Heparin with a structure similar to that of vertebrate heparin but with lower anticoagulant activity has been identified in the tissues of the ascidian Styela plicata (Chordata-Tunicata) (Cavalcante et al., 2000). An extensive structural analysis of the polymer indicated that the invertebrate heparin is composed mainly of the disaccharide α-L-IdoA(2SO₄)-1→4β-D-GlcN(SO₄)(6SO₄)-1, with a minor contribution (~25%) of the disaccharide α-L-IdoA-1→4β-D-GlcN(SO₄)(6SO₄)-1. Activated partial thromboplastin time (APTT) assays of the ascidian heparin showed that the polymer has lower anticoagulant activity than mammalian heparin. In addition, it is about 20 times less potent than mammalian heparin in stimulating the inhibition of thrombin by purified ATIII. However, S. plicata heparin activates HCII with approximately the same potency as vertebrate heparin, as indicated by comparison of the IC₅₀ values for inhibition of thrombin by purified HCII. To compare the hemorrhagic effect of the ascidian and mammalian heparin, we used a rat cut-tail bleeding model. S. plicata heparin at a dose of 4 mg/kg body weight, which is above the dose required to prevent thrombus on an animal experimental model did not increase the amount of blood loss in comparison with the saline control. In contrast, mammalian heparin, compared to the control, increased blood loss almost 2-fold (Cardilo-Reis et al., 2006).

In a TNBS-induced colitis model in rats, S. plicata heparin drastically reduced inflammation after subcutaneous administration during a 7-day period (Belmiro et al., 2009), as observed by the normalization of the macroscopic and histological characteristics of the colon. At molecular level, TNF-alpha, TGF-beta, and VEGF were reduced to normal values. Lymphocyte and macrophage recruitment and epithelial cell apoptosis were also decreased after the treatment. A drastic reduction in collagen-mediated fibrosis was also observed. No hemorrhagic events were observed after glycan treatment (Belmiro et al., 2009). These results strongly indicate a potential therapeutic use of the ascidian heparin in the treatment of colonic inflammation with a lower risk of hemorrhage, when compared with mammalian heparin.

The bivalve mollusk Nodipecten nodosus, currently cultivated in different parts of the world with an annual production of about 75,000 tons (http://www.fao.org/fishery/topic/14884/en), has been shown to contain high amounts of a heparan sulfate-like glycosaminoglycan. 1D 1^H, 2D COZY and HSQC nuclear magnetic resonance revealed characteristic signals of non-sulfated, 3- or 2-sulfated glucuronic acid, as well as N-sulfated and/or 6-sulfated glucosamine (Gomes et al., 2010). The mollusk glycan possesses an anticoagulant activity of 36 IU mg⁻¹, 5-fold lower than bovine heparin (180 IU mg⁻¹). It inhibits factor Xa (IC₅₀ = 0.835 μg mL⁻¹) and thrombin (IC₅₀ = 9.3 μg mL⁻¹) in the presence of antithrombin. Experiments in vivo, demonstrated that, the mollusk HS inhibited thrombus growth in photochemicaly injured arteries, at the dose of 1 mg Kg⁻¹. No bleeding effect, factor XIIa-mediated kallikrein activity or toxic effect on fibroblast cells were induced by the invertebrate HS at the antithrombotic dose (Gomes et al., 2010).

The ascidians S. plicata and Halocyntia pyriformis, contain dermatan sulfates formed by [α-L-IdoA(2SO₄)-1→3β-D-GalNAc(4SO₄)] disaccharide units. These oversulfated dermatans have high heparin cofactor II-mediated anticoagulant activity (Pavao et al., 1998). Due to a higher concentration of [α-L-IdoA(2SO₄)-1→3β-D-GalNAc(4SO₄)]-containing sequences, that bind to the glycosaminoglycan binding site in the inhibitor, their heparin cofactor II activities are at least 10 times higher than that of the mammalian counterpart.

The occurrence of a well-defined relationship between sulfate position on the disaccharides and biological activity can be observed studying dermatan sulfates from different species of ascidians. In the ascidian Phallusia nigra, the dermatan sulfate has the same degree of sulfation of that from S. plicata, but is composed mainly by [α-L-IdoA(2SO₄)-1→3β-D-GalNAc(6SO₄)] disaccharide units (Pavao et al., 1995). As a result of the different sulfation on the N-acetylgalactosamine (6-sulfated instead of 4-sulfated), P. nigra dermatan sulfate has very low heparin cofactor II-mediated anticoagulant activity. Overall, these results strongly suggest that binding of oversulfated dermatan sulfate polymers to heparin cofactor II requires a specific sulfation pattern on the glycans, composed by [α-L-IdoA(2SO₄)-1→3β-D-GalNAc(4SO₄)]-enriched sequences.

These unique oversulfated dermatan sulfates from ascidians, with different heparin cofactor II activities, allowed the study of the relationship between heparin cofactor II activity and antithrombotic effect. Thus, after intravascular administration S. plicata dermatan sulfate, with high heparin cofactor II activity, prevents thrombus formation in veins (Vicente et al., 2001). On the other hand, P. nigra dermatan sulfate, with a discernible heparin cofactor II activity, has no antithrombotic effect in the same venous thrombotic model and dose (Vicente et al., 2001). These results indicate that a heparin cofactor II-mediated mechanism is associated with the antithrombotic effect of dermatan sulfate polymers.

The oversulfated dermatan sulfates from the ascidian P. nigra and S. plicata, composed by [α-L-IdoA(2SO₄)-1→3β-D-GalNAc(6SO₄)] and [α-L-IdoA(2SO₄)-1→3β-D-GalNAc(4SO₄)] disaccharide unities were used in the study of mouse hippocampal neurons behavior. P. nigra dermatan possesses significant neurite outgrowth-promoting activity, which resulted specific morphological features. The ascidian dermatan sulfate induced a flattened neuronal cell soma and dendrite-like multiple neurites (Hikino et al., 2003). S. plicata dermatan sulfate, composed by [α-L-IdoA(2SO₄)-1→3β-D-GalNAc(4SO₄)], on the other hand, exhibited only a modest neurite outgrowth-promoting activity (Hikino et al., 2003; Bao et al., 2005).

Heparin has been shown to exhibit P- and/or L-selectin-mediated antimetastatic and antiinflammatory activities. P-selectin-mediated platelet-tumor cell and tumor-cell endothelium interactions facilitate the initial steps of hematogeneous metastasis (Borsig et al., 2001, 2002). The dermatan sulfates from S. plicata and P. nigra, that contain the same disaccharide core structure [α-L-IdoA(2SO₄)-1→3β-D-GalNAc]_n, but sulfated at carbon 4 or 6 of the GalNAc residues, respectively, and opposed HCII activities are potent inhibitors of P-selectin (Kozlowski and Pavao, 2011; Kozlowski et al., 2011). Both ascidian dermatan sulfates regardless of the position of sulfation on the N-acetylgalactosamine drastically attenuate metastasis of both MC-38 colon carcinoma and B16-BL6 melanoma cells, and the infiltration of inflammatory cells in a thioglycollate peritonitis mouse model (Kozlowski and Pavao, 2011; Kozlowski et al., 2011). Additionally, both ascidians glycosaminoglycans reduced thrombus size in a FeCl₃-induced arterial thrombosis model, irrespective of their HCII activities. Interestingly, the arterial thrombi demonstrated a markedly reduced platelet deposition after dermatan sulfate treatment (Kozlowski and Pavao, 2011; Kozlowski et al., 2011), suggesting that the ascidians glycosaminoglycan inhibited P-selectin and thereby the binding of activated platelets during thrombus formation. These results provide evidence that inhibition of P-selectin is a potential therapeutic target in thrombosis, inflammation and metastasis, and ascidian dermatan sulfates may serve as anti-selectin agents.

A unique natural chondroitin sulfate analog, containing sulfated fucose branches has been identified in several species of sea cucumbers (Vieira and Mourao, 1988; Vieira et al., 1991; Kariya et al., 1997). The Ludwigothurea grisea glycan has a core like that of mammalian chondroitin sulfate but substituted at the 3-position of the glucuronic acid residues with fucose-2,4 disulfated branches (Vieira and Mourao, 1988; Vieira et al., 1991). The fucosylated chondroitin sulfate has high anticoagulant and antithrombotic activities that disappear after removal of the sulfated fucose branches by mild acid hydrolysis (Mourao et al., 1996). Two anticoagulant mechanism have been proposed: activation of thrombin inhibition by heparin cofactor II, and inhibition of factor-Xa and thrombin generation by the tenase and prothrombinase complexes, respectively (Glauser et al., 2008; Buyue and Sheehan, 2009).

Interestingly, thrombosis inhibition in artery by the fucosylated chondroitin sulfate occurs at low doses, and does not modify the plasma anticoagulant activity. On the contrary, in venous thrombosis the antithrombotic activity of the fucosylated chondroitin requires high doses and occurs with an increase in the plasma anticoagulant activity (Zancan and Mourao, 2004). Additionally, daily oral doses of this glycosaminoglycan showed a decrease in thrombus weight on experimental models of venous and arterial thrombosis in experimental animals (Fonseca and Mourao, 2006).

The fucosylated chondroitin sulfate from H. grisea inhibited P- and L-selectin binding to immobilized sialyl Lewis^x, a component of leukocyte surface glycoproteins, which is also overexpressed in several tumor cells (Borsig et al., 2007). The glycan also inhibited LS180 carcinoma cell attachment to immobilized P- and L-selectins (Borsig et al., 2007). As a result of its anti-selectin effect, the intact sea cucumber glycan attenuates lung colonization by adenocarcinoma MC-38 cells in an experimental metastasis model in mice, as well as neutrophil recruitment in thioglycollate-induced peritonitis. Removal of the sulfated fucose branches abolishes the inhibitory effect in vitro and in vivo (Borsig et al., 2007). These results suggest that this glycan may be a potential therapeutic drug for blocking metastasis and inflammatory reactions.

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