Structural features and inactivation of coagulation proteases of a sulfated polysaccharidic fraction from Caulerpa cupressoides var . lycopodium ( Caulerpaceae , Chlorophyta )

Studies on biopolymers from macroalgae suggested sulfated polysaccharides (SPs) as research agents to investigate events related to haemostasis. Caulerpa cupressoides var. lycopodium is a marine green alga containing three SPs fractions (SP1, SP2 and SP3). SP2 had anticoagulant (in vitro) and antiand prothrombotic (in vivo) actions; however, its effect on the coagulation system is not fully understood. This study aimed to determine the infrared (IR) spectroscopy, chemical composition (CC), elemental microanalysis (EM), molecular weight (MW) and the effect on coagulation proteases of SP2. The presence of sulfate ester, galactose-6-sulfate, uronic acid and glycoside linkages for IR spectrum; contents of sulfate (28%), total sugars (40%) and uronic acids (7.18%) for CC; and content of carbon (21.98%), sulfate (4.27%), nitrogen (1.3%) and hydrogen (4.86%) for EM were obtained. The average molecular weights of four different SPs (SP-1, SP-2, SP-3 and SP-4) subfractions from the SP2 ranged from ~ 8 to >100 kDa. SP2 was tested on coagulation proteases (thrombin and factor Xa) in the presence of antithrombin (AT) and heparin cofactor II (HCII) using human plasma, being both thrombin and factor Xa target proteases inhibited, but requiring a concentration of about 2.5-fold higher of HCII than the thrombin inactivation by AT.


Introduction
Marine algae have been commonly consumed in the Orient (China, Japan and South Korea) as part of the daily diet, and documented as marine products in traditional medicine (WANG et al., 2008;ZHU et al., 2010).In the last decades, the interest of scientists for natural products extracted from seaweed has significantly increased.These algal products include fatty acid, protein, carbohydrate and sterol (ARAÚJO et al., 2011;MAO et al., 2011;MARINHO-SORIANO et al., 2006;SCHEVCHENKO et al., 2009), and mineral in the form of cations associated with carbohydrates (CAMPO et al., 2009).
Species of the genus Caulerpa of green seaweeds (Bryopsidales) are usually found in water tropical and subtropical marine environments.They are considered invasive and usually resulting in ecological and economical problems.The family Caulerpaceae presents a great diversity of metabolites with chemotaxonomic significance (MAO et al., 2011;RODRIGUES et al., 2012).Polysaccharides from Caulerpaceae consisted mainly of galactose and sulfate, small amount of arabinose, xylose, manose and uronic acid (HAYAKAWA et al., 2000;GHOSH et al., 2004;CHATTOPADHYAY et al., 2007;JI et al., 2008;SCHEVCHENKO et al., 2009).Some biological activities of the Caulerpa SPs have been reported.Hayakawa et al. (2000) revealed that C. okamurai and C. brachypus had SPs that interacted with heparin cofactor II (HCII) to inhibit the thrombin activity.
A hot-water extract containing SPs from C. racemosa with in vitro anticoagulant and antiviral activities was described by Ghosh et al. (2004).In addition, Ji et al. (2008) reported that SPs fractions from C. racemosa had antitumoral properties (in vitro and in vivo) against K562 and H22 cell lines.More recently, it was demonstrated that C. cupressoides var.lycopodium contained three different SPs fractions (SP 1 , SP 2 and SP 3 ), but only SP 2 had anticoagulant activity (in vitro) dependent on its high sulfate content and molecular mass distribution compared to those non-anticoagulant SP 1 and SP 3 (RODRIGUES et al., 2011d).In another study, SP 2 was tested in an assay with coagulation inhibitor using thrombin as the target protease, where an inhibitory effect mediated by antithrombin (AT) was observed (RODRIGUES et al., 2011b).This mechanism of interaction was suggested as being different when compared to other studied Caulerpa SPs (HAYAKAWA et al., 2000).Surprisingly, SP 2 also presented in vivo anti-and prothrombotic effects when assayed in a venous thrombosis model in Wistar rats (RODRIGUES et al., 2011b), similarly to red seaweeds SPs (FONSECA et al., 2008).In another study, Rodrigues et al. (2013) reported that SP 1 was lack of in vitro anti-clotting effect, but able to acts as an in vivo analgesic agent, without modify the locomotor behavior of mice.However, there are few studies concerning the structural and/or biological properties of SPs from Caulerpaceae, especially the effects on coagulation system.
Heparin is a SP member of a family of glycosaminoglycans widely used as anticlotting and antithrombotic drug, consisting of 1,4-linked residues of uronic acid and D-glucosamine.Commercial heparin is obtained from pig and bovine intestine, but at low concentrations.The continuous clinical use of heparin can be accompanied by various risk factors, such as frequent activated partial thromboplastin time monitoring, the inability to inhibit thrombin bound to the clot, hemorrhage and the occurrence of thrombocytopenia in some patients.Possibility of prions and viruses can also occur.It is also employed during extracorporeal circulation (vascular surgery and hemodialysis), exhibiting anticoagulant action mainly through potentiation of AT, a serine protease inhibitor (serpin) which controls the coagulation process, especially thrombin and factor Xa (NADER et al., 2001;QUINSEY et al., 2004).
Once the genus Caulerpa of green seaweeds has been little explored, and few descriptions of structural and biomedical properties of its SPs are still reported, we have now focused the studies with SP 2 fraction (C.cupressoides var.lycopodium) on coagulation proteases.Some molecular features of this fraction were also investigated.

Reagents
Antithrombin, heparin cofactor II, thrombin and factor Xa (Haematologic Technologies Inc., Essex Specimens of the green alga C. cupressoides var.lycopodium (West) C. Agardh (Chlorophyta, Caulerpaceae) was collected on the seashore of Flecheiras Beach on the Atlantic coast of Ceará State, Brazil.The algal samples were taken to the Carbohydrate and Lectins Laboratory (CarboLec), Department of Biochemistry and Molecular Biology, Federal University of Ceará, Brazil, and then cleaned of epiphytes, washed with distilled water and store at -20°C until use.A voucher specimen (4977) was classified and archived by Ana Cecília Fortes Xavier at the Prisco Bezerra Herbarium, Federal University of Ceará.The crude SP was extracted by papain digestion (6h, 60°C), and then separated into three fractions of SPs (SP 1 , SP 2 and SP 3 ) by anion-exchange chromatography on a DEAE-cellulose column, and eluted using a saline gradient (0.5-1 M of NaCl).The purity of SP 2 was checked by agarose gel electrophoresis procedure.The chemical composition of total sugars, sulfate, and contaminant proteins contents of the SP 2 was also determined.These procedures were performed based on Rodrigues et al. (2011d).Chemical analysis of uronic acids content of SP 2 was carried out by carbazole-sulfuric acid method using spectrophotometer (AMERSHAM BIOSCIENCES ULTROSPEC 1100) at 525 nm, using glucuronic acid as standard (DISCHE, 1962).

Infrared (IR) spectroscopy
To investigate the chemical structure, IR spectrum of SP 2 fraction was determined.Fourier transform IR spectrum (FT-IR) was recorded with a SHIMADZU IR spectrophotometer (model 8300) between 4000 and 500 cm -1 .The sample was evaluated as KBr pellet.

Polyacrylamide gel electrophoresis (PAGE)
This procedure was performed to determine the molecular weights of the SP 2 fraction based on Yoon et al. (2007).In this experiment, SP2 (10 μg) was applied to a 6% 1-mm-thick polyacrylamide slab gel in 0.02 M sodium barbital, pH 8.6, and run for 30 min.at 100 V.Then, the gel was stained with 0.1% acetic acid.The molecular masses of the SP 2 were estimated by comparison with the electrophoretic mobility of standard glycosaminoglycans chondroitin-4-sulfate (C-4-S, ~ 40 kDa), chondroitin-6-sulfate (C-6-S, ~ 60 kDa) and dextran sulfate (Dex, ~ 8 kDa).
Inhibition of thrombin or factor Xa by antithrombin (AT) and heparin cofactor II (HCII) in the presence of the C. cupressoides var.lycopodium SP2 fraction These assays were carried out based on Fonseca et al. ( 2008) using incubations in 96-well plates.The final concentrations of reactants included AT (10 nM) or HCII (15 nM), thrombin or factor Xa (both 2 nM) and SP 2 (0-1,000 μg) in 40 μL of TS/PEG buffer (0.02 M Tris/HCl, 0.15 M NaCl, and 1.0 mg mL -1 polyethylene glycol 8,000, pH 7.4).Thrombin or factor Xa was added last to initiate the reaction.After 60 s at 37°C, 25 μL chromogenic substrate (S-2238 for thrombin or S-2222 for factor Xa) was added, and the absorbance at 405 nm was recorded for 120 s.The rate of change of absorbance was proportional to the thrombin or factor Xa activity remaining in the incubation.No inhibition occurred in control experiments in which thrombin or factor Xa was incubated with AT or HCII in the absence of SP 2 .Also, no inhibition was detected when thrombin or factor Xa was incubated with SP 2 alone over the range of concentration tested.

Results and discussion
The green seaweed C. cupressoides var.lycopodium SPs were extracted by papain digestion (6h, 60°C), and then fractionated by ion-exchange chromatography (DEAE-cellulose), resulting into three different SPs fractions (SP 1 , SP 2 and SP 3 eluted at concentrations of 0.5, 0.75 and 1 M of NaCl, respectively).Fraction SP 2 presented highest charge density by agarose gel electrophoresis procedure compared with SP 1 and SP 3 , being corroborated by its highest sulfate (28%) and total sugars (40%) contents.These values were similar to those found by Rodrigues et al. (2011bRodrigues et al. ( , 2011dRodrigues et al. ( , 2013)), but highest compared with those isolated C. racemosa SPs (CHATTOPADHYAY et al., 2007;GHOSH et al., 2004;JI et al., 2008).No contaminant proteins were detected.In addition, only SP 2 has changed the in vitro normal coagulation time (APTT test, data not shown), confirming thus previous investigations (RODRIGUES et al. 2011b and d).From these previous data, it was investigated the structural
Few data have been described for SPs from Caulerpa species (CHATTOPADHYAY et al., 2007;GHOSH et al., 2004;HAYAKAWA et al., 2000;JI et al., 2008;RODRIGUES et al., 2011dRODRIGUES et al., , 2012;;SCHEVCHENKO et al., 2009).In this study, the presence of galactose-6-sulfate (819 cm -1 ) in the chemical structure of SP 2 (C.cupressoides var.lycopodium) is in accordance with SPs found in Caulerpa species (CHATTOPADHYAY et al., 2007;GHOSH et al., 2004).It has been suggested that SPs from Caulerpaceae are preponderantly made up by galactose monosaccharide (HAYAKAWA et al., 2000).Furthermore, the occurrence of arabinogalactan sulfate backbone (1074 cm -1 ) in the IR spectrum of SP 2 could also support the hypothesis of molecular markers (RODRIGUES et al., 2012) based on their structural studies as auxiliary tools in studies on morphology, anatomy and life history of marine algae (USOV, 1998).Pomin and Mourão (2008) reported that the sulfated galactans (glycosidic linkage β(1→3)) are structural sugars highly conserved in some taxonomic groups, including Rhodophyta, Chlorophyta, Angiosperms, Echinoderms and Mollusks.They occur among these phyla differing mainly in sulfation sites, with a tendency towards 4-sulfation in algae and marine angiosperms, 2-sulfation in invertebrates, and 6-sulfation, which is dispersed in minor amounts throughout phylogeny.
Also, the presence of galactose-6-sulfate (a biological precursor to transform in 3,6anhydrogalactose) in the chemical structure of SP 2 (Figure 1) could be of interest for obtaining of commercially important SPs.Mazumder et al. (2002) observed that SPs extracted with water from Gracilaria corticata (Rhodophyta) presented galactose-4-sulfate and galactose-6-sulfate.SPs obtained by alkaline treatment exhibited a signal more intense at 930 cm -1 (characteristic of 3,6-anhydrogalactose).Probably the occurrence of galactose-6-sulfate may be chemically converted to 3,6-anhydrogalactose after alkaline treatment.According to Campo et al. (2009), the alkaline extraction or treatment of red seaweeds SPs has a positive effect on the functional properties of the gel formation as thickening, gelling and stabilizing agents for food application.

Chemical analyses
The content of uronic acid of the SP 2 fraction from the green seaweed C. cupressoides var.lycopodium was 7.18%.This value is similar to those SPs extracted from the green seaweed C. racemosa (4-7.9%)(GHOSH et al., 2004;JI et al., 2008), but exhibited the lowest uronic acid content compared with those SPs from the Chlorophyta Monostroma latissimum (10.77-14.58%)(ZHANG et al., 2008).Uronic acid was also obtained in SPs extracted from the brown seaweed Lobophora variegata by Medeiros et al. (2008), whereas for a sulfated galactan from the marine green seaweed Codium cylindricum it was not detected (MATSUBARA et al., 2001).
In respect to elemental microanalysis, the analytical data for C. cupressoides var.lycopodium SP 2 fraction showed a nitrogen content (1.30%) similar to that of cold extract fraction of SPs (1.22%) from the red seaweed Gracilaria birdiae by Maciel et al. (2008).The presence of nitrogen in the SP 2 fraction could be attributed to amino acids of the proteins (GHOSH et al., 2004;JI et al., 2008) in the chemical structure of the residual polysaccharide of C. cupressoides var.lycopodium, when extracted by papain digestion, confirming perhaps the absorption signal (amide band) previously indentified (Figure 1).Marinho-Soriano et al. (2006) reported that the protein levels in marine macroalgae could be correlated with the nitrogen content.Ji et al. (2008) suggested that amino acids could be related with the bioactivity (antitumor) of C. racemosa SPs, especially aspartic acid, an amino acid that has an important role in cell energy and nitrogen metabolism.Probably, the fraction SP 2 (C.cupressoides) is a polysaccharide-protein complex (proteoglycan), contrasting with a mistaken conclusion reported by Rodrigues et al. (2011d).
The sulfate content for SP 2 (4.27%), when determined by elemental microanalysis, was also about 2-fold higher compared with aqueous SPs extract from G. birdiae (Rhodophyta) (MACIEL et al., 2008), suggesting the hypothesis of the use of enzymes to extract pharmaceutically important materials (ARAÚJO et al., 2012;RODRIGUES et al., 2011a).Fraction SP 2 had carbon and hydrogen contents of 21.98 and 4.86%, respectively.Maciel et al. (2008) found 40% of carbon content in the cold extract fraction in red seaweed G. birdiae.

PAGE
In order to estimate the average molecular size of the C. cupressoides var.lycopodium polysaccharide, the SP 2 fraction was submitted to a PAGE procedure (Figure 2).Interestingly, it was observed that SP 2 fraction contained various polysaccharides (named SP-1, SP-2, SP-3 and SP-4) of different molecular weights.SP-1 and SP-2 remained close to the origin of the gel, indicating polysaccharides of high molecular masses (> 100 and ~ 100 kDa, respectively).In respect to polysaccharides SP-3 and SP-4, the electrophoretic profile revealed a mobility similar to standards chondroitin-4-sulfate (C-4-S, ~ 40 kDa) and dextran sulfate (DexS, ~ 8 kDa), respectively (YOON et al., 2007).
In red seaweeds, SPs constitute the intercellular matrix and nonfibrillar cell walls (POMIN; MOURÃO, 2008;USOV, 1998).In this study, SPs present in SP 2 (C.cupressoides var.lycopodium) presented a dispersive migration due to a heterogeneous molecular weight, typical of these polymers (PUSHPAMALI et al., 2008;YOON et al., 2007).However, fraction SP 2 showed at least four polysaccharides of different molecular weights (Figure 2).This contrasts with the results from the seaweeds Laminaria cichorioides (Phaeophyta) (YOON et al., 2007) and Lomentaria catenata (Rhodophyta) (PUSHPAMALI et al., 2008) where a unique polysaccharide was observed.The revealed characteristics of SP 2 present in C. cupressoides var.lycopodium could perhaps be explained considering the morphology and/or coenocytes tissue structure of this species (RODRIGUES et al., 2012).Rodrigues et al. (2011c) observed SPs extracted from C. cupressoides var.lycopodium occurred as a distinct resolution degree in different portions of its tissue, when analyzed by agarose gel electrophoresis procedure.Probably, the chemical characteristics of these compounds could not be only based on their polyanionic characters, but also in molecular masses (Figure 2).Further investigations should be conducted to better understand the biological role of these compounds in species of the genus Caulerpa.

Inhibitory effects of thrombin or factor Xa activities are mediated by AT and HCII in the presence of SP2 fraction
To initially confirm the anticoagulant mechanism of thrombin (factor IIa) inactivation by AT as previously described by Rodrigues et al. (2011b), SP 2 fraction was measured.As expected, the C. cupressoides var.lycopodium SP 2 fraction interfered with the coagulation system, by inhibiting the thrombin activity mediated by AT (IC 50 ~ 18 μg mL -1 ) against IC 50 ~ 1 μg mL -1 for Acta Scientiarum. Technology Maringá, v. 35, n. 4, p. 611-619, Oct.-Dec., 2013 inactivation by unfractionated heparin.From this previous experiment and owing that each new SP purified from marine alga could be a compound with unique structure and, consequently, forming a particular complex with plasma inhibitor and the target protease (MELO; MOURÃO, 2008;PEREIRA et al., 2005;YOON et al., 2007), it was also performed two more inhibition thrombin (factor IIa) or factor Xa by AT or HCII assays in vitro.In this regard, the Figure 3A shows an inhibition thrombin (factor IIa) mediated by HCII assay.Surprisingly, the fraction SP 2 had ability on thrombin inhibition through HCIIdependent pathway (IC 50 ~ 46 μg mL -1 ) similar to other studied Caulerpa SPs (C.okamurai and C. brachypus) that also exhibited interaction with HCII and its thrombin target protease (HAYAKAWA et al., 2000).The antithrombotic action of algae SPs in animal experimentation (RODRIGUES et al., 2011b) requires the anti-factor Xa activity for a thrombosispreventing effect (FONSECA et al., 2008).In this study, it was also evaluated the influence of the SP 2 fraction from C. cupressoides var.lycopodium on factor Xa inactivation by AT.The inhibitory effect of the factor Xa in the presence of SP 2 by AT was about 24 μg mL -1 (IC 50 ).The same property was also noted for heparin (IC 50 ~ 1.7 μg mL -1 ) (Figure 3B).
These evidences found herein suggested that the interaction of SP 2 (C.cupressoides var.lycopodium) with the coagulation inhibitors could involves specific structural requirements for its anticoagulant action.Matsubara et al. ( 2001) isolated a sulfated galactan from the green alga Codium cylindricum.This polymer had effect on the normal coagulation time by the activated partial thromboplastin time (APTT) and thrombin time (TT) tests using human plasma, except for prothrombin time (TP) test.To elucidate the inhibitory mechanism of the anticoagulant, the authors observed the lack of effect of this sulfated galactan on thrombin and factor Xa in the presence of AT or HCII, seeming that its anticoagulant effect is due to inhibition of fibrin polymerization or the intrinsic pathway without potentiating plasma inhibitors (AT).Pereira et al. ( 2005) compared SPs from the red seaweeds Botryocladia occidentalis and Gelidium crinale using specific proteases and coagulation inhibitors tests.The authors found that the proportions and/or the distribution of 2,3-disulfated α-units in galactan backbone may represent a specific requirement for anticoagulant action.Based on their heterogeneities, distribution of sulfate groups and the monosaccharide composition of SPs may also be depend on for their interactions on coagulation inhibitors and the target proteases.
Our study evidenced that both thrombin and factor Xa target proteases were inhibited by SP 2 (C.cupressoides var.lycopodium), but required a concentration of about 2.5-fold higher of HCII than for thrombin inactivation by AT (Figure 3).The identification of the structural signal corresponding to presence of galactose-6-sulfate in SP 2 fraction (Figure 1) could be useful to the understanding of its structureactivity relationship in further studies.Mestechkina and Shcherbukhin (2010) reported that chemically SPs ((1→4)-β-xylan; amylase, an (1→4)-α-glucan; cellulose, an (1→4)-β-glucan; curdlan, an (1→3)-βglucan; and (1→3)-β-galactan) at the free hydroxyl groups were assayed for anticoagulant activity.The results revealed that the activity could be independent of the anomeric bond configuration, but the position of glycoside bond was important for the activity.In order to elucidate if the location of sulfate groups could be significant for anticoagulant action, the polysaccharides were desulfated, demonstrating thus that the desulfation process specifically at position C-6 in the (1→3)-linked polysaccharide the target activity was lost.When polysaccharides formed by the presence or absence of sulfate at position C-6 in the (1→4) glycoside bond were investigated, they did not exhibit anticoagulant action.
Previous studies also pointed out that the molecular size of SPs could influence their anticoagulant actions (MESTECHKINA; SHCHERBUKHIN, 2010;POMIN, 2012).High molecular weight SPs preparations (216.4-61-9kDa) had an important effect on the anticoagulant activity of the SPs obtained from green alga M. latissimum (ZHANG et al., 2008).Melo and Mourão (2008) reported that a red algal sulfated galactan (B.occidentalis), when fragmented for about 5 kDa, inhibited the venous thrombosis with discrete in vivo anticoagulant and hemorrhagic effects.More recently, a greater specificity of the activity of low molecular weight fucoidan for thrombin-induced platelet aggregation was found in L. japonica (Phaeophyta) by Zhu et al. (2010).Therefore, the fraction SP 2 from C. cupressoides var.lycopodium requires a more in-depth study with basis on the anticoagulant actions.Further investigations are in progress by our group.

Conclusion
A fraction containing sulfated polysaccharides from the green seaweed Caulerpa cupressoides var.lycopodium has chemical molecular features contrasting to other studied algal sulfated polysaccharides.The inactivation of thrombin and factor Xa may be mediated by heparin cofactor II and/or antithrombin.

Figure 1 .
Figure1.The IR spectrum of the SP 2 fraction from the green alga Caulerpa cupressoides var.lycopodium at 4000 and 500 cm -1 in kBr pellets.

Figure 3 .
Figure3.Dependence on the concentration of SP 2 fraction for inactivation of thrombin (factor IIa) (A) or factor Xa (B) by heparin cofactor II (HCII) or AT.HCII or AT (10 nM) was incubated with factor IIa or factor Xa (15 nM) under various concentrations of polysaccharide (0-1000 μg mL -1 , n = 3) from the green alga Caulerpa cupressoides var.lycopodium (SP 2 ) and heparin.Factor IIa or factor Xa activity was determined with a chromogenic substrate (∆A 405 nm min. - ).