Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

Abstract

During injury or trauma, blood coagulation is initiated by the interaction of factor VIIa (FVIIa) in the blood with freshly exposed tissue factor (TF) to form the TF⅐FVIIa complex. However, unwanted clot formation can lead to death and debilitation due to vascular occlusion, and hence, anticoagulants are important for the treatment of thromboembolic disorders. Here, we report the isolation and characterization of two synergistically acting anticoagulant proteins, hemextins A and B, from the venom of Hemachatus haemachatus (African Ringhals cobra). N-terminal sequences and CD spectra of the native proteins indicate that these proteins belong to the three-finger toxin family. Hemextin A (but not hemextin B) exhibits mild anticoagulant activity. However, hemextin B forms a complex (hemextin AB complex) with hemextin A and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Complex formation was supported by size-exclusion chromatography. Using a "dissection approach," we determined that hemextin A and the hemextin AB complex prolong clotting by inhibiting TF⅐FVIIa activity. The site of anticoagulant effects was supported by their inhibitory effect on the reconstituted TF⅐FVIIa complex. Furthermore, we demonstrated their specificity of inhibition by studying their effects on 12 serine proteases; the hemextin AB complex potently inhibited the amidolytic activity of FVIIa in the presence and absence of soluble TF. Kinetic studies showed that the hemextin AB complex is a noncompetitive inhibitor of soluble TF⅐FVIIa amidolytic activity, with a K i of 50 nM. Isothermal titration calorimetric studies showed that the hemextin AB complex binds directly to FVIIa with a binding constant of 1.62 ؋ 10 5 M ؊1 . The hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require a scaffold to mediate its inhibitory activity. Molecular interactions of the hemextin AB complex with FVIIa/TF⅐FVIIa will provide a new paradigm in the search for anticoagulants that inhibit the initiation of blood coagulation.

Non-Lethal Polypeptide Components in Cobra Venom

“…The isolation and characterization of two synergistically acting anticoagulant proteins, hemextin A and hemextin B, from the venom of H. haemachatus have been recently reported [10]. Nterminal sequences and circular dichroic spectra of the native proteins indicate that these proteins belong to the three-finger toxin family (which is discussed below in section on non-enzymatic proteins).…”

“…However, the finding of new types of proteins still continues. Thus, in 2005 ohanin [9] inducing hyperlocomotion in mice and an anticoagulant hemextin AB complex [10] were described in cobra venom. Some of these proteins are present in cobra venom only (e.g., cobra venom factor, CVF), while others can be found in venoms of snakes belonging to different genera and families (e.g., cysteine rich secretory proteins, CRISPs).…”

Denmotoxin, a Three-finger Toxin from the Colubrid Snake Boiga dendrophila (Mangrove Catsnake) with Bird-specific Activity

“…the number of ␤-strands, overall morphology of the loops, and differential lengths of turns or C-terminal tails (6), lead to the recognition of varied targets and modulate the toxicity and specificity (7). Hence, 3FTXs affect a broad range of molecular targets, including ␣ 1 -nicotinic acetylcholine receptors (nAChRs; short-and longchain ␣-neurotoxins), ␣ 7 -nAChRs (long-chain ␣-neurotoxins), and ␣ 3 -and ␣ 4 -nAChRs (-toxins) (4,5); muscarinic acetylcholine receptors (muscarinic toxins) (8); L-type calcium channels (calciseptine and FS2 toxin) (9,10); integrin ␣ IIb ␤ 3 (dendroaspin) (11,12); integrin ␣ v ␤ 3 (cardiotoxin A5) (13); acetylcholinesterase (fasciculins) (14); phospholipids and glycosphingolipids (cardiotoxins) (15); and blood coagulation protein factor VIIa (16). As the interaction with such a broad spectrum of target proteins results in a variety of pharmacological effects, the understanding of the physiological role and mechanism of action supported by the architecture of 3FTXs could be of great value in the development of novel research tools or rational drug design.…”

Toxin synergism in snake venoms

“…These two toxins combine to form a hemextin AB complex, which inhibits the TF.VIIa complex formed between freshly exposed tissue factor and factor VIIa, thereby synergistically abrogating the formation of blood clots (Banerjee et al, 2005, Kini, 2006, Kini, 2011) (conceptually similar to Figure 2D). Opposite procoagulant effects are observed for the prothrombin activator, Pseutarin C, from P.…”