Mild-acid hydrolysis of a native polysulfated fraction from Acanthophora muscoides generates sulfated oligosaccharides displaying in vitro thrombin generation inhibition

Abstract

A. muscoides (Rhodophyta) has three polysulfated fractions (-1, -2 and Am-3). Am-2 displayed anti-inflammation and serpin-independent anticoagulation effects; however, no effect of oligomers on thrombin-generation (TG) has been demonstrated. This study employed mild-acid hydrolysis to obtain low-molecular-size derivatives from Am-2 and compared in vitro inhibitory effects between intact Am-2 and its hydrolysates on a TG assay. The polysaccharidic extract was fractionated by DEAE-cellulose that revealed Am-2 eluted with 0.75-M NaCl containing sulfate (23%), hexoses (51%) and absence of proteins, and indicating, by one-dimension nuclear magnetic resonance, structure of galactan similar to that of the extract. The depolymerization with HCl (0.02 or 0.04-M, 60°C) for different times progressively reduced the charge density and the molecular-size of Am-2 based on electrophoresis in agarose and polyacrylamide gels, respectively, where at higher acid concentration and critical time up to 5h yielded fragment of ̴ 14-kDa similar to that of unfractionated heparin (UHEP). Regarding the TG assay, intact Am-2 inhibited concentration-dependent intrinsic pathway, whereas its hydrolysates abolished it like UHEP, except the analog fragment (92.87% inhibition), when in 60-fold diluted human plasma using chromogenic method in a continuous system. The results reveal an alternative approach for the production of oligosaccharides from A. muscoides with TG inhibition.

<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)

“…The combined method of electrophoreses and toluidine blue/Stains-All staining to reveal Caulerpa nonSPs was also important for distinguishing other glycans present in the fractions, when the polysaccharides were coeluted from the DEAE-cellulose column (Rodrigues et al, 2017a, b), given the difficult in the determination of sugar composition after chemically-modified polysaccharides (Rodrigues et al, 2016 Collectively, the use of this methodology could also be useful for the co-detection of acidic components naturally occurring in coenocytic structures of green seaweeds, as already reported for SPs isolated from other natural sources (Volpi & Maccari, 2002;Rodrigues et al, 2017a, b). Further studies on the sensitivity to heavy metal-acid polysaccharide complexes are also needed since the concentrations of glycans are regulated in response to environmental stresses (Cardozo et al, 2007;Wang et al, 2014).…”

“…The metachromatic fractions (F I, F II and F III) were further freeze-dried and examined for their contents of sulfate (Dogdson & Price, 1976), total sugars (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) and proteins (Bradford, 1976), while their purity and molecular mass were revealed by agarose (Dietrich & Dietrich, 1976) and polyacrylamide (Rodrigues et al, 2016) analyses, respectively, using toluidine blue/Stains-All as staining reagents (Volpi & Maccari, 2002) by comparison with the electrophoretic mobility of standards dextran sulfate ( ̴ 8 kDa), chondroitin-4-sulfate ( ̴ 40 kDa) and chondroitin-6-sulfate ( ̴ 60 kDa) (Rodrigues et al, 2016;Salles et al, 2017). Since F I and F II were available in higher quantities, more refined in vitro coagulation experiments focused only on these fractions.…”

“…Zhang et al (2014) demonstrated that the fucoidan derived from the brown seaweed Fucus vesiculosus showed both proand anticoagulant effects using calibrated automated thrombography. Studies by Rodrigues et al (2016) revealed that a native SPs fraction and its various chemically (HCl treated)-modified products from the red seaweed Acanthophora muscoides attenuated coagulation status activated by the use of cephalin in an in vitro TG continuous system. Recently, SPs isolated from Brazilian samples of Gracilaria birdiae (Gracilariales, Rhodophyceae) exerted in vitro inhibitory potential on a chromogenic TG assay in diluted human plasma (Rodrigues et al, 2017a).…”

“…Although effective, UHEP develops thrombocytopenia and bleeding being necessary to monitor its anticoagulation using the APTT test (Mourão et al, 2015) since UHEP significantly decreases TG . This therapeutic agent is mainly extracted from pig and bovine intestine and its potential risks of chemical contamination with other glycosaminoglycans for use in cardiac surgeries also justify the current efforts to develop alternatives to UHEP (Mourão & Pereira, 1999;Pomin, 2012;Rodrigues et al, 2016).…”

“…Of all algae products, extracellular matrix sulfated polysaccharides (SPs) comprise a diverse group of biomaterials exhibiting high degree of sulfation (S=O, sulfate radicals) and molecular distribution of more than 100 kDa, which are capable of displaying anticoagulant, antithrombotic and anti-inflammatory effects (Pomin, 2012;Mourão, 2015;Rodrigues et al, 2012a;Rodrigues et al, 2016). Their functionalities as gelling and stabilizing agents make them also economically attractive for hydrocolloid industry (Cardozo et al, 2007).…”