Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus

Abstract

Unwanted clots lead to heart attack and stroke that result in a large number of deaths. Currently available anticoagulants have some drawbacks including their non-specific actions. Therefore novel anticoagulants that target specific steps in the coagulation pathway are being sought. Here we describe the identification and characterization of a novel anticoagulant protein from the venom of Hemachatus haemachatus (African Ringhals cobra) that specifically inhibits factor X (FX) activation by the extrinsic tenase complex (ETC) and thus named as exactin. Exactin belongs to the three-finger toxin (3FTx) family, with high sequence identity to neurotoxins and low identity to the well-characterized 3FTx anticoagulants-hemextin and naniproin. It is a mixed-type inhibitor of ETC with the kinetic constants, Ki’ and Ki determined as 30.62 ± 7.73 nM and 153.75 ± 17.96 nM, respectively. Exactin does not bind to the active site of factor VIIa and factor Xa based on its weak inhibition (IC50 ≫ 300 μM) to the amidolytic activities of these proteases. Exactin shows exquisite macromolecular specificity to FX activation as compared to factor IX activation by ETC. Exactin thus displays a distinct mechanism when compared to other anticoagulants targeting ETC, with its selective preference to ETC-FX [ES] complex.

Multifunctional Toxins in Snake Venoms and Therapeutic Implications: From Pain to Hemorrhage and Necrosis

“…Inhibits platelet aggregation (Girish and Kini, 2016) Inhibits Factor X (McDowell et al, 1992) Cytotoxins induce necrosis in skeletal muscle (Ownby et al, 1993).…”

“…For example, αneurotoxins inhibit muscle acetylcholine receptors (nAChR) (Changeux, 1990), κ-neurotoxins inhibits neuronal AChR (Grant and Chiappinelli, 1985), muscarinic toxins inhibit muscarinic receptors (Marquer et al, 2011), fasciculins inhibit acetylcholinesterase (AChE) (Marchot et al, 1998), calciseptine modulates L-type calcium channels (De Weille et al, 1991;Garcia et al, 2001), cardiotoxins interact non-specifically with phospholipids (Konshina et al, 2017), or induce insulin secretion (Nguyen et al, 2012), mambin interacts with platelet receptors (McDowell et al, 1992), exactin inhibits Factor X (Girish and Kini, 2016), β-cardiotoxins inhibit β-adrenoreceptors (Rajagopalan et al, 2007), MTα inhibits α-adrenoreceptors (Koivula et al, 2010), mambalgins inhibit ASIC channels (Diochot et al, 2012), Tx7335 that activates potassium channels (Rivera- Torres et al, 2016) and calliotoxin activates voltagegated sodium channels (Na V ) (Yang et al, 2016). Their toxic biological effects include flaccid or spastic paralysis due to the inhibition of AChE and ACh receptors (Grant and Chiappinelli, 1985;Changeux, 1990;Marchot et al, 1998;Marquer et al, 2011), and activation of Na V 1.4 (Yang et al, 2016) and L-type calcium channels (Garcia et al, 2001) in the periphery, necrosis through the action of cardiotoxins (cytotoxins) (Konshina et al, 2017), alteration of the cardiac rate through modulation of αand β-adrenoreceptors (Rajagopalan et al, 2007;Koivula et al, 2010), and altered homeostasis through inhibition of platelet aggregation (McDowell et al, 1992) and Factor X (Girish and Kini, 2016). In addition to their multitude of bio-activities, 3FTXs can remarkably display toxicities that target distinct classes of organisms as demonstrated in non-front fanged snake venoms that produce 3FTX isoforms which are non-toxic to mice but highly toxic to lizards, and vice-versa (Modahl et al, 2018b).…”

Mechanisms Responsible for the Anticoagulant Properties of Neurotoxic Dendroaspis Venoms: A Viscoelastic Analysis

“…As displayed in Table 1, unlike most venoms derived from Elapidae snakes, Dendroaspis venoms are predominantly composed of non-enzymatic neurotoxins such as 3-finger toxins (3-FTx) and Kunitz-type serine protease inhibitors (Kunitz-type SPI), with metalloproteinases (SVMPs) comprising less than 7% [13][14][15] and PLA 2 comprising from zero to less than 0.15% [13] of the proteomes. As D. polylepis venom has a greater amount of Kunitz-type SPI than 3-FTx compared to the other three species, and given that isolated 3-FTx have acted as plasmatic anticoagulants [16,17], perhaps the predominance of 3-FTx in the other mamba species' venom that was tested could explain the differences in anticoagulant potency [11,12]. Nevertheless, given that the vast majority of enzymes in the coagulation cascade are serine proteases, the possibility that key Kunitz-type SPI may play a role in venom-mediated anticoagulation should not be discounted.…”

Toxins Are an Excellent Source of Therapeutic Agents against Cardiovascular Diseases

“…156 In contrast, exactin and ringhalexin are both mixed-type inhibitors of the extrinsic tenase complex, binding to the enzyme complex (FVIIa-tissue factor), with or without the substrate (FX). However, exactin has a better affinity for the enzyme-substrate complex, 157 whereas ringhalexin has a better affinity for the enzyme complex without substrate. 158…”