A blood bond: evolutionary, pathophysiological and biodiscovery implications of coagulotoxic snake venoms

Abstract

Snakebite is a massive global burden and is a neglected area of tropical diseases. Snake venom neurotoxins have been the subject of intense research efforts, albeit significantly less is known about coagulotoxins. Venoms can deleteriously affect any physiological system reachable by the bloodstream, including being able to directly interfere with the coagulation cascade. Such coagulopathic toxins may be either anticoagulant or procoagulant and snake venoms are unique in their use of procoagulant toxins for predatory purposes. Our limited knowledge into the mechanisms of action of snake venom hinders our understanding of the clinical pathologies and also restricts our use of these powerful natural products as lead compounds in drug design and development. Two snake families which exhibit coagulant properties and lead to a range of clinical pathologies are vipers, Viperidae, and non-front-fanged snakes, Colubridae. The primary focus of this thesis was to investigate and compare the coagulotoxicity effects and mechanisms of various species of known venomous snakes that effect coagulation. Species chosen for this investigation have had limited previous research conducted, thus a knowledge gap in the literature remains. These coagulant properties were investigated through a multi-disciplinary approach, highlighting key results from both families.Data chapters 2 and 3 explore venom from the boomslang (Dispholidus typus) and the twig snakes (Thelotornis species) - iconic African snakes belonging to the family Colubridae. Both species produce strikingly similar lethal procoagulant pathologies. Despite these similarities, antivenom is only produced for treating bites by D. typus, and the mechanisms of action of both venoms have been understudied. We found that T. mossambicanus produced a significantly stronger coagulation response compared to D. typus, governed by strong prothrombin activation through PIII-SVMPs. Despite similarities in symptoms and clinical manifestations, venom composition differs widely between the two species, recovered from a combined approach of venomics and transcriptomics. The neutralising capability of the available boomslang antivenom was also investigated on both species, with it being 11.3 times more effective upon D. typus venom than T. mossambicanus. Envenomation by Crotalinae such as Asian pit vipers can induce multiple clinical complications resulting from coagulopathic and neuropathic effects. Data chapters 4 - 8 investigate the Asian Pit Viper clade. While intense research has been undertaken for some species, such as Calloselasma rhodostoma, functional coagulopathic effects have been neglected. As these species’ venoms affect the blood coagulation cascade and are known to produce haemorrhagic shock in envenomed patients, we investigated their effects upon coagulation using venoms of 33 species from the Asian pit viper clade including Azemiops, Calloselasma, Deinagkistrodon, Gloydius, Hypnale, Protobothrops, Trimeresurus and Tropidolaemus. Fibrinogen was the main coagulation target among these species with many varying coagulotoxic effects exhibited. Various species including Calloselasma rhodostoma, Deinagkistrodon acutus, Hypnale hypnale, Protobothrops mangshanensis, Ovophis okinavensis and some Trimeresurus species produced net anticoagulant effects through pseudo-procoagulant clotting of fibrinogen, resulting in weak, unstable, transient fibrin clots. Protobothrops flavoviridis, P. tokarensis, Gloydius brevicaudus, G. saxatilis, G. ussuriensis and various Trimeresurus species exhibited a strong anticoagulant activity through the destructive cleavage of fibrinogen (at varying rates) directly resulting in an overall anticoagulant effect. Tropidolaemus wagleri was only weakly pseudo-procoagulant, clotting fibrinogen with only a negligible net anticoagulant effect. Azemiops feae and Tropidolaemus subannulatus did not affect clotting and T. subannulatus was investigated for its alternatively derived neurotoxicity effects, sharing Waglerin’s peptide propeptide region. Other clotting factors, such as FX(a), thrombin, FIX(a) and FXI(a), were only mildly affected with minimal inhibition found from varying species. Cross neutralisation of the Green Pit Viper monovalent antivenom ‘Thai Red Cross Green Pit Viper antivenin’ was also investigated among the Trimeresurus species, with T. gumprechti, T. hageni and T. mcgregori being unaffected. These results indicate that anticoagulation mediated by pseudo-procoagulant cleavage of fibrinogen is the basal state among Asian pit vipers, while anticoagulation produced by destructive cleavage of fibrinogen, such as that found in Protobothrops, is the derived state. This is the first in depth study of its kind highlighting extreme enzymatic variability, functional diversification and clotting diversification within one clade (Asian pit vipers) surrounding one target site, governed by variability in co-factor dependency. This study is also the first to examine in a phylogenetic context the coagulotoxic effects of related genera of Asiatic pit vipers. The results of these various chapters reveal substantial variation between sister genera, providing crucial information about clinical effects and implications for antivenom cross-reactivity among venomous snake species. This study increases our understanding of not only the biodiscovery potential of these medically important species but also increases our knowledge of the pathological relationship between venom and the human coagulation cascade. These results add to the body of knowledge necessary to inform clinical management of the envenomed patient, and detail the diversity of components and characteristics which enable venom research to be such a biodiscovery treasure trove in unlimited diversity, leading to novel drug design surrounding this field.