Structural and physical-chemical analyses of sulfated polysaccharides from the sea lettuce Ulva lactuca and their effects on thrombin generation

. Ulva lactuca (Chlorophyceae) has biotechnologically-important sulfated-polysaccharides (Ul-SPs), but their potentials on thrombin generation (TG) are unknown. This study analyzed the structural and physical-chemical features of the Ul-SPs as modulators of TG. Proteolytic digestion yielded (13.13%) extract containing sulfate (20.43%) and total sugars (65.72%), besides ulvan consisting of rhamnose, xylose, glucose, glucuronic acid and α -/ β -types glycosidic linkages as characterized by one-/two-dimensions nuclear magnetic resonance (NMR) experiments. Fractionation of the Ul-SPs by DEAE-cellulose chromatography yielded Ul-SP1 and Ul-SP2 (0.50 and 0.75 M NaCl, respectively) showing sulfation (15.72-18.04%) and total sugars (59.73-60.58%) consistent with the charge density pattern by combination of agarose/polyacrylamide gel eletrophoresis using sequential staining with toluidine blue and stains-all, although with slight differences in their sizes (40 and >100 kDa, respectively). By both activated partial thromboplastin time (APTT) and prothrombin time (PT) tests, anticoagulation of the fractions was virtually detected by APTT (0.39 and 0.43 IU, respectively) against heparin (193 IU). Fractions acted differently on both intrinsic/extrinsic pathways in TG using 60-fold diluted human plasma, with 50% efficacies up to 8.3 μ g, whereas at high concentrations suggested intrinsic hypercoagulability since heparin abolished both systems at low amounts. Ul-SPs block TG, but predicting thrombosis in increasing doses. de ulvana, como caracterizada por experimentos de ressonância magnética nuclear uni-/bi-dimensionais, consistindo de ramnose, xilose, glucose, ácido glucurônico e ligações glicosídicas tipos- α /- β . Fracionamento dos Ul-PSs por cromatografia de DEAE-celulose rendeu Ul-PS1 e Ul-PS2 (0,50 e 0,75 M de NaCl, respectivamente) mostrando sulfatação (15,72-18,04%) e açúcares totais (59,73-60,58%) consistentes com o grau de densidade de carga por combinação de eletroforese em gel de agarose/poliacrilamida usando coramento sequencial com azul de toluidina e "stains-all", embora com diferenças quanto aos seus tamanhos (40 e >100 kDa, respectivamente). Por ambos os testes do tempo de tromboplastina parcial ativada (TTPA) e do tempo de protrombina, anticoagulação das frações foi detectada virtualmente pelo TTPA (0,39 e 0,43 UI, respectivamente) frente heparina (193 UI). Frações atuaram diferentemente sobre ambas as vias intrínsica/extrínsica na GT usando plasma humano diluído 60 vezes, com eficácias de 50% até 8,3 μ g, enquanto em concentrações maiores sugeriram hipercoagulabilidade intrínsica visto que heparina aboliu ambos os sistemas em quantidades baixas. Ul-PSs bloqueiam GT, porém prevendo trombose em doses crescentes. Ulvales; glicanos Electrophoretic analyses reveal physical-chemical homogeneity of complex glycans

The anticoagulant effects of the seaweeds SPs are routinely measured by two tests: 1) the activated partial thromboplastin time (APTT); and 2) the prothrombin time (PT), which distinguish the anticlotting potential on both intrinsic/extrinsic coagulation pathways, respectively (Rodrigues et al., 2009;Mourão, 2015); but, they do not indicate the normal function status and thrombin generation (TG)-based coagulation assays have been introduced to develop more accurately antithrombotic drugs (Jung et al., 2014;Zavyalova & Kopylov, 2016) and for clinical prognostics (bleeding disorders and surgical procedures), and epidemiology of thrombosis (Castoldi & Rosing, 2011;Duarte et al., 2017).
Some species of tropical seaweeds have been revealed as promising sources of SPs displaying in vitro TG inhibition/activation. SPs isolated from the Phaeophyceae Ecklonia kurome (Nishino, Fukuda, Nagumo, Fujihara, & Kaji, 1999) and from the Rhodophyceae Botryocladia occidentalis (Glauser et al., 2009), Acanthophora muscoides (Rodrigues et al., 2016b) and Gracilaria birdiae (Rodrigues et al., 2017a) revealed as modulators of TG by blocking both blood coagulation pathways. Contrasting result was found for the SP isolated from the brown seaweed Fucus vesiculosus that acted as activator of TG by tissue factor pathway using calibrated automated thrombography (Zhang et al., 2014). However, studies on the biological role of the SPs isolated from the Chlorophyta species on in vitro TG inhibition assays are lacking in the literature (Rodrigues et al., 2017b;Rodrigues, Benevides, Tovar, & Mourão, 2017c;Rodrigues et al., 2018).
The scope of the present study was to analyze the structural features from SPs samples of U. lactuca collected on the coast of Ceará/Brazil using NMR technique and to characterize by agarose/polyacrylamide gels electrophoreses by sequential staining employing toluidine blue and stains-all regarding their physical-chemical properties; also, in vitro assessments were conducted with respect to its SPs on classical coagulation tests (APTT and PT) and TG assay in 60-fold diluted human plasma using a continuous detection system.

Ulva lactuca samples and physical-chemical analyses of their SPs
Specimens of the Chlorophyceae U. lactuva were collected in natural bed from the Northwestern coastline of Brazil (Flecheiras beach, Trairí-Ceará). All the samples were placed in plastic bags and conducted to the Carbohydrates and Lectins laboratory (CarboLec), Universidade Federal do Ceará. After collection, the material was cleaned and removed from macroscopic epiphytes, followed by washing with distilled water and stored at -20°C until further use (Araújo et al., 2016). A voucher specimen (#4978) was deposited in the Herbarium Prisco Bezerra of the Department of Biological Sciences, Universidade Federal do Ceará, Brazil. The experimental analyses of the U. lactuca SPs were performed at the Connective Tissue laboratory, Universidade Federal do Rio de Janero (UFRJ), Brazil.
A five grams sample of dehydrated algal tissue was cut into small pieces and subjected to papain digestion (60°C, 24 hours) in 100 mM sodium acetate buffer (pH 5.0) containing cysteine and EDTA (both 5 mM) (all Sigma Aldrich). Percent yield (%) was determined as based on the dehydrated weight of the algae obtained for papain extraction (Araújo et al., 2016). Crude SP extract (20 mg) was dissolved in 10 mL of 50 mM sodium acetate buffer (pH 5.0) and applied to a DEAEcellulose column (1.2 × 12 cm) equilibrated with the same buffer (all Sigma Aldrich). Fractionation was conducted using a stepwise of NaCl from 0 to 1.5 M NaCl in the same buffer, with intervals of 0.25 M between each concentration. Fractions of 2.5 mL were collected and analyzed for SPs using the metachromasy assay (A 525 nm ) containing dimethymethylene blue (Sigma Aldrich) with an Amersham Bioscience Ultrospec 3100 spectrophotometer at 525 nm (Farndale, Buttle, & Barrett, 1986). Extract and fractions Ul-SP1 and Ul-SP2 (eluted with 0.5 and 0.75 M NaCl, respectively) were examined for their contents of sulfate (Dodgson & Price, 1962), total sugars (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) and proteins (Bradford, 1976) according to the respective quantitative assays. SPs present in extract and fractions were checked by 0.5% agarose gel electrophoresis procedure (Dietrich & Dietrich, 1976). All these experiments were also conducted as described (Araújo et al., 2016). Next, polyacrylamide gel electrophoresis (6%) was carried out to estimate the molecular size of SPs by comparison with the electrophoretic mobility of standard compounds dextran sulfate ( ̴ 8 kDa), chondroitin-4-sulfate ( ̴ 40 kDa) and chondroitin-6-sulfate ( ̴ 60 kDa) (all Sigma Aldrich) (Rodrigues et al., 2013;Rodrigues et al., 2017a). After electrophoretic experiments, both gels were subjected to sequential staining with toluidine blue and stains-all (both Sigma Aldrich) to reveal nonSPs from the polymer samples (Volpi & Maccari, 2002;Rodrigues et al., 2017a;Rodrigues et al., 2017b;Rodrigues et al., 2017c).
Structural analysis by NMR spectroscopy 1 H and 13 C, one-dimensional and twodimensional spectra, of the crude SP extract (U. lactuca) was recorded using a Bruker DRX 600 MHz apparatus with a triple resonance probe. About 5 mg sample were dissolved in 0.6 mL of 99.9% deuterium oxide (Cambridge Isotope Laboratory, Cambridge, MA). All spectra were recorded at 25ºC with HOD suppression by presaturation. 1D 1 H-NMR spectrum was recorded with 32768 points and 8 scans and inter-scan delay set to 1 s (Tian et al., 2015;Rodrigues et al., 2014). Regarding the two dimension NMR experiment, the 1 H/ 13 C edited-HSQC spectrum (64 scans, 1024 × 512 points) was run and globally optimized alternating phase rectangular pulses (GARP) for decoupling (Quinderé et al., 2014). Chemical shifts are displayed relative to external trimethylsilylpropionic acid at 0 ppm for 1 H and relative to methanol for 13 C. All spectra were processed using the SpinWorks 3.1.8 software package (Quinderé et al., 2014;Rodrigues et al., 2016a).

Blood samples
Coagulation analyses were conducted using venous blood samples collected in citrated vacutainer tubes containing 3.2% sodium citrate from 10 different donors (University Hospital Clementino Fraga Filho, UFRJ), followed by centrifugation (2000 × g, 15 min.) prior to tests. Normal citrated human plasma aliquots of 1 mL were frozen and stored at -70°C until use (Rodrigues et al., 2017a).

APTT and PT assays
Fractions were assessed by both in vitro APTT and PT tests based on manufacturers' specifications, for measuring anti-clotting effect in a coagulometer Amelung KC4A before the in vitro TG assay. For APTT assay, a mixture of 100 μL of plasma and concentration of SPs (1 mg mL -1 ) was incubated with 100 μL of APTT reagent (kaolin bovine phospholipid reagent) (Sigma Aldrich). After 2 min of incubation at 37°C, 100 μL of 25 mM CaCl 2 was added to the mixtures, and the clotting time was recorded. Regarding the PT assay, a mixture of 100 μL plasma and concentration of SPs (1 mg mL -1 ) was incubated at 37°C for 1 min. After that, 100 μL of PT reagent (Sigma Aldrich) were added to the mixtures, and the clotting time was recorded using the same coagulation equipment. UHEP from the National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK (193 international units per mg (IU mg -1 ) of polysaccharide) was used as the standard in both tests. Data were expressed as mean ± S.E.M. (n = 3).

Results and discussion
Wall matrix SPs from the green seaweed Ulva lactuca (popularly known as sea lettuce) were extracted with papain to examine their structure, and then fractionated by DEAE-cellulose anionexchange chromatography to analyze their physicalchemical features and in vitro effects on the coagulation.
The yield of crude SP (13.13%) was about 5.95fold higher than that of the overall extract (2.21%) from the same algal species, applying the papain method (Araújo et al., 2016). This comparative examination of the total yield with a previous investigation also reflected highest levels of 20.43 and 65.72% for sulfate and total sugars, respectively, from the analytical assays of the samples, and could be the result of the drying (60°C, 24 hours) for obtaining crude SP extracted from the dehydrated raw biomass in comparison with the lyophilization procedure employed by Araújo et al. (2016), who previously examined the U. lactuca crude SP extract composition (15.10% sulfate and 54.72% total sugars).
Our observations could support the high content of total sugars prior measured these respective residues making part of the same molecule (Rodrigues et al., 2014) since enzymes help to obtain highest sugar yield and contents (Athukorala et al., 2006). Siddhanta et al. (2001) examined crude SPs extracts from Ulva species, when obtained by cold and hot water procedures. Those extracted with cold water showed to be enriched with high levels of hexose sugars, whereas the hot water extracts were rich in Rha, Xly and Gluc as parts of the structural polysaccharide. By contrast, this question deserve to be better analyzed because the Dubois, Gilles, Hamilton, Rebers, & Smith (1956)' method measures small chains of polysaccharides.
Collectively, our findings supported ulvan as characterized by infrared analysis from a more recent study (Araújo et al., 2016). On the basis of these comparative structural analyses, the current investigation suggested that the use of papain to obtain polysaccharides from the Brazilian samples of U. lactuca could also be useful as a bioprospecting strategy during comparison with others studies (Rodrigues et al., 2014;Rodrigues et al., 2016a).

Electrophoretic analyses reveal physical-chemical homogeneity of complex glycans
The profile of DEAE-cellulose anion-exchange chromatography of the crude SP confirmed the separation into two SPs fractions (Ul-SP1 and Ul-SP2) at 0.50 and 0.75 M NaCl, respectively ( Figure  2A), as documented by Araújo et al. (2016), who formerly reported U. lactuca cell wall SPs isolation.
However, as observed in Figure 1A, DEAEcellulose column-bound SPs were eluted with increasing molarity of NaCl and produced similar metachromasy between fractions, accounting the material recovered from the column almost equal between them, as also supported by their respective integrated metachromatic areas (IMAs) estimated from the chromatogram (Table 1). Siddhanta et al. (2001) extracted SPs from U. lactuca by cold and hot water and noted an important difference in the number of fractions eluted with NaCl on DEAEcellulose column.
Total fraction yield was about 6.8-fold greater than that obtained for this same algal species (Araújo et al., 2016). Similar tendency of the fractions in this study was noted for their high contents of both sulfate (15.72 and 18.04%) and total sugars (60.58 and 59.73%) and no protein contamination from the analyzed polymer samples. This compositional profile also showed to be distinct than that found by Araújo et al. (2016), who revealed 0.75 M NaCl fraction (Ul-SP2) with levels of 9.73-and 1.75-fold higher for sulfate and total sugars, respectively, than 0.5 M NaCl fraction (Ul-SP1); therefore, heterogeneous composition of the SPs as commonly isolated from seaweeds (Athukorala et al., 2006;Pomin & Mourão, 2008;Wang et al., 2014) and/or when different methods are used, when using various algal species (Siddhanta et al., 2001). The physical-chemical characteristics of the SPs from U. lactuca were further analyzed by two electrophoretic techniques. For agarose gel analysis (Figure 2Ba), extract and fractions exhibited single, homogeneous and coincident metachromasy bands co-migrating as CS, but not as UHEP, suggesting SPs with same structural conformation and total sugars/sulfate ratio (Table 1) based on their interactions with the diamine (Dietrich & Dietrich, 1976) that showed similar eletrophoretic mobilities on gel, after toluidine blue treatment (Volpi & Maccari, 2002;Fidelis et al., 2014;Rodrigues et al., 2016a;Salles et al., 2017). This presumed certain regularity on their chains of ordered helical conformation of the U. lactuca SPs as described, but more refined studies are required (Wang et al., 2014;Yaich et al., 2014). Previous report from Araújo et al. (2016) described polydispersive SPs using the same procedure. On this view, results postulated that U. lactuca could perhaps biochemically change its matrix polysaccharide composition based on collection period (Cardozo et al., 2007;Wang et al., 2014;Rodrigues et al., 2016a). The molecular distribution of the SPs from U. lactuca was clearly determined by stepwise of NaCl (DEAE-cellulose).
The characterization by polyacrylamide gel analysis evidenced 0.5 M NaCl fraction (Ul-SP1) of low molecular weights SPs as C-4-S (ca. 40 kDa) (Figure 2Ca) and similar to a SPs subfraction obtained from Caulerpa cupressoides (Chlorophyta) (Rodrigues et al., 2013), while a wide dispersion in the molecular masses (> 100 kDa) was noted for extract and fraction Ul-SP2 (0.75 M NaCl) as usually described for SPs from seaweeds (Pomin, 2012;Fidelis et al., 2014;Rodrigues et al., 2016a and2017a). Ulvales contains two major SPs populations (500-800 kDa and 150-200 kDa) (Wang et al., 2014). However, our observations did not support Yaich et al. (2014), who previously reported that enzymatic extraction does not induce a decrease in the molecular size of the SPs from U. lactuca in comparison with the acid extraction (HCl, pH 1.5, 90°C); therefore, without impacting on the structure of the algal SPs. More refined studies using chromatographic methods are needed (Yaich et al., 2014;Mourão, 2015), since similar polyanionic characters between fractions were visualized ( Figure  2Ba).
Although SPs were detected in extract and fractions by treatment with toluidine blue (Figure  2Ba), our investigations were also conducted to more precisely examine all the SPs preparations, when associated with the use of stains-all, to improve the sensitivity of the complex glycans ( Figure 2Bb). This combined strategy resulted not only in a strong increase of the staining for standards and samples (especially fraction Ul-SP1) similar to sulfated glycosaminoglycans from animal tissues (Volpi & Maccari, 2002), but also suggesting the presence of non-sulfated sugar residues from the examined material (Rodrigues et al., 2017a, b and c), reinforcing the solution NMR experiments which revealed acidic sugars residues, such as GlcA ( Figure  1), a highly conserved structural identity in Ulvaceae (Wang et al., 2014;Tian et al., 2015). Rodrigues et al. (2016) isolated and compared three crude SPs extracts obtained from the red seaweed Acanthophora muscoides. Procedures of agarose gel electrophoresis and NMR revealed charge density and structural homogeneities, respectively, of sulfated glycans extracted along the algal extracellular matrix.
In this study, such interpretations were also extended to the SPs stained with toluidine blue and stains-all from the polyacrylamide gel analysis (Figure 2Cb) (Rodrigues et al., 2017), as observed for glycosaminoglycans (Andrade, Oliveira, Tovar, Mourãos, & Vilanova, 2017). On this basis, it was also hypothesized that the elimination of minerals (like cadmium and calcium ions) associated with the algal matrix by means of protease extraction could perhaps affect the molecular weight of the U. lactuca SPs or only deducing their molecular nature, when obtained by papain protocol, as reported in Ulvales (Wang et al., 2014;Yaich et al., 2014). Further studies should be conducted to clarify this supposition (Siddhanta et al., 2001;Wang et al., 2014).
Anticoagulant-active U. lactuca SPs are displayed on TG model compared with the APTT and PT tests Evaluation of the U. lactuca SPs on both APTT and PT classical coagulation tests is shown in table 2, and their possible in vitro effects were compared with UHEP (193 IU mg -1 , 14 kDa) as plasma anticoagulant (Shanmugam et al., 2001).
Human plasma treated with the fractions (1 mg mL -1 ) discreetly delayed (0.39-0.43 IU) the normal APPT (33.5 ± 0.08 s), but did not affect the PT; therefore, without displaying both intrinsic/extrinsic pathway factor inhibitions (Rodrigues et al., 2016a;Rodrigues et al., 2017). UHEP still extended the coagulation at 2.5 (42.15 ± 0.6 s) and 100 μg mL -1 (20.13 ± 0.6 s) using APTT and PT assays, respectively. Table 2. Analysis of the SPs fractions, obtained by DEAE-cellulose chromatography, from the green seaweed Ulva lactuca on the coagulation in vitro using UHEP as a reference. These preliminary results on the coagulation were consistent with those found by Shanmugam et al. (2001), who noticed uronic acid-rich low molecular masses SPs (U. lactuca) devoid of anticoagulation according to both APTT and PT models. In fact, the presence of acid sugar residues revealed by NMR analysis in the U. lactuca SPs composition (Figure 1) had no impact on the routine clotting assays, although soluble SPs extracted with papain and highly charged fractions (Table 1 and Figure  2A). Athukorala et al. (2006) obtained an enzymatic hydrolysate containing SPs from the brown seaweed Ecklonia cava and found strong anticoagulation dependently of their high degree of sulfation in detriment to low uronic acid content. Structural variability of the seaweeds SPs may reveal different anticoagulant mechanisms making analogies with that of UHEP (Pomin, 2012;Fidelis et al., 2014;Quinderé et al., 2014), which has thrombin inactivation by AT (Pomin & Mourão, 2008;Pomin, 2012;Mourão, 2015).
Interestingly, under the conditions of our study, increasing concentrations of the SPs fractions samples of U. lactuca decreased the in vitro anticoagulant response on TG induced by cephalin vs. control without SPs (Figures 3A and B). Ul-SP1 and Ul-SP2 at 4.1 and 8.3 μg well-plate -1 more drastically attenuated TG as analyzed by the PTh and TPeak parameters, inducing more than 50% inhibition of PTh (51.02 →54.33%) compared with the positive control (TPeak: 20 and 24 min.), whereas the lag phase time (14→17 min.) remained almost unchanged. The required amount of the algal SPs was at least 4.15-fold greater than UHEP, which abolished TG at 2 μg well-plate -1 (Rodrigues et al., 2016b) as a result of its ATdependent specific mechanism absence to others SPsrich marine organisms (Glauser et al., 2009;Pomin, 2012;Mourão, 2015). TG in diluted plasma treated with 41.6 and 83.3 μg well-plate -1 of the samples only inhibited the intrinsic system by 18.36→45.08% (PTh) ( Figures  3A and B). This also indicated that the U. lactuca SPs increased the PTh as a suggestion of plasma hypercoagulability in vitro (Zhang et al., 2014) as demonstrated by direct thrombin inhibitors (Furufohri & Morshima, 2015) and similar to the in vivo experiments of venous thrombosis, in which algae SPs stimulated prothrombotic actions in rats (Rodrigues et al., 2011;Pomin, 2012;Quinderé et al., 2014) due to activation of factor XII involved in physiological formation of bradykinin, a vasoactive amine (Mourão, 2015). These observations could be connected with the analgesic and anti-inflammatory profiles on bradykinin pathway of Ul-SP2 (Araújo et al., 2016) and structure-function relationships deserve to be further examined (Pomin, 2012). Herein, SPs from U. lactuca also prolonged lag phase time (26→37 min.) from the controls, possibly predicting risk of bleeding on a prognostic view (Castoldi & Rosing, 2011), but animal studies must be conducted since seaweeds SPs do not manifest extensive bleeding effects based on those effects of UHEP inducing hemorrhage in its administration in rats (Rodrigues et al., 2011;Quinderé et al., 2014;Mourão, 2015).
TG in extrinsic pathway-activated plasma in the presence of the fractions Ul-SP1 and Ul-SP2 to evaluate their anticoagulant potential was also evaluated ( Figures 3C and D). The capacity of TG was markedly reduced in plasma treated with low concentrations of the algal SPs (4.1 and 8.3 μg wellplate -1 ) as verified by the PTh (50.90→58.18% inhibition) and TPeak (30→58 min.) parameters; but, with the lag phase time longer in Ul-SP2 (39 min.) ( Figure 3D) (Rodrigues et al., 2017c). No hypercoagulant stimulus at high doses of SPs was recorded for 60 min. based on total inhibition achieved at 83.3 μg well-plate -1 .
In fact, the similar effects of the SPs fractions on the TG response were supported by their respective total sugar and sulfate contents (Table 1), being almost equal with the increasing molarity of the salt eluents, but these combined results were not expected (Araújo et al., 2016). Furthermore, the SPs fractions samples of U. lactuca added in water did not produce viscosity in the presence of chloride calcium to display the coagulation in vitro (Table 2 and Figure 3) since that the gel formation is described as a function of pH, association between borate and free hydroxyl groups of the polysaccharide, as well as the chelation of calcium ion with borate, and sulfation pattern (Wang et al., 2014). The papain would play an important role in the solubility of these polymers in water to experimentation (Araújo et al., 2016).
Seaweeds have various chemical components (Cardozo et al., 2007;Thomas & Kim, 2013), including the possible existence of phenolic compounds as suggested for water extracts containing SPs obtained from the red seaweed Gracilaria fisheri. These findings revealed a positive correlation between the in vitro antioxidant effect and the phenolic compound content (Imjongjairak et al., 2016). However, to the best our knowledge, phenolic compounds have been widely described in higher plant showing antimalarial effect (Gadetskaya et al., 2015).
Although some phenolic compounds may influence the thrombin response (Bijak et al., 2014), it is still very premature to infer effects of these compounds derived from seaweeds on the TG system because no studies have also been done. Furthermore, the use of the papain method as mentioned above results in purity and enhanced yield of biologically active SPs for biotechnology (Athukorala et al., 2006;Mourão, 2015;Araújo et al., 2016) displaying in vitro TG inhibition (Glauser et al., 2009;Rodrigues et al., 2016b;Rodrigues et al., 2017a;Rodrigues et al., 2017b;Rodrigues et al., 2017c). The structural features revealed by NMR analysis supported the presence of ulvan-type polysaccharide in the crude SP extract (Figure 1) (Lahaye & Robic, 2007;Wang et al., 2014).
Therefore, it was observed in the present study that the U. lactuca SPs displayed their inhibitory effects more in the extrinsic pathway than in the intrinsic one, as an opposite behavior compared with a fucoidan isolated from the brown seaweed Ecklonia kurome (Nishino et al., 1999); and for the SPs isolated from the Rhodophyta Gracilaria birdiae (Rodrigues et al., 2017a); and Chlorophyta Caulerpa racemosa (Rodrigues et al., 2017c); besides of skin of Nile tilapia Oreochromis niloticus (Salles et al., 2017) on the basis of TG parameters and intrinsic inhibitory efficacy by UHEP at 2-fold higher. This peculiar role of the U. lactuca SPs fractions corroborated as activators of the intrinsic coagulation at high concentrations because UHEP only acted as a potent inhibitor of the coagulation in vitro ( Figures 3A and B) (Mourão, 2015). Collectively, our results seemed to be of practical use because in vivo models of thrombosis in experimental animals are always a laborious assay (Pomin & Mourão, 2008;Mourão, 2015), although TG in vitro no reflecting the physiological environment (Pomin & Mourão, 2008).
Overall, SPs from U. lactuca had no actions on the classical coagulation models (Table 2), but were clearly apparent using in vitro TG assay ( Figure 3) (Castoldi & Rosing, 2011;Zhang et al., 2014;Rodrigues et al., 2016b;Salles et al., 2017). The degree of inactivation on TG by algal SPs excluded a positive correlation of sulfation and molecular size (Figure 2) on these biological processes, and seemed by dependence on their stereospecific features, as also suggested for other SPs from seaweeds (Rodrigues et al., 2016b;Rodrigues et al., 2017a). SPs from seaweeds have various bioactivities (Cardozo et al., 2007;Rodrigues et al., 2012;Wang et al., 2014) and have traditionally been used in food products as thickening and gelling agents for hydrocolloid industry worldwide (Cardozo et al., 2007;Wang et al., 2014), and recently in diets for hens (Li et al., 2018). Excessive consumption of U. lactuca as raw or semi-processed material could perhaps lead to physiological reactions associated with thrombosis and bioavailability studies regarding dietary fibers have yet been scarce (Wang et al., 2014).
Regarding U. lactuca food safety, elucidation of molecular mechanisms behind the biological effects on the coagulation in vitro (Pomin, 2012;Mourão, 2015) is still needed as a critical step to antithrombotic drug formulation (Zavyalova & Kopylov, 2016).

Conclusion
The green seaweed Ulva lactuca contains ulvantype polysulfated polysaccharides with low molecular sizes (ca. 40 and > 100 kDa) and nonsulfated glycans with experimental thrombosis inhibition by both intrinsic and extrinsic pathways independently of charge and molecular size, when in 60-fold diluted human plasma using continuous method of thrombin generation in vitro, but with lower efficacies than heparin. Low amounts reveal as potent inhibitors of contact-activated thrombin generation, whereas a tendency of plasma hypercoagulability is manifested with increasing concentrations, arousing the attention of their physiological risks linked to thrombosis.