SummarySulfated polysaccharides from marine invertebrates have welldefined structures and constitute a reliable class of molecules for structure-activity relationship studies. We tested the effects of two of these polysaccharides, namely a sulfated fucan and a fucosylated chondroitin sulfate, on coagulation, thrombosis and bleeding. The compounds share similar 2,4-disulfated fucose units, which are required for high anticoagulant activity in this class of polymer. These residues occur either as branches in fucosylated chondroitin sulfate or as components of the linear chain in the sulfated fucan. These polysaccharides possess anticoagulant activity but differ significantly in their mechanisms of action. The fucosylated chondroitin sulfate inhibits thrombin by heparin cofactor II, whereas sulfated fucan inhibits thrombin by both antithrombin and heparin cofactor II. In addition, these Keywords Sulfated fucans, fucoidan, anticoagulant activity, antithrombotic activity, heparin, invertebrate polysaccharides also have serpin-independent anticoagulant activities. Fucosylated chondroitin sulfate, but not sulfated fucan, activates factor XII. As a result of the complex anticoagulant mechanism, the invertebrate polysaccharides differ in their effects on experimental thrombosis. For instance, the sulfated fucan inhibits venous thrombosis at lower doses than fucosylated chondroitin sulfate. In contrast, fucosylated chondroitin sulfate is significantly more potent than sulfated fucan in arterial thrombosis. Finally, fucosylated chondroitin sulfate increases bleeding, while sulfated fucan has only a discrete effect. In conclusion, the location of 2,4-disulfated fucose units in the polysaccharide chains dictates the effects on coagulation, thrombosis and bleeding.

We compared sulfated galactans (SGs) from two species of red algae using specific coagulation assays and experimental models of thrombosis. These polysaccharides have an identical saccharide structure and the same size chain, but with slight differences in their sulfation patterns. As a consequence of these differences, the two SGs differ in their anticoagulant and venous antithrombotic activities. SG from G. crinale exhibits procoagulant and prothrombotic effects in low doses (up to 1.0 mg/kg body weight), but in high doses (>1.0 mg/kg) this polysaccharide inhibits both venous and arterial thrombosis in rats and prolongs ex-vivo recalcification time. In contrast, SG from B. occidentalis is a very potent anticoagulant and antithrombotic compound in low doses (up to 0.5 mg/kg body weight), inhibiting venous experimental thrombosis and prolonging ex-vivo recalcification time, but these effects are reverted in high doses. Only at high doses (>1.0 mg/kg) the SG from B. occidentalis inhibits arterial thrombosis. As with heparin, SG from G. crinale does not activate factor XII, while the polysaccharide from B. occidentalis activates factor XII in high concentrations, which could account for its procoagulant effect at high doses on rats. Both SGs do not modify bleeding time in rats. These results indicate that slight differences in the proportions and/or distribution of sulfated residues along the galactan chain may be critical for the interaction between proteases, inhibitors and activators of the coagulation system, resulting in a distinct pattern in anti- and procoagulant activities and in the antithrombotic action.

SummaryFucosylated chondroitin sulfate is a potent anticoagulant polysaccharide extracted from sea cucumber. Its anticoagulant activity is attributed to the presence of sulfated fucose branches. We have shown that intravascular injection of fucosylated chondroitin sulfate inhibits thrombus formation in a venous and an arterial shunt model in rats. Since this compound resists digestion by enzymes that cleave mammalian glycosaminoglycans, we investigated the possibility that fucosylated chondroitin sulfate might be absorbed after oral administration. In fact, after oral administration of fucosylated chondroitin sulfate to rats, we observed a dose-dependent increase in the plasma anticoagulant activity, as assessed by assays for activated partial thromboplastin time (aPTT) and thrombin time (TT) (about 3-and 5-fold, respectively) and by anti-IIa activity. Furthermore, animals receiving daily oral doses of this glycosaminoglycan showeda decrease in thrombus weight on experimental models of venous and arterial shunt thrombosis. This antithrombotic action clearly has a strong relationship with anticoagulant activity. Similar doses of heparin administered orally had no effect on the plasma anticoagulant activity or on the thrombus weight. Finally, we observed that fucosylated chondroitin sulfate given orally to rats did not modify the bleeding time. Overall, our results indicate that fucosylated chondroitin sulfate is absorbed after oral administration and could become a promising oral anticoagulant.

We report the effects of a chemically oversulfated chondroitin sulfate and a naturally fucosylated chondroitin sulfate on the coagulation system. The former has been recently identified as a contaminant of heparin preparations and the latter has been proposed as an alternative anticoagulant. The mechanism of action of these polymers on coagulation is complex and target different components of the coagulation system. They have serpin-independent anticoagulant activity, which preponderates in plasma. They also have serpin-dependent anticoagulant activity but differ significantly in the target coagulation protease and preferential serpin. Their anticoagulant effects differ even more markedly when tested as inhibitors of coagulation proteases using plasma as a source of serpins. It is possible that the difference is due to the high availability of fucosylated chondroitin sulfate whereas oversulfated chondroitin sulfate has strong unspecific binding to plasma protein and low availability for the binding to serpins. When tested using a venous thrombosis experimental model, oversulfated chondroitin sulfate is less potent as an antithrombotic agent than fucosylated chondroitin sulfate. These highly sulfated chondroitin sulfates activate factor XII in in vitro assays, based on kallikrein release. However, only fucosylated chondroitin sulfate induces hypotension when intravenously injected into rats. In conclusion, the complexity of the regulatory mechanisms involved in the action of highly sulfated polysaccharides in coagulation requires their analysis by a combination of in vitro and in vivo assays. Our results are relevant due to the urgent need for new anticoagulant drugs or alternative sources of heparin.

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Increasing reports of bleeding and peri- or post-operative blood dyscrasias in Brazil were possibly associated with the use of heparin from bovine instead of porcine intestine. These two pharmaceutical grade heparins were analysed for potential differences. NMR analyses confirmed that porcine heparin is composed of mainly trisulfated disaccharides -->4-alpha-IdoA2S-1-->4-alpha-GlcNS6S-1-->. Heparin from bovine intestine is also composed of highly 2-sulfated alpha-iduronic acid residues, but the sulfation of the alpha-glucosamine units vary significantly: approximately 50% are 6- and N -disulfated, as in porcine heparin, while approximately 36% are 6-desulfated and approximately 14% N -acetylated. These heparins differ significantly in their effects on coagulation, thrombosis and bleeding. Bovine heparin acts mostly through factor Xa. Compared to porcine heparin on a weight basis, bovine heparin exhibited approximately half of the anticoagulant and antithrombotic effects, but similar effect on bleeding. These two heparins also differ in their protamine neutralisation curves. The doses of heparin from bovine intestine required for effective antithrombotic protection and the production of adverse bleeding effects are closer than those for porcine heparin. This observation may explain the increasing bleeding observed among Brazilian patients. Our results suggest that these two types of heparin are not equivalent drugs.

The anticoagulant and antithrombotic properties of three structurally correlated sea urchin-derived 3-linked sulfated α-glycans and their low molecular-weight derivatives were screened comparatively through various in vitro and in vivo methods. These methods include activated partial thromboplastin time, the inhibitory activity of antithrombin over thrombin and factor Xa, venous antithrombosis, the inhibition of platelet aggregation, the activation of factor XII, and bleeding. While the 2-sulfated fucan from Strongylocentrotus franciscanus was observed to be poorly active in most assays, the 4-sulfated fucan from Lytechinus variegatus, the 2-sulfated galactan from Echinometra lucunter and their derivatives showed multiple effects. All marine compounds showed no capacity to activate factor XII and similar low bleeding tendencies regardless of the dose concentrations used to achieve the highest antithrombotic effect observed. The 2-sulfated galactan showed the best combination of results. Our work improves the background about the structure-function relationship of the marine sulfated glycans in anticoagulation and antithrombosis. Besides confirming the negative effect of the 2-sulfated fucose and the positive effect of the 2-sulfated galactose on anticoagulation in vitro, our results also demonstrate the importance of this set of structural requirements on antithrombosis in vivo, and further support the involvement of high-molecular weight and 4-sulfated fucose in both activities.

SummaryPharmaceutical grade heparins from porcine intestine and bovine lung consist mainly of repeating tri-sulfated units, of the disaccharide →4-α-IdoA2S-1 →4-α-GlcNS6S-1 →. Heparin preparations from bovine intestine, in contrast, are more heterogeneous. Nuclear magnetic resonance (NMR) and disaccharide analysis after heparinase digestions show that heparin from bovine intestine contains α-glucosamine with significant substitutive variations: 64% are 6-O-sulfated and N-sulfated, as in porcine intestinal heparin while 36% are 6-desulfated. Desulfated α-iduronic acid units are contained in slightly lower proportions in bovine than in porcine heparin. NMR data also indicate N-, 3-and 6-trisulfated a-glucosamine (lower proportions) and α-GlcNS-1→4-α-GlcA and α-IdoA2S-1→4-α-GlcNAc (higher amounts) in bovine than in porcine heparin. Porcine and bovine heparins can be fractionated by anion exchange chromatography into three fractions containing different substitutions on the a-glucosamine units. Each individual fraction shows close disaccharide composition and anticoagulant activity, regardless of their origin (bovine or porcine intestine). However, these two heparins differ markedly in the proportions of the three fractions. Interestingly, fractions with the typical he-parin disaccharides of porcine intestine are present in bovine intestinal heparin. These fractions contain high in vitro anticoagulant activity, reduced antithrombotic effect and high bleeding tendency. These observations indicate that the prediction of haemostatic effects of heparin preparations cannot rely exclusively on structural analysis and anticoagulant assays in vitro. Minor structural components may account for variations on in vivo effects. In conclusion, we suggest that pharmaceutical grade bovine intestinal heparin, even after purification procedures, is not an equivalent drug to porcine intestinal heparin.