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, 21941590, Brazil
Received on January 27, 2002; revised on June 4, 2002; accepted on June 13, 2002
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Abstract |
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Key words: anticoagulant activity/heparin /sulfated fucan/sulfated galactan/sulfated polysaccharide
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Introduction |
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Heparin has several side effects, such as development of thrombocytopenia (Warkentin, 1999; Visentin, 1999
), hemorrhagic effect (Kelton and Hirsh, 1980
; Kakkar et al., 1986
), ineffectiveness in congenital or acquired antithrombin deficiencies, incapacity to inhibit thrombin bound to fibrin (Liaw et al., 2001
), 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., 1989; Nishino et al., 1991
; Colliec-Jouault et al., 1991
; Colliec et al., 1994
; Pereira et al., 1999
) and sulfated galactans (Potin et al., 1992
; Sem et al., 1994
; Farias et al., 2000
) 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., 1999
; Farias et al., 2000
). 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., 1994
; Pereira et al., 1999
; Farias et al., 2000
). 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 -L-fucans and sulfated
-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., 1992
; Alves et al., 1997
, 1998; Vilela-Silva et al., 1999
, 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, 1986
; Santos et al., 1992
) or involved in gamete interaction during fertilization (Alves et al., 1997
, 1998; Vilela-Silva et al., 1999
, 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 structureanticoagulant action relationship. Our aim was to identify in these compounds specific structural features necessary for activation of plasma serine-protease inhibitors.
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Results and discussion |
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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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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 -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 -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 -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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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 -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
-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 -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 IIdepleted 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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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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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 -fucans. Comparison among closely related 3-linked
-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 -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., 1995, 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 -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.
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Materials and methods |
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Clotting assays
APTT and thrombin time (TT) clotting assays were performed using normal human plasma according to the manufacturers specifications, as described by Anderson et al. (1976). 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 IIdeficient 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 01000 µg/ml sulfated polysaccharide in 100 µl 0.02 M TrisHCl, 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., 1986). 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., 1986). 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., 1992
). In addition, the polysaccharides were analyzed by 1H-NMR spectroscopy, as described (Vilela-Silva et al., 1999
, 2002).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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Alves, A.P., Mulloy, B., Diniz, J.A., and Mourão, P.A.S. (1997) Sulfated polysaccharides from the egg jelly layer are species-specific inducers of acrosomal reaction in sperms of sea urchins. J. Biol. Chem., 272, 69656971.
Alves, A.P., Mulloy, B., Moy, G.W., Vacquier, V.D., and Mourão, P.A.S. (1998) Females of the sea urchin Strongylocentrotus purpuratus differ in the structure of their egg jelly sulfated fucans. Glycobiology, 8, 939946.
Anderson, L.O., Barrowcliffe, T.W., Holmer, E., Johnson, E.A., and Sims, G.E.C. (1976) Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin-3 and by gel-filtration. Thromb. Res., 9, 575580.[ISI][Medline]
Béguin, S., Lindhout, T., and Hemker, H.C. (1988) The mode of action of heparin in plasma. Thromb. Haemostas., 60, 457462.[ISI][Medline]
Church, F.C., Meade, J.B., Treanor, R.E., and Whinna, H.C. (1989) Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin, and thrombin. J. Biol. Chem., 264, 36183623.
Colliec, S., Boisson-Vidal, C., and Jozefonvicz, J. (1994) A low molecular weight fucoidan from the brown seaweed Pelvetia canaliculata. Phytochemistry, 35, 697700.[CrossRef][ISI]
Colliec-Jouault, S., Fisher, A.M., Tapon-Bretaudière, J., Boisson, C., and Jazefonvicz, J. (1991) Anticoagulant properties of a fucoidan fraction. Thromb. Res., 64, 143154.[ISI][Medline]
Farias, W.R.L., Valente, A.P., Pereira, M.S., and Mourão, P.A.S. (2000) Structure and anticoagulant activity of sulfated galactans. Isolation of a unique sulfated galactan from the red algae Botryocladia occidentalis and comparison of its anticoagulant action with that of sulfated galactans from invertebrates. J. Biol. Chem., 275, 2929929307.
Farndale, R.W., Buttle, D.J., and Barret, A.J. (1986) Improved quantitation and discrimination of sulfated glycosaminoglycans by use of dimethylmethylene blue. Biochim. Biophys. Acta, 883, 173177.[ISI][Medline]
Kakkar, V.V. and Hedges, A.R. (1989) Use of heparin as a profilatic agent against venous thromboembolism. In: Lane, D.A., and Lindahl, U. (eds), Heparin: chemical and biological properties, clinical applications. Edward Arnold, London, 455473.
Kakkar, V.V., Kakkar, S., Sanderson, R.M., and Peers, C.E. (1986) Efficacy and safety of two regimens of low molecular weight heparin fragment (Fragmin) in preventing post-operative venous thromboembolism. Haemostasis, 16, 1924.[Medline]
Kelton, J.G. and Hirsh, J. (1980) Bleeding associated with antithrombotic therapy. Semin. Hematol., 17, 259379.[ISI][Medline]
Liaw, P.C.Y., Becker, D.L., Stafford, A.R., Fredenburgh, J.C., and Weitz, J.I. (2001) Molecular basis for the susceptibility of fibrin-bound thrombin to inactivation by heparin cofactor II in the presence of dermatan sulfate but not heparin. J. Biol. Chem., 276, 2095920965.
Lin, P.H., Sinha, U., and Betz, A. (2001) Antithrombin binding of low molecular weight heparins and inhibition of factor Xa. Biochim. Biophys. Acta, 1562, 105113.
Lindahl, U., Backström, G., and Thumberg, L. (1983) The antithrombin-binding sequence in heparin. Identification of an essential 6-O-sulfate group. J. Biol. Chem., 258, 98269830.
Maimone, M. and Tollefsen, D.M. (1990) Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity. J. Biol. Chem., 265, 1826318271.
Mulloy, B., Ribeiro, A.C., Alves, A.P., Vieira, R.P., and Mourão, P.A.S. (1994) Sulfated fucans from echinoderms have a regular tetrasaccharide repeating unit defined by specific patterns of sulfation at the O-2 and O-4 positions. J. Biol. Chem., 269, 2211322123.
Nishino, T., Aizu, Y., and Nagumo, T. (1991) Antithrombin activity of a fucan sulfate from the brown seaweed Acklonia kurome. Throm. Res., 62, 765773.[ISI][Medline]
Olson, S.T. and Shore, J.D. (1981) Binding of high affinity heparin to antithrombin III: characterization of the protein fluorescence enhancement. J. Biol. Chem., 256, 1106511072.
Pavão, M.S.G., Aiello, K.R.M., Werneck, C.C., Silva, L.C.F., Valente, A.P., Mulloy, B., Colwell, N.S., Tollefsen, D.M., and Mourão, P.A.S. (1998) Highly sulfated dermatan sulfate from ascidians: structure versus anticoagulant activity of these glycosaminoglycans. J. Biol. Chem., 273, 2784827857.
Pavão, M.S.G., Mourão, P.A.S., Mulloy, B., and Tollefsen, D.M. (1995) A unique dermatan sulfate-like glycosaminoglycan from ascidian: its structure and the effect of its unusual sulfation pattern on coagulation activity. J. Biol. Chem., 270, 3102731036.
Pereira, M.S., Mulloy, B., and Mourão, P.A.S. (1999) 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. J. Biol. Chem., 274, 76567667.
Potin, P., Patier, P., Jean-Ives, F., Jean-Claude, Y., Rochas, C., and Kloareg, B. (1992) Chemical characterization of cell-wall polysaccharides from tank-cultivated and wild plants of Delesseria sanguinea (Hudson) Lamouroux (Ceramiales-Delesseriaceae): culture patterns and potent anticoagulant activity. J. Applied Physiol., 4, 119128.
Santos, J.A., Mulloy, B., and Mourão, P.A.S. (1992) Structural diversity among sulfated -L-galactans from ascidians (tunicates): studies on the species Ciona intestinalis and Herdmania monus. Eur. J. Biochem., 204, 669677.[Abstract]
Sem, S.R., Das, A.K., Banerji, N., Siddhanta, A.K., Mody, K.H., Ramavat, B.K., Chauhan, V.D., Vedasiromani, J.R., and Ganguly, D.K. (1994) A new sulfated polysaccharide with potent blood anticoagulant activity from the red seaweed Grateloupia indica. Int. J. Biol. Macromol., 16, 279280.[CrossRef][ISI][Medline]
Streusand, V.J., Björk, I., Gettins, P.G.W., Petitou, M., and Olson, S.T. (1995) Mechanism of acceleration of antithrombin-proteinase reactions by low affinity heparin: role of the antithrombin binding pentasaccharide in heparin rate enhancement. J. Biol. Chem., 270, 90439051.
Thunberg, L., Backström, G., and Lindahl, U. (1982) Further characterization of the antithrombin-binding sequence in heparin. Carbohydr. Res., 100, 393410.[CrossRef][ISI][Medline]
Vilela-Silva, A.C.E.S., Alves, A.P., Valente, A.P., Vacquier, V.D., and Mourão, P.A.S. (1999) Structure of the sulfated -L-fucan from the egg jelly coat of the sea urchin Strongylocentrotus franciscanus: patterns of preferential 2-O- and 4-O-sulfation determine sperm cell recognition. Glycobiology, 9, 927933.
Vilela-Silva, A.C.E.S., Castro, M.O., Valente, A.P., Biermann, C.H., and Mourão, P.A.S. (2002) Sulfated fucans from the egg jellies of the closely related sea urchins Strongylocentrotus droebachiensis and Strongylocentrotus pallidus ensure species-specific fertilization. J. Biol. Chem., 277, 379387.
Visentin, G.P. (1999) Heparin-induced thrombocytopenia: molecular pathogenesis. Thromb. Haemostas., 82, 448456.[ISI][Medline]
Warkentin, T.E. (1999) Heparin-induced thrombocytopenia: a clinicopathologic syndrome. Thromb. Haemostas., 82, 439447.[ISI][Medline]