Molecular characteristics of polysulfated fractions from the green seaweed Caulerpa cupressoides and actions on thrombin generation in vitro

Caulerpa cupressoides produces sulfated polysaccharides (Cc-SPs) with serpin-dependent anticoagulant effect, but their actions on thrombin generation (TG) are unknown. This study aimed to partially characterize Cc-SPs and examine their potential as modulators of TG. Infrared analysis characterized extract containing three ulvan fractions (Cc-SP1, -SP2 and -SP3) separated by DEAEcellulose chromatography, with differences in the relative proportions of sulfate (10.99-18.38%) and total sugars (46.59-51.12%), without presenting proteins. Charge density patterns and nonSPs varying from 8 to > 100 kDa on agarose and polyacrylamide gel electrophoresis by sequential staining with toluidine blue and stains-all were also confirmed by gel permeation chromatography. The molecular weight of Cc-SP2 was not altered after treatment with 0.4 M HCl up to 5 h. Only Cc-SP2 altered the activated partial thromboplastin time (15 ± 0.3 IU) vs. heparin (193 IU) and abolished at high concentrations (> 4.1 μg) TG by intrinsic pathway in 60-fold diluted human plasma, while at 4.1 μg attenuated TG by 33.87% delaying the lag phase (32 min.) vs. control (28 min.). Cc-SP2 induced concentration-dependent TG in system without cephalin. Heparin abolished TG at 4.15-fold lower amount, but did not stimulate TG. Therefore, Cc-SPs express dual effects on thrombosis in vitro.

Extraction, physical-chemical characterization and in vitro inhibitorypotential of thrombin generation of crude sulfated polysaccharides from Brazilian tropical seaweeds / Extração, caracterização físico-química e potencial inibitório in vitro da geração detrombina de polissacarídeos sulfatados brutos de algas marinhas tropicais brasileiras

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) wereanalyzed 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 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. Withrespect to TG assay, crude SPs modified concentration-dependent and independently from molecular mass TGby both intrinsic/extrinsic pathways in 60-fold diluted human plasma, with total intrinsic inactivation using crudeSPs from A. muscoides in parallel to heparin (p < 0.05). Thrombosis in vitro is differentially modulated by distinctcrude SPs from Brazilian seaweeds.

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. Foramanalisadas e comparadas cinco espécies brasileiras de macroalgas (Gracilaria birdiae, Acanthophora muscoides, Halymenia sp., Caulerpa cupressoides e C. racemosa) quanto à abundância, às característicasfísico-químicas e os ensaios anticoagulantes in vitro de tempo de tromboplastina parcial ativada-TTPA, aotempo de protrombina-TP e a GT. A extração com papaína rendeu (p 100 kDa. Esses procedimentos,combinados ao uso de azul de toluidina/Stains-All, indicaram também polissacarídeos-não sulfatados. OsTTPAs 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 viasintrínseca/extrínseca no plasma humano diluído 60 vezes, com inativação intrínseca total usando PSs brutosde A. muscoides em paralelo à heparina (p < 0,05). A trombose in vitro é modulada diferencialmente porPSs brutos distintos de algas marinhas brasileiras.

In vitro inactivation of thrombin generation by polysulfated fractions isolated from the tropical coenocytic green seaweed Caulerpa racemosa (Caulerpaceae, Bryopsidales) / Inativação in vitro da geração de trombina por frações polissulfatadas isoladas da macroalga verde cenocítica tropical Caulerpa racemosa (Caulerpaceae; Bryopsidales)

Pharmacological efficacy of Caulerpa racemosa (Chlorophyta) sulfated polysaccharidic (SPs) fractions (F I→III) on models of coagulation and inflammation has been demonstrated, but not their effects on thrombin generation (TG). This study examined fractions for composition and physical-chemical characteristics and in vitro inactivation of TG by F I and F II in 60-fold diluted human plasma using continuous method. Papain-extraction yield of 0.7% revealed F I→III by DEAE-cellulose chromatography, with differences among the relative proportions of sulfate (17.37-24.00%), total sugars (30.03-48.34%) and absence of proteins. Charge density patterns and molecular sizes > 100 kDa of the fractions were verified by both agarose/polyacrylamide analyses, respectively. These electrophoreses combined with toluidine blue/Stains-All also indicated nonSPs. Anticoagulant effects of 4.76 (F I), 12.00 (F II) and 2.32 (F II) IU mg-1 by activated partial thromboplastin time test were recorded against heparin (193 IU mg-1), without changes in prothrombin time. Diluted plasma treated with F I and F II reduced concentration-dependent and sulfation pattern TG by both intrinsic and extrinsic pathways, with 50% inactivation by intrinsic pathway of F II even at 4.1 µg. Heparin abolished TG at least 4-fold lower. Therefore, C. racemosa produces SPs with TG inhibition.

Eficácia farmacológica de frações (F I→III) polissacarídicas sulfatadas (PSs) da Chlorophyta Caulerpa racemosa sobre modelos de coagulação e inflamação tem sido demonstrada, exceto seus efeitos sobre geração de trombina (GT). Examinaram-se frações quanto à composição, características físico-químicas e inativação in vitro de GT por F I e F II, em plasma humano diluído 60 vezes usando método contínuo. Rendimento de extração-papaína (0,7%) revelou, por cromatografia de DEAE -celulose, F I→III com diferenças entre as proporções relativas de sulfato (17,37-24,00%), açúcares totais (30,03-48,34%) e ausência de proteínas. Foram verificados, por ambas as análises agarose/poliacrilamida, graus de densidade de carga e tamanhos moleculares > 100 kDa das frações, respectivamente. Também essas eletroforeses, combinadas com azul de toluidina/Stains-All, indicaram polissacarídeos não sulfatados. Foram registrados, pelo teste do tempo de tromboplastina parcial ativada, efeitos anticoagulantes de 4,76 (F I), 12,00 (F II) e 2,32 (F II) UI mg-1 contra heparina (193 UI mg- 1), porém não modificando tempo de protrombina. Plasma diluído tratado com F I e F II reduziu GT por ambas as vias intrinsíca/extrínsica, dependente de concentração e grau de sulfatação, com F II em 4,1 µg apresentando eficácia de 50% pela via intrínsica. Heparina, quatro vezes menos, aboliu GT. Portanto, C. racemosa produz PSs com inibição de GT.