<b>Extraction, physical-chemical characterization and <i>in vitro</i> inhibitory potential of thrombin generation of crude sulfated polysaccharides from Brazilian tropical seaweeds

Abstract

ABSTRACT. The biotechnological value of macroalgae for screening assays of thrombin generation-TG using sulfated polysaccharides-SPs as substitutes to heparin has been poorly explored. Five Brazilian species of macroalgae (Gracilaria birdiae, Acanthophora muscoides, Halymenia sp., Caulerpa cupressoides and C. racemosa) were analyzed and compared for their abundance, physical-chemical characteristics and in vitro anticoagulant assays of activated partial thromboplastin time-APTT, prothrombin time-PT and TG. Papain extraction yielded (p < 0.001) from 0.66±0.03% (C. racemosa) to 41.60±1.10% (Halymenia sp.) of crude SPs varying sulfate (8.41-42.60%) and total sugars (47.80-70.53%). Crude SPs showed difference in mobility and resolution pattern by agarose electrophoresis, while polyacrylamide analysis revealed SPs of > 100 kDa. These procedures, combined with the use of Stains-All, also indicated nonSPs. APTTs ranged from 2.81 (A. muscoides) to 21.30 IU (Halymenia sp.) vs. heparin (193 IU), and were dependent on sulfation of the crude SPs. PT was not altered. With respect to TG assay, crude SPs modified concentration-dependent and independently from molecular mass TG by both intrinsic/extrinsic pathways in 60-fold diluted human plasma, with total intrinsic inactivation using crude SPs from A. muscoides in parallel to heparin (p < 0.05). Thrombosis in vitro is differentially modulated by distinct crude SPs from Brazilian seaweeds.Keywords: marine algae; polyglycans; coagulation tests; thrombin.Extração, caracterização físico-química e potencial inibitório in vitro da geração de trombina de polissacarídeos sulfatados brutos de algas marinhas tropicais brasileiras RESUMO. O valor biotecnológico das macroalgas para ensaios de varredura de geração de trombina-GT pouco tem sido explorado usando polissacarídeos sulfatados-PSs como substitutos à heparina. Foram analisadas e comparadas cinco espécies brasileiras de macroalgas (Gracilaria birdiae, Acanthophora muscoides, Halymenia sp., Caulerpa cupressoides e C. racemosa) quanto à abundância, às características físico-químicas e os ensaios anticoagulantes in vitro de tempo de tromboplastina parcial ativada-TTPA, ao tempo de protrombina-TP e a GT. A extração com papaína rendeu (p < 0,001) de 0,66±0,03% (C. racemosa) a 41,60±1,10% (Halymenia sp.) de PSs brutos variando sulfato (8,41-42,60%) e açúcares totais (47,80-70,53%). Os PSs brutos mostraram diferenças na mobilidade e resolução por eletroforese em agarose, enquanto pela análise em poliacrilamida revelou PSs brutos de >100 kDa. Esses procedimentos, combinados ao uso de azul de toluidina/Stains-All, indicaram também polissacarídeos-não sulfatados. Os TTPAs foram dependentes da sulfatação dos PSs brutos e variaram de 2,81 (A. muscoides) a 21,30 UI (Halymenia sp.) vs. heparina (193 UI). O TP não foi alterado. Com respeito ao ensaio de GT, os PSs brutos modificaram, dependente de concentração e independentemente de massa molecular, GT pelas vias intrínseca/extrínseca no plasma humano diluído 60 vezes, com inativação intrínseca ...

<b>Structural and physical-chemical analyses of sulfated polysaccharides from the sea lettuce <i>Ulva lactuca</i> and their effects on thrombin generation

“…The limited values proved by classical coagulation models on the amount of thrombin generated (Castoldi & Rosing, 2011;Duarte et al, 2017) to measure plasma anticoagulants (Table 2) (Jung et al, 2014;Rodrigues et al, 2017a;Rodrigues et al, 2017c) led us to further evaluate possible effects of SPs from U. lactuca on an in vitro TG system. In this assay, different concentrations (4.1, 8.3, 41.6 and 83.3 μg well-plate -1 ) (Rodrigues et al, 2016b;Rodrigues et al, 2017a;Rodrigues et al, 2017b;Rodrigues et al, 2017c;Rodrigues et al, 2018) of both fractions Ul-SP1 and Ul-SP2 added to 60-fold diluted human plasma attenuated TG in contact-activated system (intrinsic pathway) and thromboplastin-activated system (extrinsic pathway) (Nishino et al, 1999;Glauser et al, 2009), when the amidolytic activity of thrombin was continually measured at 37°C for 60 min., using in parallel UHEP as standard anticoagulant. No activator response of TG in plasma in the absence of cephalin or thromboplastin (negative controls) was observed in vitro for 60 min.…”

“…No activator response of TG in plasma in the absence of cephalin or thromboplastin (negative controls) was observed in vitro for 60 min. (Rodrigues et al, 2016b;Salles et al, 2017;Rodrigues et al,2018) (Figure 3).…”

“…Contrasting result was found for the SP isolated from the brown seaweed Fucus vesiculosus that acted as activator of TG by tissue factor pathway using calibrated automated thrombography (Zhang et al, 2014). However, studies on the biological role of the SPs isolated from the Chlorophyta species on in vitro TG inhibition assays are lacking in the literature (Rodrigues et al, 2017b;Rodrigues, Benevides, Tovar, & Mourão, 2017c;Rodrigues et al, 2018).…”