A prothrombin activator serine protease from the Lonomia obliqua caterpillar venom (Lopap) biochemical characterization

Lonomia obliqua venom causes a severe consumptive coagulopathy, which can l ead to a hemorrhagic syndrome. The crude bristles extract displays a procoa gulant activity due to a Factor X and to a prothrombin activating activity. Here, we describe a 69 kDa prothrombin activator serine protease purified from L. obliqua caterpillar bristle extract using gel filtration (Sephadex G 75) and HPLC (C-4 column). The purified protein was able to activate prot hrombin in a dose-dependent manner, and calcium ions increased this activit y. The prothrombin-derived fluorogenic peptide (Abz-YQTFFNPRTGSQ-EDDnp) had its main cleavage site at the Arg-Thr bond. The kinetic parameters obtaine d for this substrate were K-mapp. of 4.5 muM, k(cat) of 5.32 s(-1), and a k (cat)/K-mapp of 1.2 x 10(6) M-1 s(-1). The prothrombin fragments generated by the purified enzyme corresponded to the molecular masses of prethrombin 2, fragment 1, fragment 2, and thrombin as seen in SDS-PAGE. The thrombin g enerated was able to clot purified fibrinogen. The partial amino acid seque nce of the purified protein, named Lopap (L. obliqua prothrombin activator protease), showed no similarity to any known prothrombin activator. (C) 200 1 Elsevier Science Ltd. All rights reserved.