Anticoagulant activity of a sulfated galactan: Serpin-independent effect and specific interaction with factor Xa

Abstract

SummaryAn algal sulfated galactan has high anticoagulant and antithrombotic activities. Its serpin-dependent anticoagulant action is due to promoting thrombin and factor Xa inhibition by antithrombin and heparin cofactor II. Here, we evaluated the anticoagulant effect of the algal sulfated galactan using serpin-free plasma. In contrast to heparin, the sulfated galactan is still able to prolong coagulation time and delay thrombin and factor Xa generation in serpin-free plasma. We further investigated this effect using purified blood coagulation proteins, discovering that sulfated galactan inhibits the intrinsic tenase and prothrombinase complexes, which are critical for factor Xa and thrombin generation, respectively. We also investigated the mechanism by which sulfated galactan promotes factor Xa inhibition by antithrombin using specific recombinant mutants of the protease. We show that sulfated galactan interacts with the heparin-binding exosite of factor Xa and Arg-236 and Lys-240 of this site are critical residues for this interaction, as observed for heparin. Thus, sulfated galactan and heparin have similar high-affinity and specificity for interaction with factor Xa, though they have differences in their chemical structures. Similar to heparin, the ability of sulfated galactan to potentiate factor Xa inhibition by antithrombin is calcium-dependent. However, in contrast to heparin, this effect is not entirely dependent on the conformation of the γ-carboxyglutamic acid-rich domain of the protease. In conclusion, sulfated galactan and heparin have some similar effects on blood coagulation, but also differ significantly at the molecular level. This sulfated galactan opens new perspective for the development of antithrombotic drugs.

<b><i>In vitro</i> inactivation of thrombin generation by polysulfated fractions isolated from the tropical coenocytic green seaweed <i>Caulerpa racemosa</i> (Caulerpaceae, Bryopsidales)

“…These results allowed us to postulate that the modulation of TG by intrinsic pathway in the presence of UHEP were in concordance with the automated method (Jun et al, 2014), but occurred at concentration 5-fold lower than that of Glauser et al (2009), who reported no inhibition of TG by UHEP up to 10 μg.…”

“…In these connections, decrease in TG by C. racemosa SPs reflected a different mode of action from the other classes of polysulfated that have diverse structures and mechanisms of thrombin inhibition (Nishino et al, 1999;Mourão et al, 2001;Glauser et al, 2009;Zhang et al, 2014;Rodrigues et al, 2016) distinct from that of UHEP, which abolished TG at 2 (intrinsic pathway) (Figure 2) (Rodrigues et al, 2016;Rodrigues et al, 2017b) or 4 (extrinsic pathway) (data not shown) μg well-plate -1 (Rodrigues et al, 2017a;Salles et al, 2017) because of its specific pentasaccharide sequence with high antithrombin affinity displaying thrombin inhibition (Mourão, 2015). These results allowed us to postulate that the modulation of TG by intrinsic pathway in the presence of UHEP were in concordance with the automated method (Jun et al, 2014), but occurred at concentration 5-fold lower than that of Glauser et al (2009), who reported no inhibition of TG by UHEP up to 10 μg.…”

“…The action of these compounds would be mediated by serpins (antithrombin and heparin II cofactor) (Rodrigues et al, 2013;Wang et al, 2014) because galactose-rich polysaccharides reveal as inhibitors of thrombin (Hayakawa et al, 2000;Ghosh et al, 2004), but the relationship between structure and anticoagulation still lacks in-depth details since the existence of complexity and heterogeneity of the algae glycans make comparison difficult with UHEP at molecular level and bioactivity (Athukorala et al, 2006;Pomin, 2012;Fidelis et al, 2014;Mourão, 2015;Rodrigues et al, 2016). TG assays may measure several feedback reactions to evaluate diverse classes of SPs regarding their anticoagulant dynamics (Nishino et al, 1999;Mourão et al, 2001;Glauser et al, 2009;Zhang et al, 2014;Rodrigues et al, 2017b;Salles et al, 2017).…”

“…In summary, C. racemosa features SPs have potential applicability as novel, biomaterial to prevent thrombosis in vitro as demonstrated in the TG assay and test parameters could indicate to studies of their underlying mechanisms (Glauser et al, 2009;Rodrigues et al, 2016) when an increase of plasma prothrombin's referential values increase the TG after activation by both intrinsic and extrinsic pathways (Rao et al, 2007;Castoldi & Rosing, 2011).…”

“…For example, SPs derived from the sea cucumber Ludwigothurea grisea (Mourão et al, 2001) and from the seaweeds Ecklonia kurome (Ochrophyta) (Nishiro, Fukuda, Nagumo, Fujihara & Kaji, 1999) and Botryocladia occidentalis (Rhodophyta) (Glauser et al, 2009) inactivated TG after induction by both contact-activated and thromboplastin-activated systems and/or by the prothrombinase complex. Zhang et al (2014) demonstrated that the fucoidan derived from the brown seaweed Fucus vesiculosus showed both proand anticoagulant effects using calibrated automated thrombography.…”