Evidence for existence of a water-extractable anticoagulant in an earthworm, Lumbricus rubellus

We have isolated a water-extracted novel regulator for blood coagulation from an earthworm, Lumbricus rubellus. As a folk remedy, the earthworm has been known to facilitate blood circulation. After complete heat inactivation of endogenous proteases in the earthworm, an anticoagulant(s) was purified through ammonium sulfate fractionation and three consecutive gel permeation chromatography of Sephacryl S-300, Sephadex G-75, and G-150 by measuring activated partial thromboplastin time (APTT). The anticoagulant was further purified to 2,800 fold with a C4 reversed-phase HPLC. This activity was stable under heat (100 degrees C for 30 min) and acidic conditions (0.4 N HCl). The effects of this partially purified anticoagulant on thrombin were observed with various substrates such as N alpha-benzoyl-DL-arginine-p-nitroanilide (BApNA), H-D-phenylalanyl-L-pipecoyl-l-arginine-p-nitroanilide (S-2238), Na-p-tosyl-L-arginine methyl ester (TAME), and fibrinogen as a natural substrate. Only TAME hydrolysis, due to an esterase activity of the enzyme, was inhibited among the chromogenic substrates. In addition, the anticoagulant not only inhibited the conversion of fibrinogen to fibrin but also prolonged the fibrin clot formation monitored with the in vitro coagulation test. Based on these observations, we suggest the significance of measuring the ability of antithrombotic drugs to inhibit the esterase activity of thrombin. In this report, it was also shown that the earthworm indeed contained a water-extractable, heat- and acid-stable anticoagulant which could be used as a novel antithrombotic agent.