Abstract
Anticoagulant therapy is used for the prevention and treatment of thromboembolic disorders. Blood coagulation is initiated by the interaction of factor VIIa (FVIIa) with membrane-bound tissue factor (TF) to form the extrinsic tenase complex which activates FX to FXa. Thus, it is an important target for the development of novel anticoagulants. Here, we report the isolation and characterization of a novel anticoagulant ringhalexin from the venom of Hemachatus haemachatus (African Ringhals Cobra). Amino acid sequence of the protein indicates that it belongs to the three-finger toxin family and exhibits 94% identity to an uncharacterized Neurotoxin-like protein NTL2 from Naja atra. Ringhalexin inhibited FX activation by extrinsic tenase complex with an IC50 of 123.8 ± 9.54 nM. It is a mixed-type inhibitor with the kinetic constants, Ki and Ki’ of 84.25 ± 3.53 nM and 152.5 ± 11.32 nM, respectively. Ringhalexin also exhibits a weak, irreversible neurotoxicity on chick biventer cervicis muscle preparations. Subsequently, the three-dimensional structure of ringhalexin was determined at 2.95 Å resolution. This study for the first time reports the structure of an anticoagulant three-finger toxin. Thus, ringhalexin is a potent inhibitor of the FX activation by extrinsic tenase complex and a weak, irreversible neurotoxin.
Highlights
Inhibits the prothrombinase complex (CY Koh, RM Kini, unpublished observations)
We focused on the purification of ringhalexin as it was found to be a potent inhibitor of factor X (FX) activation by the extrinsic tenase complex
The molecular mass and homogeneity of the purified ringhalexin was determined by electrospray ionization mass spectrometry (ESI-MS)
Summary
Inhibits the prothrombinase complex (CY Koh, RM Kini, unpublished observations). We determined the mechanism of action of a novel anticoagulant protein complex, hemextin from the venom of Ringhals cobra (Hemachatus haemachatus). The tetrameric hemextin AB complex non-competitively inhibits factor VIIa (FVIIa) with nanomolar affinity[11]. On the other hand exactin isolated from the same venom inhibited the activation of factor X (FX) by extrinsic tenase complex. Exactin showed structural similarity to short-chain neurotoxins and exhibited a weak neurotoxicity (VM Girish, RM Kini, unpublished observations). We report the identification, purification and characterization of a novel anticoagulant ringhalexin (Ringhals extrinsic tenase complex inhibitor) from the venom of H. haemachatus. Ringhalexin exhibited a mixed-type inhibition to FX activation by the extrinsic tenase complex and exhibited a weak, irreversible, neurotoxicity on chick biventer cervicis muscle (CBCM) preparations. Further we determined the three-dimensional structure of ringhalexin which revealed that it has a 3FTx fold maintained by four highly conserved disulfide bonds
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