Title: Structural and Hemostatic Activities of a Sulfated Galactofucan from the Brown Alga Spatoglossum schroederi
Abstract: The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.5 kDa and contains fucose, xylose, galactose, and sulfate in a molar ratio of 1.0:0.5:2.0:2.0 and contains trace amounts of glucuronic acid. Chemical analyses, methylation studies, and NMR spectroscopy showed that the polysaccharide has a unique structure, composed of a central core formed mainly by 4-linked β-galactose units, partially sulfated at the 3-O position. Approximately 25% of these units contain branches of oligosaccharides (mostly tetrasaccharides) composed of 3-sulfated, 4-linked α-fucose and one or two nonsulfated, 4-linked β-xylose units at the reducing and nonreducing end, respectively. This sulfated galactofucan showed no anticoagulant activity on several "in vitro" assays. Nevertheless, it had a potent antithrombotic activity on an animal model of experimental venous thrombosis. This effect is time-dependent, reaching the maximum 8 h after its administration compared with the more transient action of heparin. The effect was not observed with the desulfated molecule. Furthermore, the sulfated galactofucan was 2-fold more potent than heparin in stimulating the synthesis of an antithrombotic heparan sulfate by endothelial cells. Again, this action was also abolished by desulfation of the polysaccharide. Because this sulfated galactofucan has no anticoagulant activity but strongly stimulates the synthesis of heparan sulfate by endothelial cells, we suggested that this last effect may be related to the "in vivo" antithrombotic activity of this polysaccharide. In this case the highly sulfated heparan sulfate produced by the endothelial cells is in fact the antithrombotic agent. Our results suggested that this sulfated galactofucan may have a potential application as an antithrombotic drug. The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.5 kDa and contains fucose, xylose, galactose, and sulfate in a molar ratio of 1.0:0.5:2.0:2.0 and contains trace amounts of glucuronic acid. Chemical analyses, methylation studies, and NMR spectroscopy showed that the polysaccharide has a unique structure, composed of a central core formed mainly by 4-linked β-galactose units, partially sulfated at the 3-O position. Approximately 25% of these units contain branches of oligosaccharides (mostly tetrasaccharides) composed of 3-sulfated, 4-linked α-fucose and one or two nonsulfated, 4-linked β-xylose units at the reducing and nonreducing end, respectively. This sulfated galactofucan showed no anticoagulant activity on several "in vitro" assays. Nevertheless, it had a potent antithrombotic activity on an animal model of experimental venous thrombosis. This effect is time-dependent, reaching the maximum 8 h after its administration compared with the more transient action of heparin. The effect was not observed with the desulfated molecule. Furthermore, the sulfated galactofucan was 2-fold more potent than heparin in stimulating the synthesis of an antithrombotic heparan sulfate by endothelial cells. Again, this action was also abolished by desulfation of the polysaccharide. Because this sulfated galactofucan has no anticoagulant activity but strongly stimulates the synthesis of heparan sulfate by endothelial cells, we suggested that this last effect may be related to the "in vivo" antithrombotic activity of this polysaccharide. In this case the highly sulfated heparan sulfate produced by the endothelial cells is in fact the antithrombotic agent. Our results suggested that this sulfated galactofucan may have a potential application as an antithrombotic drug. The leading causes of death in the United States are diseases that involve heart and blood vessels and, consequently, thrombosis. The incidence of death because of thrombosis is almost two times higher than the next cause, cancer (1National Vital Statistics Reports. 2003; 51: 4-9Google Scholar). Most thromboembolic processes require anticoagulant therapy. This explains the current efforts to develop specific and potent anticoagulant agents.Unfractionated heparins and low molecular weight heparins are the only sulfated polysaccharides currently used as anticoagulant drugs. However, these compounds have several side effects such as bleeding and thrombocytopenia (2Moll S. Roberts H.R. Semin. Hematol. 2002; 39: 145-157Crossref PubMed Scopus (32) Google Scholar, 3Colliec S. Fisher A.M. Tapon-Bretaudier J. Boisson-Vidal C. Durand P. Josefonvicz J. Thromb. Res. 1991; 64: 143-154Abstract Full Text PDF PubMed Scopus (142) Google Scholar). In addition, the commercial sources of heparins are mainly pig and bovine intestine. The possibility that prions and viruses could be carried by these molecules in addition to the increasing needs for antithrombotic therapies indicate the necessity to look for alternative sources of anticoagulant agents.Marine brown algae are an abundant source of anticoagulant polysaccharides. They contain a variety of sulfated l-fucans with anticoagulant activity (4Nader H.B. Lopes C.C. Rocha H.A.O. Santos E.A. Dietrich C.P. Curr. Pharm. Des. 2004; 10: 951-966Crossref PubMed Scopus (94) Google Scholar, 5Dietrich C.P. Farias G.G.M. Abreu L.R.D. Leite E.L. Silva L.F. Nader H.B. Plant Sci. 1995; 108: 143-153Crossref Scopus (56) Google Scholar, 6Leite E.L. Medeiros M.G.L. Rocha H.A.O. Farias G.G.M. Silva L.F. Chavante S.F. Dietrich C.P. Nader H.B. Plant Sci. 1998; 132: 215-228Crossref Scopus (86) Google Scholar, 7Pereira M.S. Mulloy B. Mourão P.A.S. J. Biol. Chem. 1999; 274: 7656-7667Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 8Albuquerque I.R.L. Queiroz K.C.S. Alves L.G. Santos E.A. Leite E.L. Rocha H.A.O. Braz. J. Med. Biol. Res. 2004; 37: 167-171Crossref PubMed Scopus (104) Google Scholar, 9Chevolot L. Foucault A. Chaubet F. Kervarec N. Sinquin C. Fisher A.M. Boisson-Vidal C. Carbohydr. Res. 1999; 319: 154-165Crossref PubMed Scopus (266) Google Scholar, 10Shanmugam M. Mody K.H. Curr. Sci. (Bangalore). 2000; 79: 1672-1683Google Scholar, 11Boisson-Vidal C. Haroun F. Ellouali M. Blondin C. Fischer A.M. Agostini A. Jozefonvicz J. Drugs Future. 1995; 20: 1237-1249Google Scholar). The proposed mechanisms of action of these compounds are predominantly related to the "in vitro" inhibition of factors Xa and IIa mediated by antithrombin and heparin cofactor II. Besides the anticoagulant activity, some sulfated fucans possess other important pharmacological activities such as anticomplementary, anti-inflammatory, antiproliferative, antitumoral, antiviral, antipeptic, and antiadhesive activities (12Haroun-Bouhedja F. Lindenmeyer F. Lu H. Soria C. Jozefonvicz J. Boisson-Vidal C. Anticancer Res. 2002; 22: 2285-2292PubMed Google Scholar, 13Logeart D. Prigent-Richard S. Boisson-Vidal C. Chaubet F. Durand P. Jozefonvicz J. Letourneur D. Eur. J. Cell Biol. 1997; 74: 385-390PubMed Google Scholar, 14Schaeffer D.J. Krylov V.S. Ecotoxicol. Environ. Saf. 2000; 45: 208-227Crossref PubMed Scopus (263) Google Scholar, 15Nagaoka M. Shibata H. Kimura I. Hashimoto S. Kimura K. Makimo T. Aiyama R. Ueyama S. Yokokura T. Glycoconj. J. 1999; 16: 19-26Crossref PubMed Scopus (143) Google Scholar, 16Rocha H.A.O. Franco C.R.C. Trindade E.S. Carvalho L.C.M. Veiga S.S. Leite E.L. Dietrich C.P. Nader H.B. Braz. J. Med. Biol. Res. 2001; 34: 621-626Crossref PubMed Scopus (29) Google Scholar).Most of the structural requirements for the anticoagulant activity of sulfated fucans have not yet been determined; consequently, the structure-activity relationships remain to be elucidated. Most of the difficulties for these studies arise from the fact that these compounds are very heterogeneous polysaccharides, which give complex NMR spectra with broad signals hampering resolution (17Mulloy B. Mourão P.A.S. Gray E. J. Biotechnol. 2000; 77: 123-135Crossref PubMed Scopus (113) Google Scholar). It is not always possible to define whether these algal polysaccharides have repetitive units. Furthermore, the structure of sulfated fucans varies according to the species of algae, as it is the case for heparan sulfates in vertebrates (5Dietrich C.P. Farias G.G.M. Abreu L.R.D. Leite E.L. Silva L.F. Nader H.B. Plant Sci. 1995; 108: 143-153Crossref Scopus (56) Google Scholar, 18Dietrich C.P. Tersariol I.L.S. Toma L. Moraes C.T. Porcionatto M.A. Oliveira F.W. Nader H.B Cell. Mol. Biol. 1998; 44: 417-429PubMed Google Scholar). Thus, each new sulfated polysaccharide purified from a marine alga is a new compound with unique structures and, consequently, with potential novel biological activities.Here we report the purification, structural characterization, and pharmacological activities of a new sulfated polysaccharide from the brown alga Spatoglossum schroederi. This polysaccharide has a unique structure, composed of a central core of 4-linked, partially 3-sulfated β-galactose units. Approximately 25% of these units contain branches of oligosaccharides formed by nonsulfated β-xylose and 3-sulfated α-fucose units linked to the O-2 position of the central core. Of particular significance was the finding that this sulfated galactofucan has no anticoagulant activities but shows a potent antithrombotic activity with no hemorrhagic effect. We attributed the antithrombotic activity of this sulfated polysaccharide to its potent effect stimulating the synthesis of a highly sulfated heparan sulfate by the endothelial cells of the vascular wall.MATERIALS AND METHODSReagents—Chondrointin 4-sulfate was purchased from Miles Laboratories (Elkhart, IN). Heparan sulfate from bovine pancreas and heparin from bovine mucosa were gifts from Dr. P. Bianchini (Opocrin Research Laboratories, Modena, Italy). Propylenediamine (1,3-diaminopropane) was purchased from Aldrich. Glucose, glucuronic acid, xylose, fucose, galactose, and chondroitinase AC and ABC were obtained from Sigma. Heparitinases I and II were prepared from induced Flavobacterium heparinum cells by methods described previously (20Nader H.B. Porcionatto M.A. Tersariol I.L.S. Pinhal M.A.S. Oliveira F.W. Moraes C. Dietrich C.P. J. Biol. Chem. 1990; 265: 16807-16813Abstract Full Text PDF PubMed Google Scholar). Agarose low Mr was purchased from Bio-Rad. Carrier-free [35S]inorganic sulfate was purchased from Instituto de Pesquisas Nucleares (São Paulo, SP Brazil). [3H]Methylthymidine (85 Ci/mmol) was purchased from Amersham Biosciences. Human factor Xa (FXa) 2The abbreviations used are: FXafactor XaHPLChigh pressure liquid chromatographyFCSfetal calf serumNOEnuclear Overhauser effectNOESYnuclear Overhauser effect spectroscopy. was purchased from Roche Applied Science. The synthetic substrate for FXa (benzoyl-Ile-Gly-Arg-p-nitroanilide; S2222) and for thrombin (H-d-Phe-pipecolyl-Arg-p-nitroanilide; S2238) were obtained from Chromogenix AB (Mölndal, Sweden). Antihrombin was prepared as described (19Hoffman D.L. Am. J. Med. 1989; 87 (-S26): S23Abstract Full Text PDF PubMed Scopus (42) Google Scholar).Extraction of Polysaccharides—The marine alga S. schroederi was collected on the seashore of Natal, RN, Brazil. Immediately after collection, the alga was dried at 50 °C under ventilation and was ground in a blender. The seaweed was then treated with acetone to eliminate lipids and pigments. One hundred grams of defatted, dried, and powdered alga were suspended in 500 ml of 0.25 m NaCl, and the pH was adjusted to 8.0 with NaOH. Twenty mg of maxatase, an alkaline protease from Sporobacillus (Biobras, MG, Brazil), was added to the mixture for proteolytic digestion. After 18 h of incubation at 60 °C under agitation, the mixture was filtered through cheesecloth. The filtrate was fractionated by precipitation with acetone as follows: 0.5 volumes of ice-cold acetone was added to the solution under gentle agitation and maintained at 4 °C for 24 h. The precipitate formed was collected by centrifugation (10,000 × g, 20 min), dried under vacuum, resuspended in distilled water, and analyzed. The operation was repeated by adding 0.6, 0.7, 0.9, 1.1, 1.3, and 2.0 volumes of acetone to the supernatant. The fraction precipitated with 0.9 volume of acetone (200 mg) contains the sulfated galactofucan used in the present work. This polysaccharide was further purified by ion exchange chromatography (Lewatite from Bayer, São Paulo, Brazil) eluted stepwise with increasing concentrations of NaCl (0.25-3.0 m). The eluates were precipitated with 2 volumes of methanol (18 h, 4 °C). The precipitates were collected by centrifugation (10,000 × g, 15 min), dried, and resuspended in distilled water for subsequent analysis. The fraction eluted from the resin with 2 m NaCl was further purified by molecular sieving in Sephadex G-75 (120 × 1.8 cm). About 50 mg of sulfated galactofucan, dissolved in 2 ml of water, were applied to the column and eluted with a solution of 0.2 m acetic acid and 6 m urea, and fractions of 1 ml were collected and assayed by the phenol/H2SO4 reaction.Analysis of the Acidic Polysaccharides by Agarose Gel Electrophoresis—Agarose gel electrophoresis of the acidic polysaccharides was performed in 0.6% agarose gels (7.5 × 10 cm, 0.2 cm thick) prepared in four different buffers as follows: 0.05 m 1,3-diaminopropane acetate buffer, pH 9.0; discontinuous buffer 0.04 m barium acetate, pH 4.0; 0.05 m diaminopropane acetate, pH 9.0; 0.05 m KCl-HCl buffer, pH 2.0, or 0.06 m Tris acetate buffer, pH 8.0, as described previously (21Dietrich C.P. McDuffie N.M. Sampaio L.O. J. Chromatogr. 1977; 130: 299-304Crossref PubMed Scopus (82) Google Scholar, 22Bianchini P. Nader H.B. Takahashi H.K. Osima B. Straus A.H. Dietrich C.P. J. Chromatogr. 1980; 196: 455-462Crossref Scopus (50) Google Scholar). Aliquots of the fractions (about 50 μg) were applied to the gel and subjected to electrophoresis. The gel was fixed with 0.1% cetyltrimethylammonium bromide solution for 4 h, dried, and stained for 15 min with 0.1% toluidine blue in 1% acetic acid in 50% ethanol. The gels were then destained with the same solution without the dye. The molecular weight was determined by HPLC in 0.2 m NaCl, 0.5% ethanol, using a GF-250 column (Asahipak GF series, Asahi Chemical Industry Co., Yakoo, Japan). The column was calibrated with standard glycosaminoglycans.Chemical Analyses—The polysaccharides were hydrolyzed with 5 m trifluoroacetic acid. The resulting monosaccharides were converted to their alditol acetate derivatives and analyzed by gas chromatography. Fucose, xylose, and uronic acid content of the polymers were also estimated by the methods described by Dische (23Dische Z. Methods Carbohydr. Chem. 1962; 1: 501-503Google Scholar, 24Dische Z. Methods Carbohydr. Chem. 1962; 1: 484-488Google Scholar, 25Dische Z. Methods Carbohydr. Chem. 1962; 1: 497-501Google Scholar). Total sugars were estimated by the phenol/H2SO4 reaction (26Dubois M. Gilles K.A. Hamilton J.K. Rebers P.A. Smith F. Anal. Chem. 1956; 28: 250-256Crossref Scopus (39948) Google Scholar). After acid hydrolysis of the polysaccharides (6 n HCl, 100 °C, 4 h), the sulfate content was measured by the toluidine blue method, as described previously (27Nader H.B. Dietrich C.P. Anal. Biochem. 1977; 78: 112-118Crossref PubMed Scopus (46) Google Scholar). The type of uronic acid was determined by electrophoresis in Whatman No. 3MM paper in 0.25 m ammonium formate buffer, pH 2.7 (28Kozakai M. Yosizawa Z. Anal. Biochem. 1975; 78: 425-429Google Scholar). The protein content was measured as described by Spector (29Spector J. Anal. Biochem. 1978; 86: 142-143Crossref PubMed Scopus (1354) Google Scholar).Desulfation and Methylation of Fucan—Desulfation of the polysaccharide was performed by solvolysis in dimethyl sulfoxide as used previously for desulfation of a sulfated fucan (30Vilela-Silva E.S.A. Castro M.O. Valente A.P. Biermann H.C. Mourão P.A.S. J. Biol. Chem. 2002; 277: 379-387Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The native and desulfated polysaccharides (10 mg) were subjected to three rounds of methylation, according to Patankar et al. (31Patankar M.S. Oehninger L. Barnett T. Williams R.L. Clark G.F. J. Biol. Chem. 1993; 268: 21770-21776Abstract Full Text PDF PubMed Google Scholar). The methylated polysaccharides were hydrolyzed in 5 m trifluoroacetic acid for 5 h at 100 °C and reduced with borohydride, and the alditol acetates were acetylated with acetic anhydride/pyridine (1:1, by volume) (32Kircher H.W. Anal. Chem. 1960; 32: 1103-1106Crossref Scopus (63) Google Scholar). The alditols acetates of methylated sugars were dissolved in ethanol and analyzed in a gas chromatography/mass spectrometer.NMR Experiments—1H and 13C spectra of the fucan were recorded using a Bruker DRX 600 apparatus with triple resonance probe (30Vilela-Silva E.S.A. Castro M.O. Valente A.P. Biermann H.C. Mourão P.A.S. J. Biol. Chem. 2002; 277: 379-387Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). About 15 mg of each sample was dissolved in 0.7 ml of 99.9% D2O (Cambridge Isotope Laboratory). All spectra were recorded at 60 °C with HOD suppression by presaturation. COSY, TOCSY, and 1H/13C HMQC spectra were recorded using states-times proportion phase incrementation for quadrature detection in the indirect dimension. TOCSY spectra were run with 4096 × 400 points with a spin-lock field of ∼10 kHz and a mixing time of 80 ms. HMQC spectra were run with 1024 × 256 points and globally optimized alternating phase rectangular pulses for decoupling. NOESY spectra were run with a mixing time of 100 ms. Chemical shifts are relative to external trimethysilylpropionic acid at 0 ppm for 1H and to methanol for 13C.Anticoagulant Activity of the Galactofucan—All the coagulation assays (prothrombin time, activated partial thromboplastin time; TT, thrombin time, and HEPTEST®) were performed with a coagulometer, as described earlier (33Dietrich C.P. Paiva J.F. Castro R.A.B. Chavante S.F. Jeske W. Fareed J. Gorin P.A.J. Mendes A. Nader H.B. Biochim. Biophys. Acta. 1999; 1428: 273-283Crossref PubMed Scopus (45) Google Scholar), and were measured by using human plasma from Roche Applied Science. All assays were performed in duplicate and repeated at least three times on different days (n = 6).Antithrombotic Activity—The inhibition of venous thrombosis produced after venae cavae ligature by sulfated polysaccharide was measured by the method of Reyers et al. (34Reyers I. Mussoni A.L. Donati M.B. Gaetano G. Thromb. Res. 1980; 18: 669-674Abstract Full Text PDF PubMed Scopus (120) Google Scholar). Briefly, the method consisted of exposing 1 cm of the inferior venae cavae of rats (below the left renal vein) and performing a ligature with cotton thread (number 8) 5 min after intravenous injection of the test substance. The abdominal cavity was then closed. After 2-24 h, the cavity was reopened, and the eventual thrombi formed were removed from the vein, washed, blotted with filter paper, dried under vacuum for 24 h, and weighed. At specific times, the sulfated polysaccharide was injected endovenously in a volume of 0.2 ml of saline. Ten determinations for each dose were performed. Heparin was used as control. The animal assays were approved by the Ethical Animal Research Committee of the Federal University of São Paulo.Hemorrhagic Effect—Hemorrhagic activity in a rat tail model of the polysaccharides was assayed as described previously (35Dietrich C.P. Shinjo S.K. Moraes F.A. Castro R.A.B. Mendes A. Gouvea T.C. Nader H.B. Semin. Thromb. Hemostasis. 1999; 25: 43-50Crossref PubMed Scopus (44) Google Scholar). Following anesthesia with nembutal (40 mg/kg) and urethane (0.8 g/kg), scarification with a razor blade (1-2 mm deep and 5 mm long) was made 15 mm from the distal part of the rat tail (males, 3 months old). The tail was then immersed in isotonic NaCl, scraped with gauze, and immersed again in fresh saline to observe bleeding. The duration of bleeding of the control ranged from 30 to 60 s. Grazed tails were also immersed in saline solution containing sulfated galactofucan or heparin in different concentrations for 2 min and washed extensively with saline. The treated tails were then immersed in isotonic saline solution, and the amount of blood was measured by protein determination (29Spector J. Anal. Biochem. 1978; 86: 142-143Crossref PubMed Scopus (1354) Google Scholar). The results were expressed as the sum of protein values of each tube minus the amount of blood present before exposure to the test substance.Effect of Polysaccharides on the Synthesis of Heparan Sulfate by the Endothelial Cells—The effect of polysaccharide stimulation of the synthesis (36Nader H.B. Bounassisi V. Colburn P. Dietrich C.P. J. Cell. Physiol. 1989; 140: 305-310Crossref PubMed Scopus (107) Google Scholar) of an antithrombotic heparan sulfate (37Colburn P. Buonassisi V. Biochem. Biophys. Res. Commun. 1982; 104: 220-227Crossref PubMed Scopus (53) Google Scholar) by rabbit aorta endothelial cells (38Buonassisi V. Venter J.C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 1612-1616Crossref PubMed Scopus (214) Google Scholar) was performed essentially as described for heparin and other antithrombotic compounds (39Pinhal M.A.S. Walenga J. Jeske W. Hoppensteadt D. Dietrich C.P. Fareed J. Nader H.B. Thromb. Res. 1994; 74: 143-153Abstract Full Text PDF PubMed Scopus (24) Google Scholar). Briefly, at the end of the incubation, the culture medium was removed, and the cells were washed twice with serum-free F-12 medium. Protein-free heparan sulfate and chondroitin sulfate glycosaminoglycan chains were prepared from the culture medium by incubating the sample with 0.1 mg of SUPERase for 4 h at 60 °C. At the end of the incubation, the mixture was heated for 7 min at 100 °C, and the radiolabeled glycosaminoglycans were precipitated with 2 volumes of methanol in the presence of carrier heparan sulfate. The heparan sulfate and chondroitin sulfate synthesized by these cells and secreted to the medium were quantified and characterized by their electrophoretic mobility in agarose gel and enzymatic degradation with glycosaminoglycan lyases (chondroitinases AC and ABC and heparitinases) as described previously (36Nader H.B. Bounassisi V. Colburn P. Dietrich C.P. J. Cell. Physiol. 1989; 140: 305-310Crossref PubMed Scopus (107) Google Scholar, 39Pinhal M.A.S. Walenga J. Jeske W. Hoppensteadt D. Dietrich C.P. Fareed J. Nader H.B. Thromb. Res. 1994; 74: 143-153Abstract Full Text PDF PubMed Scopus (24) Google Scholar). The radiolabeled compounds were visualized by exposure of the gel after drying and staining to a Kodak blue x-ray film. The radioactive bands were scraped from the gel and counted in a liquid scintillation counter using Ultima Gold (Packard Instrument Co.). Cell protein was estimated by a Coomassie Blue method. All the experiments were performed in triplicate for each data point. The bars of the figures indicate the ± S.E.Effect of Polysaccharide on Cell Growth—Rabbit aorta endothelial cells were grown in F-12 medium (Invitrogen) supplemented with 10% FCS (Cultilab, São Paulo, Brazil), 100 μg/ml streptomycin, and 100 IU/ml penicillin (Sigma) at 37 °C in an atmosphere of 2.5% CO2. Endothelial cells, at 1 × 105 cell/plate, were seeded in 35-mm culture plates. The cells were maintained at G0 phase for 24 h by incubation in F-12 medium without serum. Cells were released from the G0 phase by addition of F-12 medium plus 10% FCS in the absence (control) or presence of 100 μg/ml sulfated galactofucan or heparin. Proliferation was measured by daily cell count (in triplicate) for 9 days. The cell viability was checked by trypan blue exclusion (40Porcionatto M.A. Moreira C.R. Lotfi C.F. Armelin H.A. Dietrich C.P. Nader H.B. J. Cell. Biochem. 1998; 70: 563-572Crossref PubMed Scopus (25) Google Scholar).Thymidine Incorporation—The cell cycle was analyzed by [3H]thymidine incorporation. Quiescent cells were incubated with [3H]thymidine (0.25 μCi/ml) in the absence (control) or the presence of sulfated galactofucan or heparin (100 μg/ml) for various times. The cells were then washed three times with phosphate-buffered saline and harvested with 3.5 m urea in 10 mm Tris-HCl buffer, pH 8.0. The incorporated radioactivity in the cells was determined by scintillation counting as described previously (40Porcionatto M.A. Moreira C.R. Lotfi C.F. Armelin H.A. Dietrich C.P. Nader H.B. J. Cell. Biochem. 1998; 70: 563-572Crossref PubMed Scopus (25) Google Scholar).Assay for Factor Xa Activity—Confluent endothelial cells grown in 100-mm culture plates were incubated for 24 h (37 °C, 2.5% CO2) in F-12 medium without phenol red in the presence or absence of sulfated galactofucan (100 μg/ml). At the end of the incubation, conditioned media were removed, and aliquots (10-100 μl) were assayed for FXa. F-12 medium not exposed to the cells was used as a negative control. The conditioned media were also tested after incubation with heparitinases (50 μl to a final volume of 500 μl in the presence of 0.05 units of heparitinases at 30 °C, pH 7.0, overnight), and aliquots proportional to the original volume were assayed for FXa. These enzymes are free of proteolytic activity (20Nader H.B. Porcionatto M.A. Tersariol I.L.S. Pinhal M.A.S. Oliveira F.W. Moraes C. Dietrich C.P. J. Biol. Chem. 1990; 265: 16807-16813Abstract Full Text PDF PubMed Google Scholar). Briefly, the assay for FXa consisted of preincubating 5 μl of FXa (20 nm) with the different media (10-100 μl) in 96-well plate for 5 min at 37 °C. An aliquot of 5 μl of the synthetic substrate (S2222) (4.0 mm) was added to a final volume of 200 μl and incubated at 37 °C for 3000 s. The activity was continuously monitored by measurement of the absorbance at 405 nm (41Campos I.T.N. Silva M.M. Azzolini S.S. Souza A.F. Sampaio C.A.M. Fritz H. Tanaka A.S. Arch. Biochem. Biophys. 2004; 425: 87-94Crossref PubMed Scopus (16) Google Scholar) by using an enzyme-linked immunosorbent assay reader (Tecan, model Sunrise, Grödig, Austria) and the software Magellan version 5.01 (Tecan, Grödig, Austria). Three different sets of experiments were performed in duplicate for each condition investigated. The results were analyzed by nonlinear regression using GraphPad Prism version 3.0, and each point represents the mean ± S.E. The results are expressed as the ratio of the absorbance for the different experimental conditions relative to the negative control. Statistical analysis was performed using analysis of variance test of p < 0.05 and Student's t test of p < 0.05.RESULTSPurification of a Sulfated Galactofucan—In a previous work we purified a sulfated fucan from the marine alga S. schroederi, designated as "fucan A" (6Leite E.L. Medeiros M.G.L. Rocha H.A.O. Farias G.G.M. Silva L.F. Chavante S.F. Dietrich C.P. Nader H.B. Plant Sci. 1998; 132: 215-228Crossref Scopus (86) Google Scholar). However, we did not succeed in obtaining purified fractions of another sulfated polysaccharide present in high amounts in the extracts from the alga, originally d designated as "fucan B." We now applied a simple methodology for the purification of these polysaccharides, based on precipitation with different concentrations of acetone. The electrophoretic mobility of the various fractions on agarose gel, using diaminopropane/acetate buffer, is shown in Fig. 1A. Although the electrophoretic profiles show the presence of two or even three bands in the various fractions, clearly precipitation with 0.9% (v/v) acetone yields a single spot, whose electrophoretic mobility corresponds to fucan B, as we reported previously (5Dietrich C.P. Farias G.G.M. Abreu L.R.D. Leite E.L. Silva L.F. Nader H.B. Plant Sci. 1995; 108: 143-153Crossref Scopus (56) Google Scholar).The chemical composition of the fractions obtained at various concentrations of acetone is shown in TABLE ONE. The relative proportions of sugars vary among the various fractions. Thus, uronic acid is the main sugar present in the polymers precipitated with 0.5 and 0.6% of acetone, possibly due to the presence of alginic acid. Neutral sugars and sulfate are found in greater amounts in the 0.7-2.0% acetone fractions. It is clear that the relative amounts of these sugars vary according to the fraction. Of course this variation in the sugar composition may be a consequence of different types of polysaccharides found in these fractions, except for the one obtained with 0.9% acetone. This fraction shows a single electrophoretic band on agarose gel, and the sugar composition reported in TABLE ONE possibly indicates the occurrence of a sulfated polysaccharide in S. schroederi, containing galactose, fucose, sulfate, xylose, and traces of uronic acid, which we hereby will designate as sulfated galactofucan. These fractions were not contaminated with laminarans (a group of the reserve β-d-glucans found in brown algae) because glucose was not detected.TABLE ONEChemical composition of acidic polysaccharides from S. schroederi obtained by acetone precipitation The chemical composition of the sulfated galactofucan fraction is indicated by italic type.Fraction (acetone volume)PolysaccharidesMolar ratios to fucoseXyloseUronic acidGalactoseSulfate%aPercentages was determined by phenol-H2SO4 reaction0.538.00.68.01.30.635.20.64.00.31.30.712.20.90.51.51.70.95.80.60.21.63.01.15.80.70.11.32.21.32.60.90.30.72.82.00.30.40.11.12.2a Percentages was determined by phenol-H2SO4 reaction Open table in a new tab We further purified the sulfated galactofucan that is the fraction precipitated with 0.9% acetone, using ion exchange chromatography on a Lewatite resin. The polysaccharide was separated into three new fractions eluted with 1.5, 2.0, and 3.0 m NaCl. The fraction