antithrombotic activity of a fucosylated chondroitin sulphate from echinoderm: sulphated fucose...

Antithrombotic activity of a fucosylated chondroitinsulphate from echinoderm: sulphated fucose brancheson the polysaccharide account for its antithrombotic action

PAUL O A. S. MOURAO,1 MARCO A. M. GUIMARAES,1,2 BARBARA MULLOY,3 ST EPHEN THOMAS3

AND ELAINE GRAY3

1Departamento de Bioquımica, Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro,Brazil, 2Departamento de Medicina Interna, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil, and3National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire, U.K.

Received 7 January 1998; accepted for publication 26 March 1998

Summary. The antithrombotic activity of a fucosylated,chondroitin-sulphate-like polysaccharide extracted from thebody wall of sea cucumber, and of chemically modifiedderivatives of the same polysaccharide, have been assessedusing a stasis thrombosis model in rabbits. Intravenousadministration of the native polysaccharide reduced throm-bosis in a dose-dependent manner and, at a dose of 1·5 mg/kg (60 IU/kg) body weight, completely prevented thrombosisafter 10 min stasis. Removal of the sulphated fucosebranches of the polysaccharide abolished antithromboticeffectiveness.

After intravenous injection of an antithrombotic dose ofradioactively labelled polysaccharide, a correlation wasobserved between removal of radioactivity from the plasma

and decrease in ex vivo APTT values, demonstrating thatantithrombotic effectiveness depends on the level of circulat-ing polysaccharide rather than on an indirect effect of thepolysaccharide on the vascular endothelium.

Reduction of the glucuronic acid carboxyl groups in thepolysaccharide did not affect its in vitro and in vivo activities.Both partial defucosylation and desulphation of the poly-saccharide abolished all its anticoagulant or antithromboticaction.

Keywords: glycosaminoglycans, chondroitin sulphate,sulphated fucans, thrombosis, anticoagulant activity,antithrombotic action.

Unfractionated heparin and low-molecular-weight heparinsare widely used for the prophylaxis and treatment ofthrombotic diseases. These sulphated polysaccharides actby accelerating the inhibitory effects of antithrombin andheparin co-factor II on activated clotting factors (Beguin et al,1988). Other mammalian glycosaminoglycans such asdermatan sulphate have also been studied for their anti-coagulant and antithrombotic actions (Merton & Thomas,1987; Thomas et al, 1990).

There is now more interest in therapeutics prepared fromnon-mammalian sources, thus avoiding the risk of contam-ination with pathogenic agents. Hence, these novel poly-saccharides with potential clinical properties provide anattractive alternative to drugs such as heparin, which is ofbovine or porcine origin. Compounds of this type include

pentosan polysulphate, prepared from beechwood xylan, andfucoidans from algae.

Recently, we isolated novel sulphated polysaccharides fromthe body wall of a sea cucumber (Vieira & Mourao, 1988;Vieira et al, 1991; Ribeiro et al, 1994; Ruggiero et al, 1994;Mourao et al, 1996). The main fraction has a chondroitinsulphate-like structure, containing large numbers ofsulphated a-L-fucopyranose branches linked to position 3of the b-D-glucuronic acid residues (Vieira & Mourao, 1988)(Fig 1). This polysaccharide has high anticoagulant activityand is able to potentiate the inhibitory activities of bothantithrombin and heparin co-factor II (Mourao et al, 1996).

We now report the effect of this fucosylated chondroitinsulphate in prevention of experimental stasis thrombosis inrabbits. Assessments of antithrombotic activities of chemi-cally modified polymers show that the antithromboticactivity of the sea cucumber polysaccharide can be assignedmainly to sulphated fucose branches linked to thechondroitin sulphate core.

British Journal of Haematology, 1998, 101, 647–652

647q 1998 Blackwell Science Ltd

Correspondence: Dr Paulo A. S. Mourao, Departamento deBioquımica, Instituto de Ciencias Biomedicas, Universidade Federaldo Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ 21941-590, Brazil.

MATERIAL AND METHODS

Native and chemically modified polysaccharides. Nativefucosylated chondroitin sulphate was extracted from thesea cucumber Ludwigothurea grisea freshly collected fromGuanabara Bay, Rio de Janeiro. The extraction, preparation,purification and characterization of the native, partiallydefucosylated, desulphated and carboxyl-reduced chondroi-tin sulphate were as described by Mourao et al (1996). Theanticoagulant activity of these samples was measured by theactivated partial thromboplastin time (APTT) (Mourao et al,1996). The specific activity of three batches of nativefucosylated chondroitin sulphate was 23–40 IU/mg. Thecarboxyl-reduced polysaccharide retained the anticoagulantactivity of its parent compound. The partially defucosylatedand desulphated samples had undetectable anticoagulantactivity.

Assessment of antithrombotic properties. Antithromboticactivities were investigated in rabbits using a modifiedWessler model (Wessler et al, 1959; Thomas et al, 1989)with activated human serum as the thrombogenic stimulus.Briefly, New Zealand White rabbits (both sexes, 2–3 kg) wereanaesthetized intravenously via the marginal ear vein withsodium pentobarbitone. Both jugular veins were isolated andthe left carotid artery was cannulated for blood collection.Different doses of native and chemically modified fucosylatedchondroitin sulphate and of unfractionated heparin (82/502NIBSC; specific activity 193 IU/mg) were injected into themarginal ear vein and allowed to circulate for 3 min. Kaolin-activated human serum (1·32 ml/kg body weight) wasinjected over 15 s and 2 cm of the isolated jugular vein

segments were then tied off after a further 15 s. The contentsof the segments were examined after 10 min stasis.Thrombus formation was scored on a scale 0–4 as follows:a score of 0 represented completely fluid blood, 1–3represented progressively larger pieces of fibrin clot, and ascore of 4 represented a complete thrombus cast of thesegment. At least four animals were used per group. Meanthrombus scores were obtained by the average of scores fromeach group. This figure was then expressed as percentage ofthe total possible score (% thrombosis) with 100% represent-ing absence of any prevention of thrombosis. Blood sampleswere collected into 3·8% sodium citrate (9 parts blood:1 partcitrate) before infusion of drug and 3, 15, 30 and 60 minafter administration. Platelet-poor plasma was prepared bycentrifugation of the blood samples at 2000 g for 10 min.The samples were then stored at ¹408C until assay foranticoagulant activity.

Bleeding time. New Zealand White rabbits (2–3 kg) wereimmobilized by intramuscular injection of 0·3 ml of keta-mine hydrochloride. The ears were shaved and a controlbleeding time was carried out on the ear. An interconnectingvenule between the central artery and marginal vein wastransected with a Simplate Bleeding Time Device (OrganonTeknika, Belgium) and the blood adsorbed onto filter paper,carefully avoiding the site of the wound; the time untilbleeding stopped completely was recorded as the bleedingtime. Native and carboxyl-reduced fucosylated chondroitinsulphate was then injected via the marginal ear vein andallowed to circulate for 3 min before the post-injectionbleeding time was carried out on the contralateral ear. Thesites of transection on both ears were matched as closely aspossible. The bleeding time ratio was calculated by compar-ing the control bleeding time with the post-injection bleedingtime. A ratio of 1 or less indicated the injection of the drugdid not prolong the bleeding time.

Activated partial thromboplastin time (APTT). Ex vivo rabbitplatelet-poor plasma (100 ml) was incubated with 100 ml ofkaolin and 100 ml bovine phospholipid reagent (92/512,National Institute for Biological Standards and Control(NIBSC) reference reagent). After 5 min of incubation at378C, 100 ml of 0·25 M CaCl2 were added to the mixture andthe clotting time recorded. The activity was calibratedagainst the 4th International Standard for Unfractionatedheparin (82/502, NIBSC) spiked into pre-treatment rabbitplatelet-poor plasma and expressed as IU/ml.

35S-labelling of fucosylated chondroitin sulphate. The bodywalls of freshly collected sea cucumber were cut into slicesapproximately 1 mm thick. These slices were immersedimmediately in the incubation medium containing 423 mM

NaCl, 9 mM KCl, 23 mM MgCl2, 9 mM KCl and 2 mM NaHCO3

and washed three times with 5 ml of this solution. The slices(5·4 g, wet weight) were incubated at 208C for 24 h with22·2 MBq carrier-free H2

35SO4 in 10 ml of the incubationmedium. The body wall slices were then washed five timeswith ,50 ml of the incubation medium, followed byimmersion in acetone for 24 h at 48C. The samples werethen dried at 608C for 60 min before extraction andpurification as described by Vieira et al (1991). The crudeextract of the 35S-labelled polysaccharide was further

q 1998 Blackwell Science Ltd, British Journal of Haematology 101: 647–652

648 Paulo A. S. Mourao et al

Fig 1. Structure of the fucosylated chondroitin sulphate from seacucumber. The backbone is made up of repeating disaccharide unitsof alternating b-D-glucuronic acid and N-acetyl-b-D-galactosamine,the same structure as mammalian chondroitin sulphate. Some of theb-D-glucuronic acid residues bear fucose branches at the 3-position,or are 3-O-sulphated. In the defucosylated compound R1 is eitherSO3

¹ or OH; in the desulphated compound R2 is OH.

649Antithrombotic Activity of a Fucosylated Chondroitin Sulphate


purified by passage through a DEAE-cellulose column. Thefractions containing the 35S-labelled fucosylated chondroitinsulphate were identified by carbazole (Dische, 1947), meta-chromatic (Farndale et al, 1986) and radioactivity positivetests. The purified 35S-labelled fucosylated chondroitinsulphate (9440 cpm/mg dry weight) was precipitated withabsolute ethanol and dried at 608C for 1 h before use.

Administration of 35S-labelled fucosylated chondroitin sul-phate. New Zealand White rabbits (2–3 kg) were anaesthe-tized with sodium pentobarbital via the marginal ear vein.The left carotid artery was cannulated for blood collectionand the 35S-labelled fucosylated chondroitin sulphate(60 IU/kg body weight, ,28 000 cpm/rabbit) was injectedinto the marginal ear vein. Blood samples were collected into3·8% sodium citrate (9 parts of blood:1 part of citrate) beforeinfusion of the 35S-labelled polysaccharide, and 5, 10, 15,30, 45 and 60 min after administration. Samples (0·5 ml) ofplasma were added to 5 ml of 5% naphthalene, 0·5% PPO,0·01% POPOP solution in 1,4-dioxane and counted in ascintillation counter.

RESULTS

Antithrombotic activity of native and modified fucosylatedchondroitin sulphateIn the control animals the kaolin-activated human serumgave 4þ thrombus scores (100% thrombosis) after 10 min

stasis. The native fucosylated chondroitin sulphate pre-vented stasis thrombosis induced by the serum in a dose-dependent manner (Fig 2) and, at 60 IU/kg (1·5 mg/kg) bodyweight, thrombosis was not observed in any of the animals(n ¼ 5). Unfractionated heparin (Fig 2) was more effectivethan fucosylated chondroitin sulphate and completelyprevented thrombosis on the same experimental model at adose of ,12 IU/kg (0·06 mg/kg) body weight.

When tested at 60 IU/kg (1·5 mg/kg) body weight (theantithrombotic dose for the native fucosylated chondroitinsulphate), the carboxyl-reduced fucosylated chondroitin

Fig 2. Dose dependence for antithrombotic activity of fucosylatedchondroitin sulphate and of unfractionated heparin. Antithromboticactivity was investigated in rabbits according to the Wessler model ofstasis experimental thrombosis (see Methods). Different doses offucosylated chondroitin sulphate (fucCS) (X, W) and of unfractio-nated heparin (A) were injected into the marginal ear vein andallowed to circulate for 3 min. Kaolin-activated human serum(1·32 ml/kg body weight) (X, A) or saline (W) was then injected and,2 cm of the isolated jugular vein segments were tied off. Thecontents of the segments were examined after 10 min stasis. Meanthrombus scores were obtained by the average of scores from eachdose. This figure was then expressed as percentages of the totalpossible score (% of thrombosis) with 100% representing absence ofany prevention of thrombosis.

Fig 3. In vivo anticoagulant effect of native and chemically modifiedfucosylated chondroitin sulphate. (A) Blood samples (,2 ml) werecollected into 3·8% sodium citrate (9 parts blood:1 part citrate)before infusion of polysaccharide, and 3, 15, 30 and 60 min afteradministration of the indicated dose of native fucosylated chon-droitin sulphate and of saline control during the antithromboticassay. APTT was determined on the ex vivo rabbit plasma, asdescribed under Methods. (B) Fucosylated chondroitin sulphate(fucCS) before and after partial defucosylation (fucCS-defuc),desulphation (fucCS-DeSO4) or carboxy-reduction (fucCS-CR)(1·5 mg/kg body weight) and control saline were injected into therabbit marginal vein and blood samples were collected at 3, 15, 30and 60 min. APTT was determined on the ex vivo rabbit plasma.

sulphate was also able to completely prevent stasis throm-bosis. However, the partially defucosylated and the desul-phated fucosylated chondroitin sulphate were totallyineffective at the same dose by weight.

Ex vivo anticoagulant activity was assessed by the APTT.Fig 3A shows clearly that following intravenous injection ofthe native fucosylated chondroitin sulphate, there was adose-dependent increase in clotting time. At 3 min postinjection, the APTT was lengthened 2-fold with 40 IU/kgand 3-fold with 60 IU/kg body weight. The ex vivo APTTvalues for all doses tested returned to control values by60 min. Fig 3B compares the ex-vivo anticoagulant activity ofthe native fucosylated chondroitin sulphate and its threemodified derivatives. The carboxyl-reduced preparation,which has the same antithrombotic efficacy as the nativecompound, gave similar lengthening of APTT, whereas thepartially defucosylated and desulphated preparationsshowed little increase in clotting time.

Circulating levels of 35S-labelled fucosylated chondroitinsulphate following intravenous administrationAfter intravenous injection of the purified 35S-labelledfucosylated chondroitin sulphate at a dose that completelyprevented thrombosis (60 IU/kg, 1·5 mg/kg body weight;,28 000 cpm/rabbit), we observed a parallel increase anddecrease in radioactivity and APTT values in the plasmasamples (Fig 4).

Effect of the native and carboxyl-reduced fucosylatedchondroitin sulphate on bleedingThe haemorrhagic risk of the polysaccharides was assessedby the Simplate bleeding time in rabbits and compared with

that of unfractionated heparin. Infusion of 1 ml of sterileisotonic saline caused a slight shortening of the bleedingtime, giving a bleeding time ratio of 0·81.

Fig 5A shows a dose–response curve for the bleeding timeratio of native fucosylated chondroitin sulphate up to 113IU/kg, about twice the antithrombotic dose. A comparabledose–response curve for heparin is shown in Fig 5B. Thebleeding times ratios increased with increasing doses of thenative fucosylated chondroitin sulphate and at the highestdose the mean bleeding time ratio was 2·07. For heparinat 127 IU/kg the bleeding time ratio was found to be 0·89(Fig 5B).

The mean bleeding time ratio for a single dose of thecarboxyl reduced compound (75 IU/kg) is also shown, forwhich the bleeding time ratio was 1·08, less than the meanpredicted bleeding time for the native compound at thesame dose. When the bleeding time ratios of the nativeand carboxyl-reduced polysaccharides were comparedwith unfractionated heparin using random analysis



Fig 4. Time course of circulating 35S-labelled fucosylated chondroi-tin sulphate. 35S-labelled fucosylated chondroitin sulphate (1·5 mg/kg, 60 IU/kg body weight, ,28 000 cpm/rabbit) was injected intothe marginal ear vein, as described in the legend of Fig 2. Bloodsamples were collected before infusion of the 35S-labelled poly-saccharide and 5, 10, 15, 30, 45 and 60 min after administration.0·5 ml plasma was added to 5 ml of 5% naphthalene, 0·5% PPO,0·01% POPOP solution in 1,4-dioxane and counted in a scintillationcounter (X). For comparison, the data of ex vivo APTT expressed asIU/ml are also shown (W).

Fig 5. (A) Effect of native and carboxyl-reduced fucosylatedchondroitin sulphate on bleeding time. Different doses of nativefucosylated chondroitin sulphate were infused into rabbits andbleeding time measured using a Simplate device. Results areexpressed as ratios of the post- and pre-treatment bleeding time.A ratio of 1·0 or less indicates no prolongation of bleeding time.Bleeding time ratios (X) are plotted as geometric means (6SEM);n ¼ 5. The bleeding time ratio for a single dose (75 IU/kg bodyweight) of carboxyl-reduced fucosylated chondroitin sulphate (B) isalso plotted as geometric mean (6SEM); n ¼ 14. Infusion of saline(W) gave a bleeding time ratio of 0·81 (n ¼ 6). (B) Effect of heparin onbleeding time. Three doses of heparin were infused into rabbits andbleeding times measured as above. Results are expressed as ratios ofthe post- and pre-treatment bleeding time; bleeding time ratios (X)are plotted as geometric means (6SEM); n ¼ 6.

651Antithrombotic Activity of a Fucosylated Chondroitin Sulphate


(Kirkwood & Snape, 1980) on a unitage basis, the native andcarboxyl-reduced fucosylated chondroitin sulphates werefound to be respectively 6 and 2 times more haemorrhagicthan unfractionated heparin. Although the carboxyl-reduced polysaccharide was less haemorrhagic than thenative polysaccharide, the difference was not statisticallysignificant, as the 95% confidence limits predicted byrandom analysis for the two compounds overlapped.

DISCUSSION

Fucosylated chondroitin sulphate prevents experimental venousthrombosisOur previous study (Mourao et al, 1996) showed thatanticoagulant activity of fucosylated chondroitin sulphateand its carboxyl-reduced derivative was due mainly to theability of the polysaccharide to potentiate inhibition ofthrombin by both heparin co-factor II and antithrombin. Inthe present study we have evaluated its antithromboticproperties using the Wessler rabbit model, stimulating theintrinsic coagulation pathway with human activated serum.As shown in Fig 2, fucosylated chondroitin sulphate is aneffective antithrombotic agent, and prevented thrombosis ina dose-dependent manner. Unfractionated heparin wasfound to be approximately 10 times more effective. In asimilar experimental rat model which employed purifiedtissue factor to stimulate the extrinsic pathway, we alsocompared the antithrombotic action of fucosylated chon-droitin sulphate with unfractionated heparin. Fucosylatedchondroitin sulphate completely inhibited thrombus forma-tion at a concentration ,10 times greater than that ofunfractionated heparin (unpublished observations).Although not as effective as unfractionated heparin, thesedata indicate fucosylated chondroitin sulphate is efficaciousin the prevention of venous thrombosis initiated via theintrinsic and extrinsic pathways.

Following intravenous administration of native orcarboxyl-reduced fucosylated chondroitin sulphate, APTTvalues increased rapidly and then decreased slowly tonormal values (Figs 3A and 3B). This effect could beascribed to a rapid increase followed by the removal of thepolysaccharide from circulation, either by enzymatic degra-dation or internalization by other organs or even adsorptionon the vessel wall.

The infusion of 35S-labelled fucosylated chondroitinsulphate showed a good correlation between removal ofradioactivity from the plasma and decrease in ex vivo APTTvalues (Fig 4). This confirms that the decrease in ex vivoanticoagulant activity was due to rapid clearance of thepolysaccharide.

Unpublished experiments where 1·5 mg/kg of fucosylatedchondroitin sulphate was allowed to circulate beforeinduction of stasis thrombosis showed that when theconcentration of the polysaccharide was low, there was noreduction in thrombosis. This further supports the findingthat the antithrombotic effect of fucosylated chondroitinsulphate depends on the presence of the intact polysaccha-ride in the blood and not on modifications induced by thepolysaccharide in the endothelial cells of the vessel wall, as

has been suggested for heparin (Pinhal et al, 1994). Theantithrombotic effect of fucosylated chondroitin sulphatepersists for approximately the same period of time as reportedpreviously for heparin in the same experimental model andusing the same route for administration of the polysacchar-ide (Mauray et al, 1995). The echinoderm polysaccharide,when administered by a different route, may have a longerhalf life than heparin, but this aspect needs to be tested infuture experiments.

The rapid decrease in the plasma concentration offucosylated chondroitin sulphate was interesting. The che-mical structure of this polysaccharide suggests that such acompound is unlikely to be degraded by the enzymatic sys-tems of vertebrates. Fucosylated chondroitin sulphate resistsenzymes which are known to cleave chondroitin sulphatefrom vertebrate tissues (Vieira & Mourao, 1988; Vieira et al,1991). Nevertheless, we cannot exclude degradation of thispolysaccharide by mammalian enzymes or a less specificmechanism for reduction of its molecular weight.

A structural diagram of the fucosylated chondroitinsulphate and its derivatives is shown in Fig 1. Its backboneis like that of mammalian chondroitin, but it is substitutedwith sulphated fucose branches (Vieira & Mourao, 1988;Mourao et al, 1996). Of the three chemically modifiedcompounds studied, two have had these branches removedor modified; desulphation removes sulphate from the fucoseresidues and backbone, and defucosylation removes themajority of the fucose. The defucosylated sample containslittle sulphate, as the chondroitin backbone is not totallysulphated. The chondroitin backbone can be modified byreduction of glucuronic acid residues to glucose, withoutaffecting the branches.

The lack of antithrombotic activity of the desulphated anddefucosylated polysaccharides demonstrates the importanceof the sulphated fucose branches of the fucosylatedchondroitin sulphate for antithrombotic, as well as anti-coagulant, activity. Reduction of the glucuronic acidcarboxyl groups in the fucosylated chondroitin sulphatedid not affect its antithrombotic activity. The in vitroanticoagulant activities of these polysaccharides are directlyresponsible for their in vivo antithrombotic action. This isshown clearly by the striking correlation between in vitroanticoagulant activity, ex vivo anticoagulant activity asmeasured by APTT, and antithrombotic effectiveness ofnative and chemically modified fucosylated chondroitinsulphate.

Assuming the specific activity for the fucosylatedchondroitin sulphate to be 40 IU/mg, the antithromboticdose was 1·5 mg/kg. The antithrombotic action of fucosy-lated chondroitin sulphate is therefore much less effective interms of weight than unfractionated heparin, whichcompletely prevented thrombosis on a similar experimentalmodel at a dose of ,0·06 mg/kg (,12 IU/kg) body weight(Fig 2 and Thomas et al, 1989). The difference in terms of IUis not so marked. Low-molecular-weight heparin preventsonly ,45% of stasis thrombosis at the dose of 0·15 mg/kgbody weight (Thomas et al, 1989). On a unit basis theantithrombotic dose of the same low-molecular-weightheparin was reported to be around 30 IU/kg body weight.

Comparison of the thrombotic properties of othersulphated polysaccharides on the basis of units either ofweight or of activity in the APTT assay is difficult, as specificactivities vary greatly between different compounds, andthe APTT activity has not been reported for some sulphatedpolysaccharides. Compared with the fucosylated chondroitinsulphate, other sulphated polysaccharides described pre-viously as antithrombotic agents require higher doses byweight to prevent experimental thrombosis completely.Thus, sulphated fucan from brown seaweeds and dermatansulphate from mammalian tissues required up to 3·2(Mauray et al, 1995) and 2·5 (Thomas et al, 1990) mg/kgbody weight, respectively, to totally abolish stasis throm-bosis in a similar Wessler model in rabbits. Pentosanpolysulphate prevented ,70% of stasis thrombosis at adose of 0·6 mg/kg body weight (Ryn-McKenna et al,1989) and a chemically sulphated b-1,3-glucan completelyinhibited thrombus formation but only at a concentration 20times greater than that of unfractionated heparin (Albanet al, 1995).

Bleeding effects of fucosylated chondroitin sulphateFig 5 shows the bleeding time ratios obtained with nativeand carboxyl-reduced fucosylated chondroitin sulphate, anda saline control. Saline injections consistently gave a slightshortening of the bleeding time, in accordance withpublished data (Barrowcliffe et al, 1988). A linearrelationship between bleeding time ratio and dose offucosylated chondroitin sulphate was found (R2 ¼ 0·89).A single dose of carboxyl-reduced fucosylated chondroitinsulphate was studied, due to sample scarcity, and the meanbleeding time ratio was lower than would be expected for thesame dose of the native compound, though the observationfell short of statistical significance. The decreasedhaemorrhagic effect of fucosylated chondroitin afterreduction of its glucuronic acid carboxyl groups cannot bedue to a lower anticoagulant activity, since the native andcarboxyl-reduced polysaccharides have similar APTTactivities.

Overall, we observed an unequivocal effect of fucosylatedchondroitin sulphate in preventing experimental stasisthrombosis. The biological effect was assigned to sulphatedfucose branches linked to the polysaccharide core. The effectoccurred in the same range of concentration observed forother antithrombotic polysaccharides and was dependent onthe level of circulating polysaccharide.

ACKNOWLEDGMENTS

This work was supported by grants from Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq: FNDCT,PADCT and PRONEX), Financiadora de Estudos e Projetos(FINEP), Fundacao de Amparo a Pesquisa do Estado do Riode Janeiro (FAPERJ) and Mizutani Foundation for Glyco-science (to P.A.S.M.). We are grateful to Adriana A. Eira fortechnical assistance.

REFERENCES

Alban, S., Jeske, W., Welzel, D., Franz, G. & Fareed, J. (1995)Anticoagulant and antithrombotic actions of a semisynthetic b-1,3-glucan sulfate. Thrombosis Research, 78, 201–210.

Barrowcliffe, T.W., Merton, R.E., Gray, E. & Thomas, D.P. (1988)Heparin and bleeding: an association with lipase release.Thrombosis and Haemostasis, 60, 434–436.

Beguin, S., Lindhout, T. & Hemker, H.C. (1988) The mode of action ofheparin in plasma. Thrombosis and Haemostasis, 60, 457–462.

Dische, Z. (1947) A new specific color reaction of hexuronic acids.Journal of Biological Chemistry, 167, 189–198.

Farndale, R.W., Buttle, D.J. & Barret, A.J. (1986) Improvedquantitation and discrimination of sulfated glycosaminoglycansby use of dimethylmethylene blue. Biochimica et Biophysica Acta,883, 173–177.

Kirkwood, T.B. & Snape, T.J. (1980) Biometric principles in clottingand clot lysis assays. Clinical and Laboratory Haematology, 2, 155–167.

Mauray, S., Sternberg, C., Theveniaux, J., Millet, J., Sinquin, C.,Tapon-Bretaudiere, C. & Fischer, A.M. (1995) Venous anti-thrombotic and anticoagulant activities of a fucoidan fraction.Thrombosis and Haemostasis, 74, 1280–1285.

Merton, R.E. & Thomas, D.P. (1987) Experimental studies on therelative effectiveness of dermatan sulfate and heparin asantithrombotic agents. Thrombosis and Haemostasis, 58, 839–842.

Mourao, P.A.S., Pereira, M.S., Pavao, M.S.G., Mulloy, B., Tollefsen,D.M., Mowinckel, M.C. & Abildgaard, U. (1996) Structure andanticoagulant activity of a fucosylated chondroitin sulfate fromechinoderm: sulfated fucose branches on the polysaccharideaccounts for its high anticoagulant action. Journal of BiologicalChemistry, 271, 23973–23984.

Pinhal, M.A., Walenga, J.M., Jeske, W., Hoppensteadt, D., Dietrich,C.P., Fareed, J. & Nader, H.B. (1994) Antithrombotic agentsstimulate the synthesis and modify the sulfation pattern of aheparan sulfate proteoglycan from endothelium cells. ThrombosisResearch, 74, 143–153.

Ribeiro, A.C., Vieira, R.P., Mourao, P.A.S. & Mulloy, B. (1994)A sulfated a-L-fucan from sea cucumber. Carbohydrate Research,255, 225–240.

Ruggiero, J., Vieira, R.P. & Mourao, P.A.S. (1994) Increased calciumaffinity of a fucosylated chondroitin sulfate from sea cucumber.Carbohydrate Research, 256, 275–287.

Ryn-McKenna, J., Gray, E., Weber, E., Ofosu, F.A. & Buchanan, M.R.(1989) Effects of sulfated polysaccharides on inhibition ofthrombus formation initiated by different stimuli. Thrombosis andHaemostasis, 61, 7–9.

Thomas, D.P., Gray, E. & Merton, R.E. (1990) Potentiation of theantithrombotic action of dermatan sulphate by small amounts ofheparin. Thrombosis and Haemostasis, 64, 290–293.

Thomas, D.P., Merton, R.E., Gray, E. & Barrowcliffe, T.W. (1989) Therelative antithrombotic effectiveness of heparin, a low molecularweight heparin, and a pentasaccharide fragment in an animalmodel. Thrombosis and Haemostasis, 61, 204–207.

Vieira, R.P. & Mourao, P.A.S. (1988) Occurrence of a unique fucose-branched chondroitin sulfate in the body wall of a sea cucumber.Journal of Biological Chemistry, 263, 18176–18183.

Vieira, R.P., Mulloy, B. & Mourao, P.A.S. (1991) Structure of a fucose-branched chondroitin sulfate from sea cucumber: evidence for thepresence of 3-O-sulfo-b-D-glucuronosyl residues. Journal ofBiological Chemistry, 266, 13530–13536.

Wessler, S., Reimer, S.M. & Sheps, M.C. (1959) Biologic assay of athrombosis inducing activity in human serum. Journal of AppliedPhysiology, 14, 943–946.



antithrombotic activity of a fucosylated chondroitin sulphate from echinoderm: sulphated fucose...

Documents