Zinc-dependent prothrombin activator from Rhabdophis tigrinus venom with extended substrate specificity

Rhabdophis tigrinus, a common venomous snake in Japan, produces venom with potent procoagulant activity through activation of human prothrombin. However, the biochemical properties and classification of its prothrombin activator remain unclear. In this study, we partially purified the prothrombin activator from R. tigrinus venom (provisionally termed "Rhabdarin") and characterized its enzymatic and structural properties. The enzyme was partially purified using gel filtration and hydroxyapatite chromatography, yielding a disulfide-linked homodimer (∼110 kDa non-reducing; ∼55 kDa reducing). Rhabdarin induced clotting in human citrated plasma without the need for calcium, phospholipids, or factor V, consistent with a Group A-type activator. SDS-PAGE analysis revealed dose- and time-dependent cleavage of prothrombin into meizothrombin desF1, α-thrombin, and fragments 1 and 2, with cleavage confirmed at Arg320. Notably, unlike typical serine proteases, Rhabdarin failed to cleave the small chromogenic substrates S-2222 and S-2765 but showed clear activity toward a synthetic fluorescent peptide mimicking the native cleavage site. This strictly sequence-context-dependent specificity distinguished it from classical coagulation proteases. Inhibitor studies demonstrated complete loss of activity with ethylenediamine-N,N,N',N'-tetraacetic acid, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid, and the zinc chelator 1,10-phenanthroline, but not with phenylmethylsulfonyl fluoride, confirming that Rhabdarin is a zinc-dependent metalloprotease. Consistent with this biochemical classification, LC/MS analysis of the partially purified protein identified peptide fragments from conserved regions of snake venom metalloproteinases, including the ZnMc_adamalysin_II-like domain. Together, these findings demonstrate that Rhabdarin is a zinc-dependent Group A prothrombin activator exhibiting extended substrate recognition, offering new insights into the classification and mechanism of this unique procoagulant enzyme.