SFs are a class of SPs composed mostly of α-L-fucopyranosyl (Fucp) units. SFs can be extracted mostly from marine organisms, such as sea-urchins (echinoidea), sea-cucumbers (holothuroidea) and brown algae (phaeophyta). These molecules are also known as fucoidans when isolated from brown algae. Besides Fucp, the fucoidans are also composed of other sugars, such as xylose and uronic acids. This heterogeneous monosaccharide composition, associated with the lack or an unclear structural pattern of regularity and the presence of branching residues, makes the establishment of the structure-function relationships for algal molecules very hard (Pereira et al., 1999). On the other hand, the SFs isolated from invertebrates can reveal structures quite regular (Figures 1A–I). This type of structural pattern helps to achieve advanced structure-function correlations about their anticoagulant properties. Using the invertebrate SFs, we are able to understand which structural features are important for the anticoagulant activity of these molecules (Pereira et al., 1999; Pomin, 2009, 2012b,c). Below, some of these influential structural features on coagulation of the MSPs of well-defined chemical structures will be described.

SGs are a class of SPs composed of α-L-, α-D-, or β-D-galactopyranosyl (Galp) units. SGs can be found in green algae (clorophyta), red algae (rodophyta), sea-urchins and ascidians which are also known as tunicates (ascidiacea). Like SFs, algal SGs have structures more complex than the regular and simpler invertebrate counterparts. In green algae, for example, the sulfation pattern is usually complex, with additional substitutions like pyruvates, and the possibility of branches (Farias et al., 2008). In red algae, although the SGs show heterogeneous sulfation patterns, they are usually composed of disaccharide repeating units of 3-linked β-D-Galp and 4-linked α-Gal units in their backbones. Sometimes, the latter unit can be seen forming an extra carbon ring which results in an anhydro-sugar (Quinderé et al., 2014). This is an additional heterogeneity that enhances structural complexity in the red algal molecule. However, some red algal species can show very simple structures whose sulfation patterns vary accordingly to the species of extraction. Two examples of these structures are shown at panels N and O of Figure 1. Advanced structure-function correlations can be reached when these red algal SGs are used, as opposed to the more heterogeneous SGs from red or green algae. Conversely, the invertebrate SGs are very often composed of well-defined chemical structures (Figures 1J–M), which allow accurate structure-function correlations.

The information in the two previous paragraphs have made clear the advantages of the structures of the invertebrate SFs and SGs over the algal molecules, except few cases of red algal molecules (Figures 1N,O). These advantages come from the fact that the invertebrate molecules and the regular red algal SGs (Figure 1), which are composed of well-defined chemical structures, can be successfully used in structure-function relationship studies (Table 1). These studies allow prediction of the most influential structural combinations of the MSPs to achieve desirable anticoagulant responses. This knowledge is relevant to the development of these molecules as future therapeutic candidates.

Marine GAGs have different structures than those present in common mammal GAGs. For example, dermatan sulfate (DS) isolated from the ascidian species Styela plicata is composed of [→4)-α-L-IdoA-(2R¹,3R²)-(1→3)-β-D-GalNAc-(4R³,6R⁴)-(1→]_n, where IdoA is iduronic acid, GalNAc is N-acetyl galactosamine. The R¹, R², R³, and R⁴ are sulfate groups at 66, < 5, 94, and 6 percent, respectively (Pavão et al., 1998). The S. plicata is mostly 2-sulfated at the IdoA unit but largely 4-sulfated at the GalNAc unit. Conversely, the commonest mammalian DS is mostly composed of 2-sulfated IdoA units together with occasional C4 sulfation at GalNAc units. Another different GAG from marine invertebrates is the fucosylated chondroitin sulfate (FucCS) isolated from sea-cucumbers. These molecules are composed of the following structure {→3)-β-D-GalNAc-(1→4)-[α-L-Fucp-(1→3)]-β-D-GlcA-(1→}_n (Figure 2) (Pomin, 2014a). The branching Fucp unit can be sulfated at the 2, and/or 3 and/or 4-positions within different percentages according to the species of occurrence (Table 2). Conversely, the commonest chondroitin sulfates (CSs) of mammals are made of the following structure [→4)-β-D-GlcA-(1→3)-β-D-GalNAc-(1→]_n, in which the GalNAc units can be either mostly 4-sulfated (CS-A) or predominantly 6-sulfated (CS-C) (Pomin et al., 2012).

The effects of MSPs on hemostasis are the mostly studied medical activities of these compounds. The mechanisms of action in this particular clinical activity reside basically on the inhibition of some coagulation proteases like thrombin citation(IIa) and factor Xa, via their natural inhibitors, named serpins citation(serine-protease inhibitors). The most common serpins of this system are antithrombin citation(AT) and heparin cofactor II citation(HCII). Although at different degrees of response, the majority of the MSPs described in this review, the ascidian DS (Pavão et al., 1998; Vicente et al., 2004; Kozlowski et al., 2011), the sea-cucumber FucCS (Mourão et al., 1996; Mourão, 2004), the algal SFs and SGs (Pereira et al., 1999; Farias et al., 2000; Mourão, 2004) and the invertebrate SFs and SGs (Pereira et al., 1999, 2002; Pomin, 2012c) have either negligible or impressive effects in this serpin-dependent mechanism. The resultant anticoagulant activities of these MSPs (Tables 1, 2) are intimately and ultimately dependent on their structural features, as described below.

Besides the common serpin-dependent anticoagulant mechanism, the FucCS from the sea-cucumber Ludwigothurea grisea, which its fucosyl unit is mostly 2,4-di-sulfated (Figure 2; Table 2), and the SG from the red alga Botryocladia occidentalis (Figure 1N) have also shown serpin-independent anticoagulant mechanism (Glauser et al., 2008, 2009). Initially, their anticoagulant actions were primarily attributed by their capacity in potentiate the inhibition of factors Xa and IIa, via either AT or HCII. However, the sea-cucumber FucCS and the red algal SG are also known to inhibit the generation of factor Xa and IIa by interfering in the formation of the blood cofactor complexes at the surface of the cells. Factor Xa is activated mainly by the intrinsic tenase complex, while IIa is converted from II by the prothrombinase complex. FucCS and SG were shown the ability to inhibit the activation of these tenase and prothrombinase complexes (Glauser et al., 2008, 2009). The formation of these complexes is a key step for the generation and amplification of the coagulation cofactors. This serpin-independent mechanism has also been reported for other types of SGs (Quinderé et al., 2014). Unfortunately, advanced structure-function correlations are yet to be established for this novel mechanism. Therefore, the structural requirements necessary to achieve the anticoagulant activity via the serpin-independent mechanism are still unknown. For this reason, we will keep this new anticoagulant mechanism out of discussion here.

The anticoagulant structural features responsible for the anticoagulant activity of the MSPs can be determined from systematic analyses using the MSPs of well-defined chemical structures (Figure 1). On this way, comparing all the structures in Figure 1, one can discern structural similarities and differences in these molecules. For example, both the SF from Strongylocentrotus franciscanus (Figure 1H), and the SG from Echinometra lucunter (Figure 1J) present the same sulfation pattern (exclusive and entirely 2-sulfated), the same anomeric configuration (α-form), the same glycosidic linkage (1→3), and based on previous works (Pereira et al., 2005a), the same molecular mass (~100 kDa). Their single difference is the sugar type (Fucp or Galp). Interestingly, this single structural difference is itself enough to promote great changes in the anticoagulant outcomes of these homopolysaccharides. The 2-sulfated α-galactan from E. lucunter exhibits a significant anticoagulant activity monitored by the activated partial thromboplastin time (aPTT) method (Pomin, 2014b). The anticoagulant potential of this SG was determined to be 20 IU mg, although almost 10-fold less than unfractionated heparin (UFH) (Table 1). The specific anticoagulant assay with the purified proteases revealed that this SG enhances both IIa and factor Xa inhibition by either AT or HCII (Table 1). Conversely, the anticoagulant effect of 2-sulfated α-fucan from S. franciscanus is exclusively based on catalysis of AT inhibition over factor Xa, although it is 12.5-fold less active than the α-SG. This single effect on the Xa/AT system explains the much lower activity of the compound from S. franciscanus (aPTT of ~2 IU mg⁻¹, 100-fold less active than UFH) since the anti-Xa activity has a relatively minor influence on the aPTT. This is an illustrative and typical example of a sugar-type-dependent biological effect of polysaccharides. The structural combination of 2-sulfation with 3-linked α-Galp units results therefore in the anticoagulant response.

Based on this same systematic and comparative analysis, the SGs from the red algae Botryocladia occidentalis and Gelidium crinale were seen to exhibit identical backbones, and the same chain sizes (Farias et al., 2000; Pereira et al., 2005b). However, there are slight differences in their sulfation patterns (Figures 1M,O). As summarized in Table 1, these structural differences account for the 30% difference in anticoagulant activity as observed by the aPTT values of these algal macromolecules and the even greater difference in catalytic effect of the SP from B. occidentalis on the HCII-mediated anti-IIA activity, which was approximately 25-fold more than the catalytic effect of the SP from G. crinale. When the backbones of the red algal SGs are identical but bearing sulfation as their main difference, the anticoagulant activity seems to be proportionally dependent of the sulfation content (Table 1).

The structural requirements for the interaction of the MSP of well-defined chemical structures with the coagulation cofactors and their target proteases and inhibitors were determined to be very stereospecific (Mourão, 2004; Pomin and Mourão, 2008; Pomin, 2009). The site of sulfation has a major impact on the activity. This can be illustrated by the fact that 2,4-di-sulfated units have an amplifying effect on the AT-mediated anticoagulant activity in the series of 3-linked α-L-fucans (Figure 1; Table 1). Specific sulfation sites are required for the interaction with plasma serine-protease inhibitors. Note the occurrence of the 4-sulfated unit content in the 3-linked α-L-fucans vs. their anticoagulant activities (Table 1). L. variegatus (a single 4-sulfated unit/tetrasaccharide, Figure 1B, has 3 IU/mg of activity), S. pallidus (a double 4-sulfated unit/tetrasaccharide, Figure 1C, has 18 IU/mg of activity), and S. purpuratus isotype II (a double 4-sulfated unit/trisaccharide, Figure 1F, has 76 IU/mg of activity). This 4-sulfation is the structural motif required to enhance the inhibition of IIa by HCII. In contrast, the presence of 2-sulfated residues seems to have a deleterious effect on HCII-mediated anti-IIA activity of the polysaccharides (Mourão, 2004).

In the studies using the ascidian DS, Pavão and co-authors demonstrated that 4-sulfation at GalNAc units together with some 2-sulfation at the IdoA units is a structural motif of anticoagulant properties in these molecules (Pavão et al., 1998). This was observed based on the fact that DS from the species Styela plicata and Halocynthia pyriformis have large amounts of 4-O-sulfated GalNac units (94 and 99%, respectively), together with the reasonable amounts of 66 and 70% of 2-sulfation at the IdoA units. Their anticoagulant actions were measured by aPTT and shown to be 11 and 8 IU/mg, respectively; while the activity of the DS from bovine mucosa was shown to be only 2 units/mg (Pavão et al., 1998). Unlike the ascidian DSs, the mammalian DS is largely 2-sulfated at the IdoA unit and much less sulfated at the 4-position of the GalNAc units, as above-mentioned.

The sulfated fucosyl branches in the sea-cucumber FucCS were shown to be essential to their anticoagulant activities. This statement is based on the fact that when these branching units are removed, for example, by mild acid hydrolysis, or desulfated, by desulfation reactions, their anticoagulant effects disappears (Pomin, 2014a). Besides the necessary existence of these branching units, their sulfation patterns are also influential to the levels of anticoagulant activity. This can be seen from the structures and aPTT values of Table 2. The 2,4-di-sulfation in the fucosyl branching units appears to be the best sulfation pattern to the anticoagulant activity of this class of SPs. The importance of the 2,4-di-sulfation in Fucp units was already pointed out in the work of Fonseca et al. (2009), as above for the AT-mediated anticoagulant activities of the series of 3-linked α-L-fucans (Figure 1, Table 1). In the work of Fonseca et al. (2009), the 2,4-di-sulfated Fucp units were reported to be crucial to the anticoagulant activity of both sea-cucumber FucCS molecules and the linear SFs from invertebrates (Figure 1, Table 1).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.