Oreochromis niloticus has skin anticoagulant glycosaminoglycans (GAGs), but their effects on thrombin generation (TG) are unknown. This study partially characterized skin GAGs and analyzed as inhibitors of TG. Papain-extraction yield of 0.1% contained two fractions separated by DEAE-cellulose chromatography, differing on charge density and carboxylated groups by a combination of agarose/polyacrylamide electrophoresis and sequential toluidine blue/stains-all staining, presenting molecular sizes ca. 40 kDa. Depolymerization of the fractions with chondroitin ABC lyase showed dermatan sulfate (DS) as the unique GAG by agarose analysis. Both activated partial thromboplastin time (APTT) and prothrombin time tests only showed anticoagulation by fractions and mammalian DS by APTT (0.61, 0.47 and 1.72 IU, respectively) against heparin (193 IU). Fractions acted concentrationdependent on both intrinsic/extrinsic pathways in TG using 60-fold diluted human plasma, with more than 50% inactivation (41.6 and 83.3 µg), whereas DS and heparin entirely abolished at low amounts. DS from O. niloticus skin blocks in vitro TG in human plasma.Key words: Cichlidae; freshwater fish; sulfated glycans; thrombosis in vitro; waster Inibição da geração de trombina por dermatam sulfato isolado da pele de Oreochromis niloticus RESUMOOreochromis niloticus possui glicosaminoglicanos (GAGs) anticoagulantes de pele, porém desconhecidos são seus efeitos sobre geração de trombina (GT). Caracterizou-se parcialmente GAGs de pele e analisou-se como inibidores de GT. Rendimento de 0,1% da extração com papaína, conteve duas frações separadas por cromatografia de DEAE-celulose, e por combinação de eletroforese de agarose/poliacrilamida e coramento sequencial azul de toluidina/"stains-all", diferiram quanto à densidade da carga e grupos carboxilados, apresentando tamanhos moleculares ca. 40 kDa. Depolimerização das frações com condroitinase ABC mostrou, por análise em agarose, dermatam sulfato (DS) como GAG único. Ambos os testes do tempo de tromboplastina parcial ativada (TTPA) e do tempo de protrombina mostraram anticoagulação, das frações e DS de mamífero, somente pelo TTPA (0,61; 0,47 e 1,72 UI, respectivamente) contra ao da heparina (193 UI). Frações atuaram sobre ambas vias intrínsica/ extrínsica dependente de concentração na GT usando plasma humano diluído 60 vezes, com inativação mais que 50% (41,6 e 83,3 µg), enquanto em quantidades baixas DS e heparina aboliram totalmente. DS da pele de O. niloticus bloqueia GT in vitro no plasma humano. Palavras-chave:Cichlidae; peixe dulcícola; glicanos sulfatados; trombose in vitro; resíduo
Ulva lactuca (Chlorophyceae) has biotechnologically-important sulfated-polysaccharides (Ul-SPs), but their potentials on thrombin generation (TG) are unknown. This study analyzed the structural and physicalchemical features of the Ul-SPs as modulators of TG. Proteolytic digestion yielded (13.13%) extract containing sulfate (20.43%) and total sugars (65.72%), besides ulvan consisting of rhamnose, xylose, glucose, glucuronic acid and α-/β-types glycosidic linkages as characterized by one-/two-dimensions nuclear magnetic resonance (NMR) experiments. Fractionation of the Ul-SPs by DEAE-cellulose chromatography yielded Ul-SP1 and Ul-SP2 (0.50 and 0.75 M NaCl, respectively) showing sulfation (15.72-18.04%) and total sugars (59.73-60.58%) consistent with the charge density pattern by combination of agarose/polyacrylamide gel eletrophoresis using sequential staining with toluidine blue and stains-all, although with slight differences in their sizes (40 and >100 kDa, respectively). By both activated partial thromboplastin time (APTT) and prothrombin time (PT) tests, anticoagulation of the fractions was virtually detected by APTT (0.39 and 0.43 IU, respectively) against heparin (193 IU). Fractions acted differently on both intrinsic/extrinsic pathways in TG using 60-fold diluted human plasma, with 50% efficacies up to 8.3 μg, whereas at high concentrations suggested intrinsic hypercoagulability since heparin abolished both systems at low amounts. Ul-SPs block TG, but predicting thrombosis in increasing doses.
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 ...
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