by heparin cofactor II. In this case occurrence of single 4-O-sulfated fucose units is enough to achieve the inhibitory effect. Thus, a fucan without 2,4-di-O-sulfated residues but with 4-O-sulfated units (as in S. pallidus) has the same level of activity as sulfated fucan II from S. purpuratus (Figure 4B, Table II). However, an exclusively 2-O-sulfated fucan (as in S. franciscanus) is almost devoid of activity.

Finally, we tested the anticoagulant activity of the sulfated galactan from E. lucunter and the sulfated fucan I from S. purpuratus using antithrombin- and heparin cofactor II–depleted plasma (Table III). Both polysaccharides lose the anticoagulant effects on the modified plasma and assure their activities are dependent on these two cofactors.


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Table III. Thrombin time (TT) of sulfated galactan and sulfated fucan on normal and antithrombin (AT) + heparin cofactor II (HCII)–depleted plasma
 
Is the anticoagulant activity of the sulfated galactan ascribed to local specific structure or to the bulk pattern of sulfation?
An additional aspect we need to clarify is related to the possibility that the anticoagulant activity of the sulfated galactans and sulfated fucans is related to local specific structure in the polysaccharide chain, rather than with the bulk pattern of sulfation or monosaccharide composition. This point has been well characterized in the case of heparin. Thus, heparin preparations with a similar bulk structure may vary their anticoagulant activity due to different proportions of a specific pentasaccharide sequence, almost undetectable on analysis of the native molecule (Thunberg et al., 1982Go; Lindahl et al., 1983Go). This specific sequence induces a conformational activation of the antithrombin (Olson and Shore, 1981Go; Lin et al., 2001Go). However, heparin still has an additional anticoagulant mechanism, resulting from bridging the protease and the antithrombin molecules. Thus, a heparin preparation, devoid of the specific pentasaccharide sequence, still potentiates thrombin inactivation in the presence of antithrombin, although with a reduced magnitude (Streusand et al., 1995Go). Therefore, heparin accelerates the antithrombin–protease reaction by a combined bridging and conformational activation mechanisms. The latter effect requires a specific local structure, whereas the first is determined by the size and bulk structure of the heparin chain.

We attempted to clarify these aspects in the case of the new anticoagulant polysaccharides we reported. Our approach was to decrease the molecular size of the sulfated galactan from B. occidentalis by mild acid hydrolysis and to separate the fragments by gel filtration. Four different subfractions were obtained, F1, F2, F3, and F4. Their average molecular masses were estimated by polyacrylamide gel electrophoresis (Table IV). The four subfractions and the native polysaccharide have similar 1H-NMR spectra (data not shown), which show that the pattern of sulfation was not modified in the course of acid hydrolysis.


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Table IV. IC50 of fragments obtained from the sulfated galactan of B. occidentalis for thrombin or factor Xa in the presence of antithrombin or heparin cofactor II
 
A slight decrease in the molecular mass of the sulfated galactan dramatically reduced its effect on thrombin in the presence of antithrombin, as noted when we compared the native polysaccharide and subfraction F1 (Table IV). This was especially noticeable when comparing the average molecular masses of subfraction F1 and of low-molecular-weight heparin (45 and 5 kDa, respectively) and their effect on thrombin inhibition in the presence of antithrombin (IC50 > 10 and 0.03 µg/ml, respectively). Even when we replaced thrombin by factor Xa or antithrombin by heparin cofactor II, the sulfated galactan always required fragments with much higher molecular masses than did heparin to inactivate the protease. These results suggested that the sulfated galactan accelerates antithrombin–proteinase reactions mostly by bridging mechanisms, as determined by the bulk polysaccharide structure. The effect of the sulfated galactan on antithrombin conformational activation may have a reduced magnitude compared to heparin. Such a proposal was further supported by the observation that when the native or the various subfractions of sulfated galactan were added to an antithrombin solution, they did not induce the change of intrinsic tryptophan fluorescence, as observed for heparin (data not shown). This methodology is widely used to follow the antithrombin conformational change induced by heparin or similar compounds (Olson and Shore, 1981Go; Lin et al., 2001Go).

Structure versus anticoagulant activity
Our results indicated that the anticoagulant activity of sulfated galactans and sulfated fucans was not merely a consequence of their charge density and sulfate content. The structural requirements for interaction of these polysaccharides with coagulation cofactors and their target proteases are stereospecific. The major conclusions from our experiments are summarized.

The nature of sugar residue modifies markedly the anticoagulant activity. This conclusion comes from comparison between the active galactan from E. lucunter and the almost inactive fucan from S. franciscanus. Both polysaccharides are 3-linked, 2-O-sulfated (Figures 1A and 1D) and have similar molecular masses (Figure 3) but differ in their sugar composition. In addition, a 2,3-di-O-sulfated galactan from the red algae B. occidentalis (Table I) is significantly more active than a 2,4-di-O-sulfated fucan from the invertebrate S. purpuratus (Table II).

Occurrence of 2,4-di-O-sulfated units has an amplifying effect on the anticoagulant activity of 3-linked {alpha}-fucans. Comparison among closely related 3-linked {alpha}-L-fucans, which differ exclusively in their sulfation patterns, indicates 2,4-di-O-sulfation is an amplifying motif for these compounds enhance thrombin inhibition by antithrombin, and single 2-O-sulfated units have a deleterious effect (Figure 4A, Table III). This is not merely a consequence of increased charge density. The anticoagulant activity increases ~38-fold from the sulfated fucan of S. franciscanus to sulfated fucan I of S. purpuratus (based on APTT assays, Tables I and II), and their sulfate content increases ~1.8-fold.

Specific sulfation sites are required for interaction with plasma serine-protease inhibitors. The occurrence of single 4-O-sulfated units is the structural motif for 3-linked {alpha}-L-fucans enhanced inhibition of thrombin by heparin cofactor II. Again, the presence of exclusively 2-O-sulfated residues has a deleterious effect. This conclusion is based on comparison between the effect of a totally 2-O-sulfated fucan (as in S. franciscanus) and two other fucans, containing either intercalate 4-O-sulfated units (as in S. pallidus) or unsulfated residues (as in A. lixula) (Figures 2B and 4B). As the content of exclusively 4-O-sulfation increases, or the proportion of 2-O-sulfation decreases, a more potent inhibitory effect is achieved.

Overall, our results extend the structural stringency for interaction with coagulation cofactors to the sulfated galactans and  sulfated fucans as already reported for mammalian glycosaminoglycans. For example, oversulfated dermatan sulfate showed only discrete, selected sites competent for interaction with heparin cofactor II (Pavão et al., 1995Go, 1998).

The conformational analysis of these sulfated polysaccharides is an important route to follow. The differences in chemical structure may in fact determine the spacing between sulfate groups required to match the interval between basic amino acid residues in the protein chain. Conformational analysis may explain the drastic differences in biological activity between sulfated galactan and sulfated fucan, in spite of the same positions of sulfation and glycosidic linkage. Similarly, changes in biological activity may reflect dramatic modifications in the conformation of the polysaccharide as a consequence of 2-O- and/or 4-O-sulfation of the 3-linked {alpha}-L-fucans.

Finally, our results demonstrated that combining structural analysis of sulfated polysaccharides with specific biological assays is a useful tool to investigate anticoagulant activity in mammals. These studies may help delineate a closer relationship between structure and biological activity of sulfated polysaccharides. New compounds with obvious practical applications may be found.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Sulfated galactans and sulfated fucans from invertebrates
Sulfated galactans and sulfated fucans were extracted from the tunic of ascidians or from the egg jelly coat of sea urchins by protease digestion (Albano and Mourão, 1986Go). These polysaccharides were purified by anion exchange and/or gel filtration chromatography, and the purity of each fraction was checked by agarose gel electrophoresis and NMR spectroscopy, as described (Santos et al., 1992Go; Alves et al., 1997Go, 1998; Vilela-Silva et al., 1999Go, 2002).

Clotting assays
APTT and thrombin time (TT) clotting assays were performed using normal human plasma according to the manufacturer’s specifications, as described by Anderson et al. (1976)Go. The clotting times were recorded in a coagulometer (Amelung KC4A). For the APTT assays, the activity was expressed as international units/mg using a parallel standard curve based on the 4th International Heparin Standard (193 IU/mg). In some experiments, the clotting assays were performed with antithrombin + heparin cofactor II–deficient plasma obtained from Affinity Biologicals (Ontario, Canada).

Inhibition of thrombin or factor Xa by antithrombin and heparin cofactor II in the presence of sulfated polysaccharides
Incubations were preformed in disposable semi-microcuvettes. The final concentrations of reactants included 68 nM heparin cofactor II or 50 nM antithrombin, 15 nM thrombin, or factor Xa (all from Diagnostica Stago, Asnières, France) and 0–1000 µg/ml sulfated polysaccharide in 100 µl 0.02 M Tris–HCl, 0.15 M NaCl, and 1.0 mg/ml polyethylene glycol (pH 7.4) (TS/PEG buffer). Thrombin or factor Xa was added last to initiate the reaction. After 60 s incubation at room temperature, 500 µl 100 µM chromogenic substrate S-2238 for thrombin or S-2222 for factor Xa (Chromogenix AB, Molndal, Sweden) in TS/PEG buffer was added, and the absorbance at 405 nm was recorded for 100 s. The rate of change of absorbance was proportional to the thrombin activity remaining in the incubation. No inhibition occurred in control experiments, in which thrombin was incubated with antithrombin or heparin cofactor II in the absence of sulfated polysaccharide. Nor did inhibition occur when thrombin was incubated with sulfated polysaccharide alone over the range of concentrations tested.

Gel filtration chromatography
Sulfated galactan from E. lucunter or sulfated fucan from S. franciscanus (5 mg of each) was applied to a Superose-6 (HR 10/30) column, linked to an FPLC system from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom), equilibrated with 0.2 M NH4HCO3 (pH 8.0). The column was eluted with the same solution at a flow rate of 0.5 ml/min, and fractions of 0.5 ml were collected and assayed by metachromasia using 1,9-dimethylmethylene blue (Farndale et al., 1986Go). The various fractions were pooled, dialyzed against distilled water, and lyophilized.

Preparation of fragments from the sulfated galactan of B. occidentalis with reduced molecular masses
Sulfated galactan from B. occidentalis (40 mg) was dissolved in 1.0 ml 0.1 M HCl, and the solution was incubated at 60°C for 60 min. Thereafter the mixture was neutralized with 1.0 ml 0.1 M NaOH. The partial hydrolyzed sulfated galactan was applied to a Sephacryl S-400/HR column (220 x 0.75 cm) and equilibrated with 0.2 M NH4HCO3 (pH 7.0). The column was eluted with the same solution, at a flow rate of 28 ml/h, fractions of 4 ml were collected and assayed by metachromasia (Farndale et al., 1986Go). The various fractions were pooled as four different subfractions, designated F1, F2, F3, and F4, and lyophilized. The molecular masses of the subfractions were estimated by polyacrylamide gel electrophoresis (Santos et al., 1992Go). In addition, the polysaccharides were analyzed by 1H-NMR spectroscopy, as described (Vilela-Silva et al., 1999Go, 2002).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This investigation was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: FNDCT, PADCT, and PRONEX), Financiadora de Estudos e Projetos (FINEP), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Coordenação de Aperfeiçoamento do Pessoal de Nível Superior (CAPES). We are grateful to Adriana A. Piquet for technical assistance. We thank Dr. Victor D. Vacquier (Scripps Institution of Oceanography) and Dr. Christiane H. Biermann (Friday Harbor Laboratories) for the supply of sea urchin egg jellies. P.A.S.M. is a fellow from the John Simon Guggenheim Memorial Foundation.


    Abbreviations
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
APTT, activated partial thromboplastin time; NMR, nuclear magnetic resonance; TS/PEG, a solution containing 0.02 M Tris–HCl, 0.15 M NaCl, and 1.0 mg/ml polyethylene glycol; TT, thrombin time.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: pmourao@hucff.ufrj.br Back


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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