Red algae sulfated polysaccharides (SPs) have been widely described as anticoagulant and antithrombotic agents; however no description of antithrombotic activity regarding green algae SPs has been reported. Caulerpa cupressoides (Chlorophyta) has three different SPs fractions (SP1, SP2 and SP3). We investigated the effects of SP2 on thrombin activity by antithrombin and in an experimental model of venous thrombosis in rats. The inhibition of thrombin assay was evaluated using antithrombin (AT) in the presence of SP2 and the antithrombotic activity was investigated in rats with thromboplastin as the thrombogenic stimulus. The anticoagulant effects of SP2 are suggested be due to the potentiation of thrombin inhibition by antithrombin (IC50 ~ 10.0µg mL-1) and this mechanism of interaction is different when compared to other studied Caulerpa polysaccharides. SP2 exhibited antithrombotic effects at doses of 1.0 and 2.0mg kg-1 body weight, but at higher doses (>2.0mg kg-1 body weight) this polysaccharide revert the antithrombotic property. No hemorrhagic effect (2.0mg kg-1) was observed. As occurs with red algae SPs, these results indicate that green algae SPs are also capable of exhibiting different in vivo properties.
Heparin is an intracellular product of vertebrate mast cell currently used as exogenous anticoagulant. Despite of the potent biological activities of exogenous heparin, its physiological function has not been clearly established yet. Here, a heparin with similar structure and anticoagulant properties to the mammalian counterpart was shown to occur as the intracellular product of test cells, a cell monolayer that surrounds egg of the invertebrate Styela plicata (Chordata-Tunicata). As in the case of mammalian mast cells, heparin from the ascidian test cells is removed from the intracellular granules after incubation with compound 48/80. Following fertilization, the test cells surrounding the developing larva still retain heparin as metachromatic granulation. In the adult invertebrate, heparin occurs as intracellular granules at the apical tip of epithelial cells surrounding the lumen of both intestine and pharynx, in close contact with the external environment. This is the first description of the presence of heparin in cytoplasmic granules of epithelial-like cells around the lumen of sites exposed to external agents. This arrangement may reflect the participation of heparin in defense mechanisms in this invertebrate.
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.
The results presented in this paper show that collagen fibers can be clearly distinguished from reticular fibers using the picrosirius-polarization method. A morphologic and morphometric study of these two types of fibers with electron microscopy shows that reticular fibers are characterized by the smaller diameter of their fibrillar components and the higher content of interfibrillar material, resulting in a loose arrangement of the fibrils. The evidences presented suggest that the amorphous matrix in which fibrils are embedded is responsible for the silver impregnation of reticular fibers. Our results show that the matrix of reticular fibers is characteristically rich in heparitin sulfate, and that the glycosaminoglycans present show a high interaction with the fibrillar component of these fibers.
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
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