Glicosaminoglicano do camarão Farfantepeneaus brasiliensis: caracterização físicoquímica e atividades anticoagulante e antitrombótica

Abstract

Glycosaminoglycans from marine animals are compounds with great therapeutic potential due their structural variability. The aim of this study was to perform the extraction, Chemical characterization, and to investigate the anticoagulante and antithrombotic activities of heparam sulfate (HS) obtained from the shrimp Farfantepeneaus brasiliensis. The F2M fraction was obtained by step-wise ion Exchange resin chromatography, analyzed by agarose gel electrophoresis in two buffer sustems (PDA and Ba/PDA) its Chemical contente was determined by Chemical assays (hexosamine contente, uronic acid, sulfate and residual protein). The modal molecular weight was estimated by electrophoresis and polyacrylamide gel. The structural characteristics of HS were obtained by enymatic depolymerization using lyases from Flavobacterium heparinum (heparinase and haparitinases) and by means of RAMAN and FT-IR spectroscopic techniques. The anticoagulante activity was investigated in vitro by the USP 1965 method, aPTT, Fibrinogen Time and inhibition of factors Xa and IIa. In RAEC, the ability of HS to stimulate the synthesis of antithrombotic heparam sulfate proteoglycan (PGHS) was evaluated using metabolic sulfate labeling [S35]. Antithrombotic activity was evaluated in vivo (CEAUA/UFJF 014/2019) by the model of thrombosis induced by ligation of the inferior vena cava, in male Wistar rats, amd the aPTT in the blood of rats exposed F2M fraction was evaluated. The in vivo hemorragic activity of the F2M fraction of F. brasiliensis shrimp was performed using male Wistar rats, using the caudal scarification model. The results showed that the F2M fraction showed electrophoretic migration bands with metachromasia similar to HS, high concentration of sulfates, molecular weight of 56.86 kDa, and is composed of the disaccharides U-GlcNS, 6S e U-GlNS, commonly found in HS of terrestrial animals. The F2M fraction at a concentration of 55 μg/μl prolonged the aPTT by 8 times in relation to the negative control, significantly inhibiting factors Xa and IIa, 60% and 90%, respectively. RAEC stimulated with F2M fraction (13.7 and 27.5 μg/ mL) showed dose-dependent increase in PGHS synthesis. In the analysis of antithrombotic activity in vivo, the F2M fraction (27.5 μg/ mL) reduced the trombus formed after 1 hour of exposure by 80%. However, no prolongation on aPTT was observed When evaluated in the blood of these animals. In addition, the F2M fraction showed no significant residual bleeding when compared to heparina. The results showed that the HS obtained from the cephalothorax of the shrimp F.brasiliensis shows promissing antithrombotic and anticoagulant activity and suggest that the antithrombotic activity may not be correlated only with the inhibition of factors involved in the intrinsic pathway of blood clotting.