Wu M , Xu L , Zhao L , Xiao C , Gao N , Luo L , Yang L , Li Z , Chen L , Zhao J .

	Figure 1. FT-IR spectrum of the sulfated fucan from sea cucumber.
	Figure 2. 1H (A,B) and 13C (C) one-dimensional NMR spectra at 500 MHz of the sulfated fucan from H. edulis. The spectra were recorded at 300 K for samples in D2O solution. Chemical shifts are relative to external trimethylsilylpropionic acid at 0 ppm. The residual water has been suppressed by pre-saturation. The anomeric signals assigned by 1H/13C HSQC (see Figure 5) are labeled A–E in the sulfated fucan. Expansion of the 4.9–5.6 ppm region of the 1H spectrum is shown in the inset in (A). The integrals were listed under the anomeric signals (B).
	Figure 3. 1H/1H COSY spectra of the sulfated fucans from H. edulis (A) and L. grisea (B). The spectra were recorded at 300 K for samples in D2O solution. Chemical shifts are relative to external trimethylsilylpropionic acid at 0 ppm. The residual water has been suppressed by pre-saturation. The anomeric signals assigned by 1H/13C HSQC (see Figure 5) are labeled A–E in the sulfated fucans.
	Figure 4. Expansions of the TOCSY (A,B) and ROESY (C,D) spectra of two sulfated fucans from H. edulis and L. grisea (B,D).The TOCSY spectra (A,B)show some cross-peaks used in the assignment of the fucose residue, especially positions bearing sulfate esters. The ROESY spectra (B,D) show ROEs, the sequence-defining A1–B3, B1–C3, C1–D2, D1–A3 and E1–D4. The five fucose residues in the repeating unit are marked A–E as described in the legend of Figure 6.
	Figure 5. 1H/13C HSQC (A,B) and HMBC (C,D) spectra of two sulfated fucans from two sea cucumbers H. edulis (A,C) and L. grisea (B,D). The assignments were based on TOCSY and COSY spectra. The anomeric signals were identified by the characteristic carbon chemical shifts and are marked A–E. The HMBC spectra (C,D) also show the sequence-defining A1–B3, B1–C3, C1–D2, D/D'1–A3 and E1–D4. The five fucose residues in the repeating unit are marked A–E as described in the legends of Figure 6.
	Figure 6. Proposed regular repeating units of sulfated fucan isolated from two sea cucumbers H. edulis (A) and L. grisea (B). The five fucose residues in the repeating unit are marked A–E as described in the legends.
	Figure 7. Inhibitory effects of the sulfated fucans, heparin, low molecular weight heparin (LMWH) and dermatan sulfate (DS) on thrombin mediated by heparin cofactor II. (A) Shows the time course of thrombin inhibition. HCII (~1 μM) was incubated with thrombin (20 NIH/mL) in the presence of 30 μL (625 ng/mL) samples at 37 °C. After 2 min, 30 μL of 4.5 mM CS-01 (38) was added, the residual thrombin activity was recorded by absorbance at 405 nm; (B) Shows the dependence on the sulfated polysaccharide concentration for thrombin inactivation in the presence of HCII. The reaction mixtures were as described in (A), except that different concentrations of sulfated polysaccharides were used. Results are shown as means of duplicates. See Table 4 for IC50 values.
	Figure 8. Inhibitory effects of the sulfated fucans, heparin, LMWH and DS on thrombin in the presence of antithrombin. (A) Shows the time course of thrombin inhibition. Mixed samples of 30 μL of polysaccharides (625 ng/mL) and 30 μL of 0.25 IU/mL AT were incubated at 37 °C for 2 min, and 30 μL of 24 NIH/mL IIa was then added. After incubation for 2 min, 30 μL of 1.25 mM CS-01 (38) was added, the residual factor IIa activity was recorded by absorbance at 405 nm; (B) Shows the dependence on the sulfated polysaccharide concentration for thrombin inactivation mediated by AT. The reaction mixtures were as described in (A), except that different concentrations of sulfated polysaccharides were used. Results are shown as means of duplicates. See Table 4 for IC50 values.
	Figure 9. Inhibitory effects of the sulfated fucans, heparin, LMWH and DS on factor Xa in the presence of antithrombin. (A) Shows the time course of Xa inhibition. Mixed samples of 30 μL of polysaccharides (625 ng/mL) and 30 μL of 1 IU/mL AT were incubated at 37 °C for 2 min, and 30 μL of 8 μg/mL bovine Xa was then added. After incubation for 1 min, 30 μL of 1.20 mM CS-11(65) was added, the residual Xa activity was recorded by absorbance at 405 nm; (B) Shows the dependence on the sulfated polysaccharide concentration for Xa inactivation in the presence of AT. The reaction mixtures were as described in (A), except that different concentrations of sulfated polysaccharides were used. Results are shown as means of duplicates. See Table 4 for IC50 values.
	Figure 10. Profile of the platelet aggregation induced by the sea cucumber polysaccharides: the sulfated fucan from L. grisea (A); the sulfated fucan and fucosylated glycosaminoglycan from H. edulis (B). The profile showed that the sulfated fucans from sea cucumber do not cause platelets to aggregate at several concentrations.

BORN, THE AGGREGATION OF BLOOD PLATELETS. 1963, Pubmed

BORN, THE AGGREGATION OF BLOOD PLATELETS. 1963, Pubmed
BORN, Aggregation of blood platelets by adenosine diphosphate and its reversal. 1962, Pubmed
Bordbar, High-value components and bioactives from sea cucumbers for functional foods--a review. 2011, Pubmed , Echinobase
Chen, Sequence determination and anticoagulant and antithrombotic activities of a novel sulfated fucan isolated from the sea cucumber Isostichopus badionotus. 2012, Pubmed , Echinobase
Ciucanu, Elimination of oxidative degradation during the per-O-methylation of carbohydrates. 2003, Pubmed
Dietrich, Structure of heparan sulfate: identification of variable and constant oligosaccharide domains in eight heparan sulfates of different origins. 1998, Pubmed
Duus, Carbohydrate structural determination by NMR spectroscopy: modern methods and limitations. 2000, Pubmed
Gao, Preparation and characterization of O-acylated fucosylated chondroitin sulfate from sea cucumber. 2012, Pubmed , Echinobase
Holtkamp, Fucoidans and fucoidanases--focus on techniques for molecular structure elucidation and modification of marine polysaccharides. 2009, Pubmed
Jackson, The growing complexity of platelet aggregation. 2007, Pubmed
Kariya, Isolation and partial characterization of fucan sulfates from the body wall of sea cucumber Stichopus japonicus and their ability to inhibit osteoclastogenesis. 2004, Pubmed , Echinobase
Li, Mechanism of rabbit platelet agglutination induced by acidic mucopolysaccharide extracted from Stichopus japonicus Selenka. 1988, Pubmed , Echinobase
Lian, Anti-HIV-1 activity and structure-activity-relationship study of a fucosylated glycosaminoglycan from an echinoderm by targeting the conserved CD4 induced epitope. 2013, Pubmed
Luo, Comparison of physicochemical characteristics and anticoagulant activities of polysaccharides from three sea cucumbers. 2013, Pubmed , Echinobase
Mourão, Structure and anticoagulant activity of a fucosylated chondroitin sulfate from echinoderm. Sulfated fucose branches on the polysaccharide account for its high anticoagulant action. 1996, Pubmed , Echinobase
Mourão, Use of sulfated fucans as anticoagulant and antithrombotic agents: future perspectives. 2004, Pubmed
Mourão, Searching for alternatives to heparin: sulfated fucans from marine invertebrates. 1999, Pubmed
Mulloy, Sulfated fucans from echinoderms have a regular tetrasaccharide repeating unit defined by specific patterns of sulfation at the 0-2 and 0-4 positions. 1994, Pubmed , Echinobase
Mulloy, Structure/function studies of anticoagulant sulphated polysaccharides using NMR. 2000, Pubmed , Echinobase
Nagase, Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions: antithrombin III- and heparin cofactor II-independent inhibition of factor X activation by factor IXa-factor VIIIa complex and heparin cofactor II-dependent inhibition of thrombin. 1995, Pubmed , Echinobase
Patankar, A revised structure for fucoidan may explain some of its biological activities. 1993, Pubmed
Pereira, Is there a correlation between structure and anticoagulant action of sulfated galactans and sulfated fucans? 2002, Pubmed
Pomin, Selective cleavage and anticoagulant activity of a sulfated fucan: stereospecific removal of a 2-sulfate ester from the polysaccharide by mild acid hydrolysis, preparation of oligosaccharides, and heparin cofactor II-dependent anticoagulant activity. 2005, Pubmed , Echinobase
Ribeiro, A sulfated alpha-L-fucan from sea cucumber. 1994, Pubmed , Echinobase
Rocha, Structural and hemostatic activities of a sulfated galactofucan from the brown alga Spatoglossum schroederi. An ideal antithrombotic agent? 2005, Pubmed
Sheehan, Depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic tenase complex by a common antithrombin-independent mechanism. 2006, Pubmed
Streiff, Venous thromboembolic disease. 2011, Pubmed
Tsukamoto, Determination of the molecular mass of new L-fucose-containing glycosaminoglycan and its distribution by high-performance gel-permeation chromatography with laser light-scattering detection. 2001, Pubmed
Ustyuzhanina, Influence of fucoidans on hemostatic system. 2013, Pubmed
Vieira, Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues. 1991, Pubmed , Echinobase
Vilela-Silva, Sulfated fucans from the egg jellies of the closely related sea urchins Strongylocentrotus droebachiensis and Strongylocentrotus pallidus ensure species-specific fertilization. 2002, Pubmed , Echinobase
Volpi, Low molecular weight heparins (5 kDa) and oligoheparins (2 kDa) produced by gel permeation enrichment or radical process: comparison of structures and physicochemical and biological properties. 1992, Pubmed
Volpi, Dermatan sulfate from beef mucosa: structure, physicochemical and biological properties of fractions prepared by chemical depolymerization and anion-exchange chromatography. 1994, Pubmed
Warkentin, Heparin-induced thrombocytopenia in patients treated with low-molecular-weight heparin or unfractionated heparin. 1995, Pubmed
Wu, Anticoagulant and antithrombotic evaluation of native fucosylated chondroitin sulfates and their derivatives as selective inhibitors of intrinsic factor Xase. 2015, Pubmed
Yang, Hyperbranched acidic polysaccharide from green tea. 2010, Pubmed
Zhu, Structural identification of (1→6)-α-d-glucan, a key responsible for the health benefits of longan, and evaluation of anticancer activity. 2013, Pubmed
de Azevedo, Heparinoids algal and their anticoagulant, hemorrhagic activities and platelet aggregation. 2009, Pubmed