<b>Structural features and inactivation of coagulation proteases of a sulfated polysaccharidic fraction from <i>Caulerpa cupressoides</i> var. <i>lycopodium</i> (Caulerpaceae, Chlorophyta)</b> - doi: 10.4025/actascitechnol.v35i4.16709

Abstract

ABSTRACT. Studies on biopolymers from macroalgae suggested sulfated polysaccharides (SPs) as research agents to investigate events related to haemostasis. Caulerpa cupressoides var. lycopodium is a marine green alga containing three SPs fractions (SP 1 , SP 2 and SP 3 ). SP 2 had anticoagulant (in vitro) and anti-and prothrombotic (in vivo) actions; however, its effect on the coagulation system is not fully understood. This study aimed to determine the infrared (IR) spectroscopy, chemical composition (CC), elemental microanalysis (EM), molecular weight (MW) and the effect on coagulation proteases of SP 2 . The presence of sulfate ester, galactose-6-sulfate, uronic acid and glycoside linkages for IR spectrum; contents of sulfate (28%), total sugars (40%) and uronic acids (7.18%) for CC; and content of carbon (21.98%), sulfate (4.27%), nitrogen (1.3%) and hydrogen (4.86%) for EM were obtained. The average molecular weights of four different SPs (SP-1, SP-2, SP-3 and SP-4) subfractions from the SP 2 ranged from ~ 8 to >100 kDa. SP 2 was tested on coagulation proteases (thrombin and factor Xa) in the presence of antithrombin (AT) and heparin cofactor II (HCII) using human plasma, being both thrombin and factor Xa target proteases inhibited, but requiring a concentration of about 2.5-fold higher of HCII than the thrombin inactivation by AT.Keywords: Caulerpaceae, polysulfated, chemical analysis, coagulation, modulator action.Características estruturais e inativação de proteases da coagulação de uma fração polissacarídica sulfatada de Caulerpa cupressoides var. lycopodium (Caulerpaceae; Chlorophyta) RESUMO. Estudos sobre biopolímeros de macroalgas sugeriram polissacarídeos sulfatados (PSs) como agentes de pesquisa para investigar eventos relacionados à hemostasia. A alga marinha verde Caulerpa cupressoides var. lycopodium contém três frações de PSs (PS 1 ; PS 2 e PS 3 ). A PS 2 apresentou ações anticoagulantes (in vitro), anti-e pró-trombótica (in vivo). Entretanto, carece de investigações de seus efeitos sobre o sistema de coagulação. Objetivou-se determinar de PS 2 a espectrocopia por infravermelho (IV), composição química (CC), microanálise elementar (ME), peso molecular (PM) e os efeitos sobre proteases da coagulação. Foi obtida a presença de éster sulfato, galactose-6-sulfato, ácido urônico e ligações glicosídicas para o espectro de IV; conteúdos de sulfato (28%), açúcares totais (40%) e ácidos urônicos (7,18%) para a CC; e conteúdos de carbono (21,98%), sulfato (4,27%), nitrogênio (1,3%) e hidrogênio (4,86%) para a ME. Os pesos moleculares médios das quatro diferentes subfrações de PSs (PS-1; PS-2; PS-3 e PS-4) de SP 2 variaram de ~ 8 a >100 kDa. A PS 2 foi testada sobre proteases da coagulação (trombina e fator Xa) na presença de antitrombina (AT) e cofator II da heparina (CIIH) usando plasma humano, sendo inibidas ambas as proteases alvo trombina e fator Xa. Entretanto, requerendo uma concentração de aproximadamente 2,5 vezes maior de CIIH do que para inativação de trombina por AT.

<b>Extraction and structural properties of <i>Acanthophora muscoides </i>(Rhodophyceae) extracellular matrix sulfated polysaccharides and their effects on coagulation

“…Algae bioprospection for novel potentially active natural chemicals have revealed innumerable sulfated polysaccharides (SPs) with health-related benefits (Cardozo et al, 2007;Rodrigues et al, 2012;Rodrigues et al, 2013), generating biotechnological innovation (Prajapati et al, 2014) and economical impact (Cardozo et al, 2007;Pereira & CostaLotufo, 2012). These marine glycans of high molecular weights (Pomin, 2012;Fidelis et al, 2014) are abundantly available in the cell-walls and intercellular space as structural components of the algae (Estevez, Ciancia, & Cerezo, 2008;Rodrigues, Torres, Alencar, Sampaio, & Farias, 2009;Miguel et al, 2014).…”

<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)

“…Such observations contrasted with those found for two SPs fractions from the green seaweed C. cupressoides var. lycopodium, where average molecular masses varying from 8 to > 100 kDa were observed by polyacrylamide gel electrophoresis (Rodrigues et al, 2013;Rodrigues et al, 2017b). Ghosh et al (2004) obtained a hot water crude SP from the red seaweed C. cupressoides and found apparent molecular weights of two major peaks (F 1 and F 2) to be 120 and 70 kDa, respectively, when a size exclusion chromatography (Sephacryl S-100 column) was used.…”

“…Although no correlation with the molecular masses of the SPs fractions (C. racemosa) was attributed on the TG inactivation because the polysaccharide chains were not affected after protease treatment as visualized by polyacrylamide gel electrophoresis (Figure 1) (Athukorala et al, 2006;Rodrigues et al, 2013;Fidelis et al, 2014), our study clearly evidenced SPs as blockers of TG dependently of charge, contrasting with the SPs from the red seaweed G. birdiae that suggested stereospecific mechanisms (Rodrigues et al, 2017a) to modulate the active coagulation factors converted by thrombin in plasma (Rau et al, 2007).…”

“…This distinct inhibitory profile of the C. racemosa fractions on TG by the coagulation reaction of the intrinsic pathway stimulated by cephalin (contactactivation) could perhaps be revealed as serpinsdependent thrombin inhibitors (Rodrigues et al, 2013;Wang et al, 2014) at differential inactivation patterns to modulate thrombosis in vitro (Nishino et al, 1999;Mourão et al, 2001) because antithrombin-dependent anticoagulants do not enhance TG in plasma and have ability to attenuate thrombin activity (Furugohri, Sugiyama, Morishima & Shibano, 2011). On the contrary, the impact of sulfation on the extrinsic-pathway induced TG did not appear using both fractions F I and F II, showing that the sulfation level was not relevant in this process because the algal SPs had only a discrete inhibitory action from the positive control (thromboplastine); therefore, a significant response was not found in plasma treated with the polymers (data not shown).…”

“…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).…”