Sulfated agaran with 4,6-pyruvated form from red seaweed Acanthophora muscoides attenuates thrombin formation: in vitro and ex vivo studies

Abstract

In vitro studies have described the sulfated agaran from Acanthophora muscoides as an intrinsic inhibitor of thrombin generation (TG), but not in ex vivo assay. This investigation partially characterized a pyruvate fraction with in vitro and ex vivo effects on an intrinsic/extrinsic pathway-induced thrombin generation (TG) continuous model using 36 or 60-fold diluted mice or defibrinated, normal human plasma. Fraction separated by DEAE-cellulose chromatography exhibited charge homogeneity and non-sulfated polysaccharides (<100 kDa) by agarose and polyacrylamide gel electrophoresis, respectively, using Stains-all alone. Fourier Transform Infrared and Nuclear Magnetic Resonance studies indicated a 4,6-pyruvated agaran-structure. The fraction and heparin had no effect on prothrombin time, but there was a preponderant intrinsic rather than extrinsic pathway inhibition in TG assay; themselves, acting on both free and fibrin bound thrombin activity without chromogenic substrate interaction. Both fractions, desulfated and native, anticipated and induced thrombin formation in activators-devoid or normal plasma. In addition, mice pretreated with fraction (20 mg kg^-1, intraperitoneally) reduced intrinsically plasma TG ex vivo after 2h. Heparin suppressed TG in vitro, but induced it ex vivo. Therefore, agaran from A. muscoides blocks TG on in vitro and ex vivo studies, suggesting to evaluate the blood coagulability status.