Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis

Biochimica et Biophysica Acta 1790 (2009) 1377–1381

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis Hatem Majdoub a,⁎, Mohamed Ben Mansour b, Frédéric Chaubet c, Mohamed S. Roudesli a, Raoui M. Maaroufi b a b c

Laboratoire Polymères, Biopolymères et Matériaux Organiques, Faculté des Sciences, Monastir, 5000 Monastir, Tunisia Laboratoire de Pharmacologie, Faculté de Médecine, Monastir, 5000 Monastir, Tunisia Laboratoire de Bio-ingénierie des Polymères Cardiovasculaires, Institut Galilée, F-93430 Villetaneuse, Université Paris 13, France

a r t i c l e

i n f o

Article history: Received 18 May 2009 Received in revised form 10 July 2009 Accepted 16 July 2009 Available online 23 July 2009 Keywords: Anticoagulant activity Arthrospira platensis Calcium spirulan Heparin cofactor II Thrombin Ultrafiltrated polysaccharide

a b s t r a c t Background: The polysaccharide of culture medium from Arthrospira platensis was extracted by ultrafiltration, partially characterized and assayed for anticoagulant activity. Methods: The crude polysaccharidic fraction was fractionated by anion exchange chromatography on DEAEcellulose, subjected to acetate cellulose electrophoresis and characterized by physicochemical procedures. The anticoagulant effect of the ultrafiltrated polysaccharide was checked by several coagulation tests. Results: Anion exchange chromatography revealed in the whole ultrafiltrated polysaccharidic fraction the occurrence of a sulfated spirulan-like component designated PUF2. The average molecular weight of PUF2 was determined by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) and viscosimetry and was 199 kDa and the sulfate content was 20% weight/dry weight. The physicochemical characterization indicated the occurrence of rhamnose (49.7%), galacturonic and glucuronic acid (32% of total sugar). The anticoagulant effect of this sulfated polysaccharide was mainly due to the potentiation of thrombin inhibition by heparin cofactor II and was 4-times higher than that of the porcine dermatan sulfate whereas it had no effect on anti-Xa activity. Conclusions: An ultrafiltrated sulfated polysaccharide, likely a calcium spirulan was obtained from the culture medium of A. platensis and showed an anticoagulant activity mediated by heparin cofactor II. General significance: Old culture medium of A. platensis may represent an important source for the spirulanlike PUF2 which was endowed with potentially useful anticoagulant properties whereas its obtention by ultrafiltration may represent an extraction procedure of interest. © 2009 Published by Elsevier B.V.

1. Introduction Sulfated polysaccharides include a group of complex macromolecules endowed with various biological activities. These anionic polymers are widespread in nature, occurring in a great variety of organisms such as vertebrates [1,2], invertebrates [3,4] and marine algae [5]. Marine algal sources contain a variety of sulfated polysaccharides such as fucans [6] and sulfated galactans [7–9] with heterogeneous structures and biological properties as well as anticoagulant activities mediated by antithrombin (AT) and/or heparin cofactor II (HCII) [7]. In particular, Calcium spirulan (Ca-Sp) a sulfated polysaccharide with a novel structure was isolated from blue-green alga Spirulina platensis [5]. Ca-Sp consists of rhamnose, 3-O-methylrhamnose (acofriose), 2,3-di-O-methylrhamnose, 3-O-methylxylose, uronic acids, sulfate and calcium ion [10] and includes two types of ⁎ Corresponding author. Tel.: +216 22995799; fax: +216 73500278. E-mail address: [email protected] (H. Majdoub). 0304-4165/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.bbagen.2009.07.013

disaccharide repeating units, O-rhamnosyl-acofriose and O-hexuronosyl-rhamnose (aldobiuronic acid) [11]. It has been reported that this sulfated polysaccharide was endowed with several biological activities such as antiviral activity against enveloped viruses [5,12,13], and acts on cultured human fetal lung fibroblasts [14] and bovine endothelial cells [15] by modulating their metabolism and proliferation, respectively. Ca-Sp also exhibited anticoagulant activity by means of thrombin inhibition through interacting with heparin cofactor II, a physiological inhibitor of this serine-protease, and increased the reaction rate more than 1000-times [16,17]. These heterogeneous activities were probably due to the presence of sulfate groups in varying amounts and different positions along the macromolecular backbone. The clinical use of glycosaminoglycans and particularly heparin is associated with various side effects such as bleeding and heparininduced thrombocytopenia [18] and therefore alternative drugs are sought. The aim of this work was an investigation of a novel source i.e. culture medium for polysaccharide extraction from Arthrospira

1378

H. Majdoub et al. / Biochimica et Biophysica Acta 1790 (2009) 1377–1381

platensis and preliminary studies of the structure and anticoagulant activity of the ultrafiltrated polysaccharide extract. 2. Material and methods 2.1. Materials 30 day old culture medium (pH 12) of A. platensis was purchased from Bioalgal Society (El Alya Tunisia). Unfractionated heparin H 108 (173 UI/mg) (Choay, Sanofi, France), chondroitin sulfate from bovine trachea and hyaluronic acid were from Fluka, Germany, chondroitin sulfate B sodium salt (dermatan sulfate) ∼90%, glucuronic acid and 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Sigma, USA, galactose from Park Scientific Ltd. USA, alcian blue was from Sigma, USA; human heparin cofactor II from Biogenic, human antithrombin and human thrombin (3000 NIH units/mg) were purified respectively according to Bezeaud et al. [19]; chromogenic substrate for thrombin, S2238 (H-D-Phe-Pip-Arg-pNA) was from Kabivitrum, Stockholm, Sweden. Chromogenic substrate CBS (Lys-ProArg-pNA) for factor Xa and bovine factor Xa were purchased from Diagnostica Stago, Asnières, France. Platelet poor plasma (PPP) was prepared from whole blood, drawn on 0.13 M sodium citrate (9:1, v/v), obtained by venipuncture of healthy volunteers. 2.2. Polysaccharide extraction A sample of 10 L of the old culture medium of A. platensis (pH 12) was filtered in vacuum through on sintered glass funnel G2, G3 and G4 successively. The resulting filtrate was purified by ultrafiltration using a Minitan cell equipped with a series of membranes with molecular weight cut off 100 kDa in order to eliminate compounds of low molecular weight and salts. The ultrafiltration was conducted against deionised water (Milli Q process) for about 1 week until the conductivity become as low as that of deionised water [20]. The extensively ultrafiltrated polysaccharide fraction (weight cut off 100 kDa) was lyophilized and called PUF. 2.2.1. Anion exchange chromatography 200 mg polysaccharide was dissolved in 4 mL sodium acetate 0.5 M pH6 and then applied to a DEAE-cellulose column (1 × 15 cm). The column was first eluted by sodium acetate 0.5 M pH6 and then by sodium acetate 0.5 M, NaCl 1 M pH6 at flow rate 10 mL/h. 2.5 mL fractions were collected and assayed by the phenol–sulfuric method for neutral hexoses according to Dubois et al. [21]. The fraction containing neutral hexoses were pooled separately, dialyzed against distilled water, and lyophilized.

column as protection (Shodex SBG, Japan) and two columns connected in series (SB804HQ and SB803 HQ, Shodex, Japan). The polymer was directly dissolved (about 15 g/L) in the 0.1 μm filtered carrier (PBS 0.15 M, pH 7.4) then filtered through 0.45 μm type membrane (Millipore, France). The detections were carried out with TDA Model 301 (refractive index, light scattering and viscosimetry) (Viscotek, Great Britain). Molecular weights were obtained after collecting and processing data with Omnisec 4.0 software (Viscotek, Great Britain). Fourier-transform infrared (FTIR) spectra of KBr pellets of polysaccharides were recorded in a Thermo Nicolet FTIR-spectrometer AVATAR 370 FT-IR, scanning between 4000 and 400 cm− 1. The sugar content was determined by methanolyse and silylation followed by gas chromatography according to M'sakni et al. [24]. Briefly, polysaccharide fractions (0.5–1 mg) were methanolysed in 2 M anhydride acid in methanol (24 h, 80 °C) for measurement of individual sugars, using myoinositol as internal standard. They were sylilated (4 °C overnight in 1% trimethylchlorosilane in N, O-bis (trimethylsilylfluoroacetamide)) and analysed by gas phase chromatography capillary columns DB 225 (J.W. Instruments) with nitrogen as vector gas and air-hydrogen mixture as fuel. 2.4. Anticoagulant activity Activated partial thromboplastin time (aPTT, sec) was performed using Platelin LS reagent (Trinity Biotech PLC, Cowicklow, Ireland) and thrombin time (TT, sec) using bovine thrombin 1.5 NIH/mL (Dade Behring, Marburg, Germany) performed on the STAR analyzer (Diagnostica Stago, Asnières, France), according to the manufacturer's protocol. Coagulation tests were determined on DS samples at various concentrations diluted in a pool of frozen normal plasma. Heparin sodium Choay (Sanofi-Aventis, France) and DS from porcine intestinal mucosa were used as references. 2.5. Inhibition of thrombin by AT and HCII in the presence of sulfated polysaccharide from alga A. platensis Incubations were performed in microtitration plates. The reactants at final concentrations included 50 nM antithrombin or 68 nM heparin cofactor II, 15 nM thrombin and 0–250 μg/mL sulfated polysaccharide in HEPES buffer (10 mM, pH 7.5, containing 150 mM NaCl and 1.0 mg/ mL polyethylene glycol 8000). Thrombin was added at last to initiate the reaction. After 1 min incubation at room temperature, 100 μL of 0.4 mM chromogenic substrate S-2238 in HEPES buffer was added, and the absorbance at 405 nm was recorded for 2 min. The rate of change in absorbance was proportional to the thrombin activity. 2.6. Anti-Xa assay

2.3. Physico-chemical characterization Neutral hexoses were evaluated by the phenyl–sulfuric method according to Dubois et al. [21]. The sulfate content was determined by turbidimetry according to Dodgson and Price using a Quantichrom™ Sulfate Assay Kit [22]. The cellulose acetate electrophoresis was performed as follows. Two microliters of the polysaccharide solution containing about 2 μg of uronic acids was placed at the origin (10 mm from the cathode side) of a cellulose acetate strip (Sartorius). Electrophoresis was carried out in Zn-acetate 0.1 M pH6 buffer and run at 200 V at room temperature, for 1 h. After electrophoresis, the cellulose acetate strip was stained by alcian blue [23]. Molecular weight determination was carried out according to Ben Mansour et al. [2]. Phosphate buffered saline (PBS) (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4 used as carrier, was filtered through 0.45 μm filter unit under vacuum (Millipore), eluted at 0.5 mL/min flow rate (Flom HPLC pump, GPC Max VE2001, Viscotek, Great Britain) and through 0.5 μm filter upstream of columns. The sample was injected in a 100 μL full loop. The size exclusion chromatography line consisted of a guard

The anti-Xa activity of the sulfated polysaccharide was assessed as follows, 100 μL citrated PPP containing or not polysaccharide at concentrations ranging from 0 to 250 μg/mL were mixed with 125 μL chromogenic substrate CBS and 125 μL bovine factor Xa 1.1 UI/mL. The absorbance at 405 nm was recorded and was proportional to the amount of residual Xa. The anti-Xa activity was expressed as units/mg. 3. Results 3.1. Extraction and purification of polysaccharides from alga A. platensis The crude polysaccharide fraction extracted by ultrafiltration (PUF) from old culture medium of A. platensis was subjected to anion exchange chromatography on DEAE-cellulose, where the column-bound polysaccharide was eluted step-wise with NaCl. Two fractions were obtained, the first fraction (PUF1) was eluted with 0.5 M NaCl in sodium acetate buffer pH6 whereas the second (PUF2) was eluted with 1 M NaCl in the same buffer. The presence of

H. Majdoub et al. / Biochimica et Biophysica Acta 1790 (2009) 1377–1381

1379

Table 1 Sugar composition of polysaccharides.

Fig. 1. Acetate cellulose electrophoresis of sulfated polysaccharides stained with alcian blue. (A) Crude polysaccharidic fraction extracted by ultrafiltration (PUF) (track 1), hyaluronic acid (HA) (track 2) and chondroitin sulfate (CS) (track 3). (B) Ultrafiltrated anion exchange chromatography fractions eluted at 1.5 M (PUF2) (track 1) and at 0.5 M (PUF1) (track 2) and HA (track 3). The arrow indicates the direction of the electrophoretic migration.

polysaccharides in the chromatographic fraction was monitored by a total hexose assay according to Dubois et al. [21]. The fractions eluted at different ionic strength values (PUF1 and PUF2) were pooled separately, exhaustively dialyzed to remove salts and then lyophilized. Subsequently, the yield, on weight to weight basis, for PUF2, was 50%. The analysis of these fractions with acetate cellulose electrophoresis in Zn-acetate migration buffer at pH 6 (Fig. 1A) revealed two main bands for the crude polysaccharidic fraction which were separated after ion exchange chromatography. PUF1 migrated less far than hyaluronic acid (HA), whereas PUF2 migrated as chondroitin sulfate (CS) (Fig. 1B). 3.2. Physico-chemical characterization The number (Mn), weight (Mw), viscosity (Mv) average molecular masses and polydispersity index (Ip) of the sulfated fraction (PUF2) were determined by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) and viscosimetry and were respectively 152, 198.7, 165 kDa and 1.3. Infrared spectrum of the fraction PUF2 within the range 4000– 400 cm− 1 displayed the characteristic features of sulfated polysaccharides and showed a strong OH stretch at 3200–3500 cm− 1 and a strong OH bend at 1620–1660 cm− 1 [25] (Fig. 2). Thin and strong absorbance bands at 1013, 1052 and 1147 cm− 1, corresponded to C–O– C, C–OH and C–C ring vibrations in the osidic cycles [26,27]. The

Fractions

PUF2

Exopolysaccharide from Spirulina platensisa

Rhamnose (%) Xylose (%) Mannose (%) Galactose (%) Glucose (%) Galacturonic acid (%) Glucuronic acid (%) Not identified (%)

49.7 5.9 0.9 5.8 4.3 16.9 15.1 1.4

0.3 1.3 Traces 2 2 40 –

The monosaccharide composition of the sulfated polysaccharides was determined as percent weight of fraction dry weight by gas chromatography after methanolyse and silylation according to M'sakni et al. [24]. PUF2: ultrafiltrated sulfated polysaccharide from old culture medium of Arthrospira platensis. a Data from Filali et al. [30].

occurrence of uronic acids was suggested by the strong absorbance band at 1417 cm− 1 (O–CfO bending) [28]. The intensity of the absorbances at 1240 cm− 1 and at 869 cm− 1 were attributed to the bending of SfO and C–O–S groups, respectively and evidenced sulfate residues as substituents [27,29]. The monosaccharide composition of the sulfated polysaccharides was determined by gas chromatography after methanolyse and silylation. The results obtained indicate that the sugar composition qualitatively consists in five neutral sugars: rhamnose, xylose, galactose, glucose and mannose and two uronic acids: glucuronic and galacturonic acids (Table 1). Quantitatively, the major component in the structure of PUF2 was rhamnose with about 49.7% of total sugars. Uronic acids were found to represent 32% of the total sugars in PUF2 (Table 1). Its sulfate content determined by turbidimetry according to Dodgson and Price [22] was 20%. 3.3. Anticoagulant activity The anticoagulant activity of PUF2 was investigated by the classical coagulation assays aPTT and TT using heparin and DS from porcine intestinal mucosa as references. The PUF2 prolonged aPTT and TT in a concentration-dependent manner (Fig. 3). 5-times and 7-times higher concentrations of PUF2 were necessary to obtain the same effect in comparison with heparin in the aPTT (Fig. 3A) and in the TT (Fig. 3B)

Fig. 2. Infrared spectrum of the PUF2. The infrared spectrum %T (transmittance) = f (wavelength number cm− 1) of sulfated polysaccharide fraction. Some characteristic vibrations are depicted by arrows (see text).

1380

H. Majdoub et al. / Biochimica et Biophysica Acta 1790 (2009) 1377–1381

amounts of DS from porcine intestinal mucosa were required to obtain the same prolongation time for aPTT and TT, in comparison with PUF2. 3.4. Thrombin inhibition by heparin cofactor II and antithrombin in the presence of PUF2 Fig. 3 illustrates the thrombin inhibition by heparin cofactor II (HCII) in the presence of the ultrafiltrated sulfated polysaccharide, PUF2, from A. platensis. The PUF2 concentration that yielded 50% thrombin inhibition (IC50) by HCII was 1.7 μg/mL (Table 2). As for the DS from porcine intestinal mucosa, a higher concentration (IC50 = 200 μg/mL) was required to achieve the same effect. The sulfated polysaccharide from A. platensis did not have any effect on thrombin inhibition by antithrombin (AT) (not shown). 3.5. Factor Xa inhibition in poor platelet plasma in the presence of PUF2 Anti-Xa activity remained practically lower than 0.1 IU/mg for the highest PUF2 concentration used (250 μg/mL) and suggested that there was no Xa inhibition (not shown). 4. Discussion The crude polysaccharide extract obtained by ultrafiltration (PUF) from old culture medium of A. platensis, formerly called S. platensis, was fractionated by anion exchange chromatography on DEAEcellulose. The fraction eluted at high ionic strength 1.5 M was designated PUF2 and subjected to acetate cellulose electrophoresis which indicated the occurrence of a negatively charged polysaccharide in this fraction migrating as far as the chondroitin sulfate standard. This was in agreement with data from FTIR spectra which indicated that PUF2 contained sulfate groups in addition to carboxylate groups which contributed to a higher overall negative charge and resulted in a farther migration on acetate cellulose electrophoresis in comparison with PUF1 and even hyaluronic acid. Indeed, the physicochemical characterization of the ultrafiltrated product PUF2 evidenced relatively high sulfate content (20%). It was particularly higher when compared to that of an exopolysaccharide obtained from the culture medium of S. platensis (5%) [30]. In addition, the majors components in the structure of the sulfated polysaccharide extracted from the culture medium of A. platensis, were respectively rhamnose (49.7%) and uronic acids (32% of total sugar), glucuronic acid and galacturonic acid (Table 1). These results were in agreement with those obtained by Hayashi et al. as for abundance of rhamnose residues and occurrence of uronic acids in the sugar composition of PUF2 [5] and with the results of Filali et al. as for the occurrence of galactose, glucose and the abundance of uronic acids (40%) residues in the exopolysaccharide extract from culture medium of S. platensis [30]. The structural differences, in sugar

Fig. 3. Analysis of the anticoagulant activity of PUF2 (■) as measured by (A) the activated partial thromboplastin time (aPTT) and (B) thrombin time (TT). DS from porcine intestinal mucosa (●) and heparin (▲) were used as references. (C) Inactivation of thrombin by HCII in the presence of various concentrations of PUF2 (▲) and DS from porcine intestine (●).The assays were performed with a chromogenic substrate for thrombin as referred to in the section methods. Thrombin was added to DS and HCII. The remaining thrombin activity was determined after 1 min incubation (ΔA405 nm/min).

Table 2 Anticoagulant effect of sulfated polysaccharides. Sources

Polysaccharides

IC50 (μg/mL) thrombin/HC II

Blue-green algae Sea urchins

Ca-Sp from Arthrospira platensis (PUF2)a Sulfated α-galactanb Sulfated α-fucanb Sulfated D-galactan F3c Sulfated polysaccharidesd

1.7 6 N 500 0.23 0.02–0.26

Red algae (B. occidentalis) Green algae

assays, respectively. No inhibition occurred when thrombin was incubated with the sulfated polysaccharide alone over the range of concentrations tested. Using a parallel standard curve based on the aPTT activity of heparin (173 U/mg), the specific anticoagulant activity of the old culture medium extract of A. platensis (PUF2) was estimated as 35 U/mg. Moreover, this sulfated polysaccharide was more effective in prolonging aPTT and TT than the porcine DS. Indeed, 4-times higher

IC50 of sulfated polysaccharides for thrombin inhibition in the presence of heparin cofactor II. IC50 values represent the concentration of sulfated polysaccharides that produced 50% inhibition of thrombin under the conditions of experiments shown in Fig. 2. a This work. b Data from Pereira et al. [8]. c Data from Farias et al. [7]. d Data from Hayakawa et al. [31].

H. Majdoub et al. / Biochimica et Biophysica Acta 1790 (2009) 1377–1381

composition and sulfate content, between the sulfated polysaccharide of the culture medium from A. platensis and S. platensis respectively, in our results and those of Filali et al. might be explained by differences in extraction procedures, sample origins and culture age and conditions. On the other hand, the differences in the sugar composition between the species of green alga can be explained by production of different types of sulfated polysaccharides. These findings indicated that the A. platensis sulfated polysaccharide isolated by ultrafiltration was a spirulan-like substance but with higher sulfate content. It is noteworthy that the ultrafiltrated spirulan-like polysaccharide studied in this work exhibited a remarkable anticoagulant activity higher than that of mammalian dermatan sulfate from porcine intestine considered as a reference. Moreover, it was only 5-fold lower than standard unfractionated heparin in the aPTT and TT assays. In addition, this sulfated polysaccharide enhanced thrombin inhibition by heparin cofactor II with an IC50 much lower than that of mammalian DS. In fact, it was lower in comparison with a variety of sulfated polysaccharides from different origins including sulfated fucan and galactan [8], but was higher than the IC50 of sulfated polysaccharides from green [31] and red alga [7] (Table 2) which were reported to enhance thrombin inhibition by antithrombin and/or heparin cofactor II. In contrast, neither PUF2 nor the mammalian dermatan sulfate stimulated the inhibition of thrombin by AT. In summary, our results indicated that the ultrafiltrated spirulanlike polysaccharide PUF2 from old culture medium had an effective anticoagulant activity based on specific potentiation of thrombin inhibition by heparin cofactor II. Our results also indicate that old culture medium of A. platensis may constitute a cheap and abundant source for the extraction of the sulfated polysaccharide PUF2 which was thus endowed with potentially useful anticoagulant properties – in particular, PUF2 was more effective than dermatan sulfate from porcine origin – whereas its obtention by ultrafiltration may represent an extraction procedure of interest. Acknowledgments We thank Mr. Youssef Krichen from Bioalgae Company (Tunisia) for the raw material of alga. We thank also Isabelle Bertholon for technical help in molar mass characterization. References [1] M.L.S Souza, J.M.M. Dellias, F.R. Melo, L.C.F. Silva, Structural composition and anticoagulant activity of dermatan sulfate from the skin of the electric eel Electrophorus electricus (L), Comp. Biochem. Phys. B 147 (2007) 387–394. [2] M.B. Mansour, H. Majdoub, I. Bataille, et al., Polysaccharides from the skin of ray Raja radula. Partial characterization and anticoagulant activity, Thromb. Res. 123 (2009) 671–678. [3] M.S.G. Pavão, K.R. Aiello, C.C. Werneck, et al., Highly sulfated dermatan sulfates from ascidians structure versus anticoagulant activity of these glycosaminoglycans, J. Biol. Chem. 273 (1998) 27848–27857. [4] J.C. Santos, J.M.F. Mesquita, C.L.R. Belmiro, et al., Isolation and characterization of a heparin with low antithrombin activity from the body of Styela plicata (ChordataTunicata). Distinct effects on venous and arterial models of thrombosis, Thromb. Res. 121 (2007) 213–223. [5] K. Hayashi, T. Hayashi, I. Kojima, A natural sulfated polysaccharide, calcium spirulan, isolated from Spirulina platensis: in vitro and ex vivo evaluation of anti-

[6]

[7] [8] [9]

[10] [11]

[12]

[13] [14]

[15]

[16] [17] [18] [19]

[20]

[21] [22] [23] [24]

[25]

[26] [27] [28]

[29]

[30]

[31]

1381

herpes simplex virus and anti-human immunodeficiency virus activities, AIDS Res. Hum. Retrov. 12 (1996) 1463–1471. M.S. Pereira, B. Mulloy, P.A.S. Mourão, 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 (1999) 7656–7667. W.R.L. Farias, A.P. Valente, M.S. Pereira, P.A.S. Mourão, Structure and anticoagulant activity of sulfated galactans, J. Biol. Chem. 275 (2000) 29299–29307. M.S. Pereira, A.C. Vilela-Silva, A.P. Valente, P.A.S. Mourão, A 2-sulfated, 3-linked-Lgalactan is an anticoagulant polysaccharide, Carbohyd. Res. 337 (2002) 2231–2238. M.S. Pereira, N.M.B. Benevides, M.R.S. Melo, A.P. Valente, F.R. Melo, P.A.S. Mourão, Structure and anticoagulant activity of sulfated galactan from the red alga, Gelidium crinale. Is there a specific structural requirement for the anticoagulant action? Carbohyd. Res. 340 (2005) 2015–2023. J.B. Lee, T. Hayashi, K. Hayashi, et al., Further purification and structural analysis of calcium spirulan from Spirulina platensis, J. Nat. Prod. 61 (1998) 101–104. J.B. Lee, T. Hayashi, K. Hayashi, U. Sankawa, Structural analysis of calcium spirulan (Ca-SP)-derived oligosaccharides using electrospray ionization mass spectrometry, J. Nat. Prod. 63 (2000) 136–138. J.B. Lee, P. Srisomporn, K. Hayashi, T. Tanaka, U. Sankawa, T. Hayashi, Effect of structural modification of calcium spirulan, a sulfated polysaccharide from Spirulina platensis, on antiviral activity, Chem. Pharm. Bull. 49 (2001) 108–110. S. Rechter, T. Konig, S. Auerochs, et al., Antiviral activity of Arthrospira-derived spirulan-like substances, Antivir. Res. 72 (2006) 197–206. Y. Hayakawa, T. Hayashi, K. Hayashi, T. Ozawa, K. Niiya, N. Sakuragawa, Calcium spirulan as an inducer of tissue-type plasminogen activator in human fetal lung fibroblasts, Biochim. Biophys. Acta 1355 (1997) 241–247. T. Kaji, Y. Fujiwara, Y. Inomata, et al., Repair of wounded monolayers of cultured bovine aortic endothelial cells is inhibited by calcium spirulan, a novel sulfated polysaccharide isolated from Spirulina platensis, Life Sci. 70 (2002) 1841–1848. Y. Hayakawa, T. Hayashi, J.B. Lee, T. Ozawa, N. Sakuragawa, Activation of heparin cofactor II by calcium spirulan, J. Biol. Chem. 275 (2000) 11379–11382. Y. Hayakawa, Y. Hirashima, H. Yamamoto, et al., Mechanism of activation of heparin cofactor II by calcium spirulan, Arch. Biochem. Biophys. 416 (2003) 47–52. B.H. Chong, Heparin-induced thrombocytopenia, J. Thromb. Haemost. 1 (2003) 1471–1478. A. Bezeaud, M.H. Denninger, M.C. Guillin, Interaction of human alpha-thrombin and gamma-thrombin with antithrombin III, protein C and thrombomodulin, Eur. J. Biochem. 153 (1985) 491–496. H. Majdoub, M.S. Roudesli, L. Picton, D. Le Cerf, G. Muller, M. Grisel, Prickly pear nopals pectin from Opuntia ficus-indica physico-chemical study in dilute and semi-dilute solutions, Carbohyd. Polym. 46 (2001) 69–79. M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination for sugars and related substances, Anal. Chem. 28 (1956) 350–356. K.S. Dodgson, R.G. Price, A note on the determination of the ester sulphate content of sulphated polysaccharides, Biochem. J. 84 (1962) 106–110. Y. Wegrowski, F.X. Maquart, Cellulose acetate electrophoresis of lycosaminoglycans, Methods Mol. Biol. 171 (2001) 175–179. N.H. M'sakni, H. Majdoub, M.S. Roudesli, et al., Composition, structure and solution properties of polysaccharides extracted from leaves of Mesembryanthenum crystallinum, Eur. Polym. J. 42 (2006) 786–795. S.C. Wang, S.W.A. Bligh, S.S. Shi, et al., Structural features and anti-HIV-1 activity of novel polysaccharides from red algae Grateloupia longifolia and Grateloupia filicina, Int. J. Biol. Macromol. 41 (2007) 369–375. N.P. Camacho, P. West, P.A. Torzilli, R. Mendelsohn, FTIR microscopic imaging of collagen and proteoglycan in bovine cartilage, Biopolymers 62 (2001) 1–8. T. Toida, T. Maruyama, Y. Ogita, et al., Preparation and anticoagulant activity of fully O-sulphonated glycosaminoglycans, Int. J. Biol. Macromol. 26 (1999) 233–241. V. Vicente-Garcia, E. Rios-Leal, G. Calderon-Dominguez, R.O. Canizares-Villanueva, R. Olvera-Ramirez, Detection, isolation and characterisation of exopolysaccharide produced by a strain of Phormidium 94a isolated from an arid zone in Mexico, Biotech. Bioeng. 85 (2004) 306–310. W. Garnjanagoonchorn, L. Wongekalak, A. Engkagul, Determination of chondroitin sulfate from different sources of cartilage, Chem. Eng. and Processing 46 (2006) 465–471. R. Filali Mouhim, J.F. Cornet, T. Fontaine, B. Fournet, G. Dubertret, Production, isolation and preliminary characterization of the exopolysaccharide of the cyanobacterium Spirulina platensis, Biotechnol. Lett. 15 (1993) 567–572. Y. Hayakawa, T. Hayashi, J.B. Lee, et al., Inhibition of thrombin by sulfated polysaccharides isolated from green algae, Biochim. Biophys. Acta 1543 (2000) 86–94.