Ringhalexin from Hemachatus haemachatus: A novel inhibitor of extrinsic tenase complex.

Snake venoms constitute a pharmacological repertoire of various proteins and polypeptides. Venom usually contains hundred or more different proteins that belong to various structural super-families like three-finger toxins (3FTxs), C-type lectin like proteins, phospholipase A2s, serine proteases and metalloproteases1,2,3. 3FTxs are a well-characterized family of non-enzymatic polypeptides containing 60 to 74 amino acid residues4,5. These proteins are abundant in elapid (cobras, kraits and mambas), hydrophiid (sea snakes) and colubrid venoms6 and have also been identified in viper venoms7,8. They contain four to five disulfide bridges, four of which are strictly conserved. They show an analogous pattern of folding, in which three β-stranded loops extend from a central core containing the four conserved disulfide bridges resembling the three outstretched fingers of a hand4,5. Due to this appearance, this family of proteins is named as 3FTxs. Despite their structural similarity, 3FTxs display a wide range of functional diversity2,5. They can be broadly classified into neurotoxins9, cardiotoxins/cytotoxins10 and anticoagulants11 based on their mechanism of action. Snake venom neurotoxins target the neuromuscular junctions in the peripheral and central nervous system thereby interfering with cholinergic transmissions12. They can be categorized into α-neurotoxins, κ-toxins and muscarinic toxins that target muscle nicotinic acetylcholine receptors (nAChR), neuronal nAChR and various subtypes of muscarinic receptors, respectively. The short-chain and long-chain α-neurotoxins bind to muscle αβγδ nAChR with equipotency. However, only long-chain but not short-chain neurotoxin bind to neuronal α7 receptor with high affinity13,14. κ-Toxins bind specifically to neuronal (α3β4) nAChR whereas muscarinic toxins specifically and selectively targets various subsets of muscarinic acetylcholine receptors15,16. Cardiotoxins/cytotoxins though structurally resemble to short-chain neurotoxins exhibit cardiotoxic and cytolytic effects by targeting phospholipid membranes10,17,18 whereas anticoagulant 3FTxs inhibits a specific protease or coagulation complex in the coagulation cascade. We are specifically interested to study anticoagulant 3FTxs because of the paucity of knowledge on their structure-function relationships. The anticoagulant and antiplatelet effects of 3FTxs was first described from the venom of Naja nigricollis crawshawii19. However, the mechanisms of anticoagulant activity of 3FTx were unknown. Recently, we have characterized a 3FTx, naniproin from N. nigricollis crawshawii venom, which specifically inhibits the prothrombinase complex (CY Koh, RM Kini, unpublished observations). We also 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 affinity11. On the other hand exactin isolated from the same venom inhibited the activation of factor X (FX) specifically by extrinsic tenase complex. Interestingly, exactin showed structural similarity to short-chain neurotoxins and exhibited a weak neurotoxicity (VM Girish, RM Kini, unpublished observations). Here 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 also 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.