Extraction , physical-chemical characterization and in vitro inhibitory potential of thrombin generation of crude sulfated polysaccharides from Brazilian tropical seaweeds

The biotechnological value of macroalgae for screening assays of thrombin generation-TG using sulfated polysaccharides-SPs as substitutes to heparin has been poorly explored. Five Brazilian species of macroalgae (Gracilaria birdiae, Acanthophora muscoides, Halymenia sp., Caulerpa cupressoides and C. racemosa) were analyzed and compared for their abundance, physical-chemical characteristics and in vitro anticoagulant assays of activated partial thromboplastin time-APTT, prothrombin time-PT and TG. Papain extraction yielded (p < 0.001) from 0.66±0.03% (C. racemosa) to 41.60±1.10% (Halymenia sp.) of crude SPs varying sulfate (8.41-42.60%) and total sugars (47.80-70.53%). Crude SPs showed difference in mobility and resolution pattern by agarose electrophoresis, while polyacrylamide analysis revealed SPs of > 100 kDa. These procedures, combined with the use of Stains-All, also indicated nonSPs. APTTs ranged from 2.81 (A. muscoides) to 21.30 IU (Halymenia sp.) vs. heparin (193 IU), and were dependent on sulfation of the crude SPs. PT was not altered. With respect to TG assay, crude SPs modified concentration-dependent and independently from molecular mass TG by both intrinsic/extrinsic pathways in 60-fold diluted human plasma, with total intrinsic inactivation using crude SPs from A. muscoides in parallel to heparin (p < 0.05). Thrombosis in vitro is differentially modulated by distinct crude SPs from Brazilian seaweeds.

There is an increasing demand for seaweeds extracellular matrix complex sulfated polysaccharides (SPs) and the main biomaterials of economic value are agar and carrageenans, representing a billionaire market of over US$ 6 billion dollars year (Pereira & Costa-Lotufo, 2012).Regarding their chemical classes, Rhodophyceae and Phaephyceae are the most common sources of sulfated galactans and fucoidan or fucan, respectively (Pomin, 2012;Mourão, 2015), while sulfated heteropolysaccharides frequently occur in Chlorophyceae (Pomin & Mourão, 2008;Rodrigues et al., 2013;Arata et al., 2015).Although revealing a high structural diversity among algal species, these marine glycans of large molecular masses (> 100 kDa), with variable sulfation on their chains, different linkages and high degree of branching, which contribute to their heterogeneity and complexity, have high levels of effectiveness for screening assays of anticoagulation of extracts from seaweeds to the identification of novel SPs with potent inhibitory actions of the coagulation (Athukorala, Jung, Vasanthan, & Jeon, 2006;Pomin, 2012;Mourão, 2015).To obtain seaweeds SPs, enzyme-assisted extraction methods allow high yield and enhanced quality on the physical-chemical and biological properties of these molecules (Athukorala et al., 2006), considering the effort to obtain a commercial product on a large scale (Pereira & Costa-Lotufo, 2012).
With the advance of glycomics, a substantial number of structurally diverse SPs from seaweeds displaying anticoagulant action has been extensively described (Shanmugam, Ramavat, Mody, Oza, & Tewari, 2001;Athukorala et al., 2006;Fonseca et al., 2008;Pomin, 2012;Mourão, 2015;Mansour et al., 2017).Their effects are stereospecifics and complexes and do not display only as a consequence of negative charge density and sulfate content, but also of structural composition, with anticoagulant mechanisms distinct from those revealed for heparin (HEP), a commercial drug obtained from pig and bovine tissues primarily used in anticoagulant therapy (e.g., thromboembolic disorders), as well as in extracorporeal circulation and hemodialysis, but known due to its episodes of extensive bleeding and HEP-induced thrombocytopenia.This functional class of glycosaminoglycan has specific pentasaccharide sequence with high antithrombin affinity, which is not present in other SPs-expressing living organisms to achieve the same effect of HEP (Mourão, 2015).Both the activated partial thromboplastin time (APTT) and the prothrombin time (PT) tests are conventionally employed, respectively, for intrinsic and extrinsic coagulation pathway examination.However, these screening assays have limited value to predict risk of circulatory disorder (e.g., bleeding and thrombosis) and, in the last years, thrombin generation (TG)based coagulation methods have constituted as more representatives of the total clot formation process (Castoldi & Rosing, 2011;Duarte, Ferreira, Rios, Reis, & Carvalho, 2017) to give additional data about plasma alternative anticoagulants (Nishino, Fukuda, Nagumo, Fujihara, & Kaji, 1999;Mourão et al., 2001;Glauser et al., 2009;Zhang et al., 2014;Rodrigues et al., 2016a;2017;Mansour et al., 2017;Salles et al., 2017).
The present study was designed to enzymatically extract and compare the physical-chemical properties of anticoagulant crude SPs derived from five Brazilian macroalgae species (G.birdiae, A. muscoides, Halymenia sp., C. cupressoides and C. racemosa) using APTT and PT-based coagulation models.In addition, crude SPs were analyzed for their in vitro inhibitory effects of TG.

Marine macroalgae samples and analyses of the crude SPs
The Brazilian samples of Rhodophyceae G. birdiae Plastino & Oliveira, A. muscoides (Linnaeus) Bory de Saint-Vicent and Halymenia sp.J. Agardh; and of Chlorophyceae C. cupressoides var.lycopodium C. Agardh and C. racemosa (Forsskal) J. Agardh were collected in September 2011 on seashore from the Flecheiras beach, Trairí, Ceará State, and then they were taken to the Carbohydrates and Lectins Laboratory (CarboLec), Universidade Federal do Ceará, in plastic bags.Voucher of each algal species (# 40781, 46093, 56149, 4977 and 52418, respectively) was also deposited in the Herbarium Prisco Bezerra of the Department of Biological Sciences, Universidade Federal do Ceará, Brazil.After collection, the algal samples were washed with distilled water and cleaned to remove residues and macroscopic epiphytes, followed by dehydration at room temperature (25±0.05ºC)(Rodrigues et al., 2011;Quinderé et al., 2013;Ribeiro et al., 2014).The experimental analyses of the macroalgae crude SPs were performed at Connective Tissue Laboratory, Universidade Federal do Rio de Janeiro (UFRJ), Brazil.
Two grams of each dehydrated algal tissue were cut into small pieces and then incubated with papain (60°C, 24 hours) in 100 mM sodium acetate buffer (pH 5.0) containing cysteine and EDTA (both 5 mM), as previously published elsewhere (Rodrigues et al., 2011;Vanderlei et al., 2011;Rodrigues et al., 2012c;Quinderé et al., 2013).Briefly, the incubation mixture was then filtered through a nylon membrane and the homogenate was saved.The residue was washed with 50 mL distilled water and the SPs in mixture were precipitated with 6.4 mL of 10% cetylpyridinium chloride (CPC) solution.After 24 hours at room temperature, the mixture was centrifuged at 2,560 × g at 5°C for 30 min.The SPs in the pellet were washed with 200 mL of 0.05% CPC solution, dissolved with 100 mL of a 2 M NaCl: Ethanol (100: 15, v v -1 ) mixture and precipitated with 200 mL of absolute ethanol.After 24 hours at 4°C, the precipitate was collected by centrifugation (2,560 × g at 5°C for 30 min), washed twice with 200 mL of 80% ethanol and washed once with 150 mL absolute ethanol.The final precipitate was oven dried at 60°C for 24 hours.After that, the crude SPs extract obtained for each algal species was quantified as a percentage (%) of the dehydrated matter and analyzed for its contents of sulfate (Dogson & Price, 1962), total sugars (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) and proteins (Bradford, 1976).

In vitro anticoagulant assays APTT and PT tests
Crude SPs were assessed by both the in vitro APTT and PT tests, using normal citrated human plasma (10 different donors, University Hospital Clementino Fraga Filho, UFRJ) according to the manufacturers' specifications, for evaluating a possible anti-clotting effect in a coagulometer Amelung KC4A before the in vitro TG assays.For the APTT test, a mixture of 100 μL plasma and different concentrations of SPs (0-1 mg mL -1 ) was incubated with 100 μL APTT reagent (kaolin bovine phospholipid reagent) from Wiener Lab (Rosario, Argentina).After 2 min of incubation at 37°C, 100 μL of 25 mM CaCl 2 from Wiener Lab (Rosario, Argentina) was added to the mixtures, and the clotting time was recorded.Regarding the PT assay, a mixture of 100 μL plasma and various concentrations of SPs (0-1 mg mL -1 ) was incubated at 37°C for 1 min.After that, 100 μL PT reagent from Wiener Lab (Rosario, Argentina) was added to the mixtures, and the clotting time was recorded using the same coagulation equipment.The polysaccharide concentrations and the curve of unfractionated heparin (UHEP) (with 193 international units per mg (IU mg -1 ) from Europharma Lab, São Paulo, Brazil, on both tests were determined as described by Rodrigues et al. (2011).Tests were performed in triplicate.

Statistical analysis
Analyses were normalized and performed using a statistical software program (GraphPad Prism version 5.0.1,USA).Values (mean±S.E.M., n = 3) of the classical coagulation tests and the TG parameters were subjected to Analysis of Variance (One or Two-way ANOVA), followed by Tukey's test for unpaired data, at level of p < 0.05.

Results and discussion
In this study, samples of five different Brazilian seaweed species [G.birdiae, A. muscoides and Halymenia sp.(Rhodophyta); C. cupressoides var.lycopodium and C. racemosa (Chlorophyta)] collected in Flecheiras beach were digested with papain to obtain crude SPs and compare their respective physical-chemical characteristics by electrophoresis.After incubation, followed by both CPC and ethanol precipitations and drying, the percentage of crude SPs recovered from the dehydrated matter varied among the benthic macroalgae species (p < 0.05), as shown in Figure 1A.
The crude SPs yields ranged from 0.66±0.03% in the green seaweed C. racemosa to 41.60±1.10% in the red seaweed Halymenia sp.(Table 1).Depending on the phylum, yields from 13.57±1.72 to 41.60±1.10% in Rhodophyta species were considered higher compared with those of Chlorophyta species (p < 0.001), as a typical biosynthetic profile of these marine organisms (Pomin & Mourão, 2008).
The highest yield in Halymenia sp. was consistent with that of Rodrigues et al. (2012b), who previously demonstrated a high level of crude SPs from this species (46% yield); it was also at least 2.77-fold higher than those obtained by aqueous (18%) and alkaline (15%) conditions containing 43-44% sulfate content from the red seaweed Halymenia durvillei (Fenoradosoa et al., 2009).However, similar yields (p > 0.05) for G. birdiae and A. muscoides (Rhodophyta) were observed; likewise, for samples of A. muscoides collected in the same region by Quinderé et al. (2013), who obtained 11.7% extraction yield (Table 1).G. birdiae presented a yield 3.12-fold higher than that of Vanderlei et al. (2011), while varying levels of yields (0.52-8.26%) and total sugars/sulfate ratio (5.10-15.00)were found by Fidelis et al. (2014), applying proteolysis, NaOH, ultrasound or water under different extraction conditions, to obtain crude SPs from G. birdiae collected in Rio Grande do Norte State, Brazil.
Electrophoretic analyses of the crude SPs extracts on agarose or polyacrylamide gel are illustrated in Figure 1B and C, and all algal extracts revealed SPs with toluidine blue staining.
As shown in Figure 1Ba, agarose analysis showed distinct mobility among the seaweeds crude SPs, as well as in polydispersion and metachromasy.Bands corresponding to crude SPs from G. birdiae and A. muscoides migrated as dermatan sulfate, where that of A. muscoides revealed a single band; therefore, a homogeneous crude SPs extract in charge density than that of Quinderé et al. (2013) and that from G. birdiae (Vanderlei et al., 2011) which showed polydispersion and relatively weaker metachromasy; while that of Halymenia sp. had mobility close to the heparan sulfate, and highly charged (Rodrigues et al., 2012b).Caulerpa species showed polydisperse crude SPs extracts with relatively low charge density, as expected (Rodrigues et al. 2012c).
These data confirmed the differences on the physical-chemical aspects among the macroalgae crude SPs (Figure 1 and Table 1), but some revealed contrasting characteristics compared to previously described results for both Rhodophyta A. muscoides (Quinderé et al., 2013) and Halymenia sp.(Rodrigues et al., 2012b), which showed essentially heterogeneous crude SPs by agarose analysis.As the diamine interacted with the algae crude SPs through their sulfated groups, distinct crude SPs were suggested with basis on their differences in charge/mass ratio (Table 1) and structural conformation, as showed by respective electrophoretic profiles (Figure 1Ba) (Dietrich & Dietrich, 1976).
Our conflicting observations are justified by the influence of climatic conditions on biosynthesis of other SPs, life cycle of the algae, and/or use of various protocols for obtaining these compounds (Pereira & Costa-Lotufo, 2012;Rodrigues et al., 2016b;2017).As presented in Figure 1Ab, sequential staining with toluidine blue and Stains-All led to a improved sensitivity of detection not only SPs, but also non SPs, speculating the presence of carboxylated groups in their chemical structures, as also demonstrated for glycosaminoglycans (Volpi & Maccari, 2002;Salles et al., 2017).
Using the polyacrylamide technique, low molecular masses SPs (C.cupressoides) were also detected (Figure 1Cb and Cc) (Rodrigues et al., 2013), although with low amounts of polysaccharide species in complex mixtures (Volpi & Maccari, 2002).Taking with literature data, previous studies revealed acid polysaccharides in G. birdiae (Fidelis et al., 2014) and in C. cupressoides (Rodrigues et al., 2013;2017); and pyruvate in H. durvillei (Fenoradosoa et al., 2009) and in A. muscoides (Quinderé et al., 2014;Rodrigues et al., 2016b).Our combined findings led to a suggestion that Stains-All could be useful to detect other structural components in the algae extracellular matrix polysaccharides, as well as in analysis of purity of these complex glycans preparations (Volpi & Maccari, 2002;Rodrigues et al., 2017;Salles et al., 2017).
Evaluation of in vitro anticoagulant effect of the macroalgae crude SPs using APTT, PT and TG assays Samples of each macroalgae crude SPs extract were further assessed for their in vitro anticoagulant effects by APTT and PT tests compared with that of 193 IU mg -1 standard UHEP before the in vitro TG assays.As shown in Figure 2, the APTT coagulation test revealed intrinsic pathway inhibition by algae crude SPs extracts (p < 0.05), except for G. birdiae crude SPs extract (32.95±0.31s, p > 0.05) that at a concentration of 1 mg mL -1 did not modify the normal APTT values (33.5±0.08 s).The most extended APTT effects had positively related to the sulfation (Table 1) and were of the order of 21.30 (C.cupressoides) and 15.90 IU mg -1 (Halymenia sp.) (p > 0.05), but 9-to 12-fold lower than that of UHEP, which had a marked increment on intrinsic system already at low concentrations.Using these same macroalgae species, Rodrigues et al. (2011;2012b) recorded effects of 24.62 and 72.66 IU mg -1 from major polysaccharidic fractions, respectively.The tested samples of A. muscoides and C. racemosa showed modest inhibitory effects corresponding to 2.81 and 7.88 IU mg -1 , respectively, as described by Quinderé et al. (2014) and Rodrigues et al. (2016b;2012c), who previously reported reduced anticoagulation induced by their SPs extracted with papain.The serpin-dependent or independent anticoagulation of both C. cupressoides (Rodrigues et al., 2013) and A. muscoides (Quinderé et al., 2014) SPs was previously demonstrated in purified systems.According to Fonseca et al. (2008) and Mourão (2015), slight differences in the proportion and/or distribution of sulfated residues in polysaccharide chains could be critical for the interaction of proteases, inhibitors and activators of the coagulation system, resulting in a distinct pattern of anti-and pro-coagulant effects and in antithrombotic action.Regarding the PT test, treatment of the human plasma with crude SPs extracts (1 mg mL -1 ) did not modify the normal values in the extrinsic coagulation pathway (data not shown - Rodrigues et al., 2011).
An intriguing observation of our study was the lack of in vitro anticoagulant effect by APTT assay of the crude SPs extract from G. birdiae based on a more recently published report by Fidelis et al. (2014), who detected with the same standard method anti-clotting effect of different crude SPs from this species, when obtained by various experimental conditions.The inhibitory response by crude SPs decreased in parallel with their molecular sizes and a molecular mass of over 45 kDa was required to display anticoagulation.In our case, the length of the sugar chain (> 100 kDa) was not affected by protease digestion (Figure 1C -Pomin, 2012;Mourão, 2015).
Additionally, a high solubility of the crude SPs preparations from the seaweeds in water was obtained (Athukorala et al., 2006) because the gel formation in solution is greatly dependent on the amount of 3,6-anhydrogalactose and sulfation, as well as associated cations with carbohydrates, as calcium (Fenoradosoa et al., 2009;Paula et al., 2015).As the addition of calcium chloride to display the APTT had no influence on the evaluation by in vitro testing (Figure 2), limited values of detection by in vitro APTT test in human plasma treated with G. birdiae crude SPs extract could be speculated because this classical test measures only 5% of thrombin formed in plasma, and the production of thrombin and fibrin could be still occurring in vitro (Castoldi & Rosing, 2011;Duarte et al., 2017).
Although no considering as an automated method (Duarte et al., 2017), our alternative technique have shown able to evaluate TG in vitro (Rodrigues et al., 2016a;Salles et al., 2017).
The accuracy of the algae crude SPs on a TG system in 60-fold diluted human plasma triggered at 37°C for 60 min, using in parallel the HEP as a function of its inhibitory reference, was further examined in this study.Differential inhibition patterns in correspondence to the TG parameters (PTh and TPeak) were observed under the conditions used (Figure 3 and Table 2).
The respective crude SPs extracts added to plasma modified the total amount of thrombin formed and, in the presence of the crude SPs extract from A. muscoides, it was totally abolished on the range of concentrations used (4.1-83.3μg plate-well -1 , 100% inhibition, p < 0.05) vs. others classes of algal polyglycans and at almost the same level of concentration than UHEP in the contact-activated pathway (Figure 3A and Table 2), which has an antithrombin-specific pentasaccharide sequence not found in other SPs-rich sources (Mourão, 2015).The inhibition of the extrinsic pathway (63.68%PTh inhibition) by its crude SPs extract required a concentration of at least 4.1 μg plate-well -1 since the ∆ abs of control was 2-fold higher than that from intrinsic system (Figure 3A and D) and UHEP added to plasma abolished TG at same concentration in this assay (Figure 3D).
The species Halymenia sp.(Figure 3B and E) and C. racemosa (Figure 3C and F) had crude SPs extracts that acted in different dose-response curves on both cephalin-induced and thromboplastin-induced systems, with ranges for TPeak and PTh inhibition from 32 to 45 min and from 51.88 to 81.58%, respectively (Table 2), based on maximum ∆ abs of active thrombin generated (Figure 3B and E, 3C and F) (Mourão et al., 2001).By contrast, lower inhibitory effects of TG in plasma in the presence of both crude SPs extracts from G. birdiae and C. cupressoides were recorded (p > 0.05) (Table 2).The reduced TG in system reflected the strength of anticoagulation independently from molecular mass and sulfate content of the diverse classes of seaweeds SPs (Figure 1C and Table 1) (Salles et al., 2017), except G. birdiae and C. cupressoides that had the lowest sulfate contents among all macroalgal species (Table 1).Indeed, the complex effects of the crude SPs extracts on TG varied with macroalgal species and stimuli in vitro (Nishino et al., 1999;Glauser et al., 2009;Zhang et al., 2014), possibly due to their different anticoagulant mechanisms (Rodrigues et al., 2013;Quinderé et al., 2014).Recently, Mansour et al. (2017) examined a fucosylated chondroitin sulfate from the sea cucumber Holothuria polii body wall on TG using calibrated automated thrombography.This glycan manifested a dual effect, with a procoagulant tendency for low doses and an anticoagulant action when at higher doses.
Elevated TG is associated with the risk of venous thrombosis (Castoldi & Rosing, 2011;Duarte et al., 2017) and thrombin interplays between coagulation and inflammation (Pomin, 2012).The TG method led us to a more precise analysis of macroalgae anticoagulants than traditional methods (Castoldi & Rosing, 2011;Duarte et al., 2017).The diverse classes of SPs prevented the human plasma coagulability induced by both cephalin and thromboplastin and TG parameters would have potential to postulate their mechanisms underlying in vitro, due to the lack of structure-function relationship data of these compounds, as a biotechnological perspective (Nishino et al., 1999;Mourão et al., 2001;Glauser et al., 2009;Zhang et al., 2014).Furthermore, our data could be useful to analyze the molecular mechanisms behind the anti-inflammatory actions of macroalgae SPs, which involve multiple points during the leukocyte recruitment into inflamed tissues in order to regulate the inflammatory process (Vanderlei et al., 2011;Rodrigues et al., 2012d;Quinderé et al., 2013;Ribeiro et al., 2014) because an increase of TG is also associated to exposure of blood to tissue factor in the subendothelium (Rau, Beulieu, Huntington, & Church, 2007).These hypotheses combined in a more critical study deserve to be investigated for a mechanistic characterization of the polymers.

Conclusion
Availability of crude sulfated polysaccharides from five Brazilian tropical seaweed species shows Rhodophyta (Gracilaria birdiae, Acanthophora muscoides and Halymenia sp.) to contribute more than Chlorophyta (Caulerpa cupressoides and C. racemosa).The combined technique of agarose/polyacrylamide gel electrophoresis with sequential toluidine blue/Stains-all staining partially characterizes distinct sulfated polysaccharides and non-sulfated polyglycans presenting large molecular masses.The model of thrombin generation exhibits accurately anti-clotting effects by algae crude SPs than classical assays.Actions occur dependently of concentration regardless of molecular size on both intrinsic/extrinsic coagulation pathways, differentially preventing thrombosis in diluted human plasma, but with lower potential than heparin.

Figure 2 .
Figure 2. In vitro anticoagulant effect by APTT assay of the crude SPs extracts, obtained by papain digestion, from Brazilian tropical seaweeds.

Table 1 .
Chemical composition (%) of crude SPs extracts obtained from the Brazilian tropical seaweeds collected at Flecheiras beach compared with other studies.Dosage by Dodgson and Price' method using NaSO 3 as standard; b Dosage by Dubois et al.' (1956) method using D-galactose as standard; c Ratio of hexose and sulfate of each crude SPs extract; d Dosage by Bradford' method using bovine serum albumin (-not detected).
a * Not determined.

Table 2 .
TG parameters in diluted human plasma (peak thrombin [% PTh]) and time to peak (TPeak.min) in the presence of different concentrations of crude SPs from the Brazilian tropical seaweeds compared to UHEP.
Figure 3.Effect of different concentrations of crude SPs extracts from the Rhodophyta A. muscoides (Am) (A and D) and Halymenia sp.(Hsp) (B and E); and from the Chlorophyta C. racemosa (Cr) (C and F) on cephalin-activated or thromboplastin-activated TG systems in 60-fold diluted human plasma using chromogenic method by a continuous detection system (405 nm, at 37°C for 60 min).Acta Scientiarum.BiologicalSciences, v. 40, e34463, 2018