Effects of Australian snake venoms on coagulation: Differential potency, biochemistry, and antivenom efficacy Christina N. Zdenek BSc., MPhil A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2020 School of Biological Sciences Abstract Venomous snakes are a valuable bio-resource for potential therapeutic drug design and development yet are also a major health hazard for society. Snakebite is a globally neglected tropical disease which disproportionately affects the poor and can cause debilitation and death. Snake antivenom is the only recommended treatment for snakebite, yet antivenom efficacy is often untested against many species for which it may be prescribed. In lieu of unethical testing in vivo, venom and antivenom tests in vitro can inform clinicians treating human and pet (domestic and livestock) snakebite victims. Australian venomous snakes (family: Elapidae; subfamily Hydrophiinae) are a specious group of some of the most venomous snakes in the world, whose bites can cause life-threatening coagulopathy (ie. disruption of blood haemostasis). This thesis focuses on Australian elapid venom effects on plasma—primarily human plasma—and the efficacy of currently available antivenoms. Chapter 1 introduces the main concepts and coagulotoxic theme and includes some redundancy with the introductions of individual chapters because all data chapters have either been published as papers or are prepared as such. For this same reason, knowledge gaps are identified within each chapter’s introduction, rather than in Chapter 1. Chapter 2 focuses on the two most coagulotoxic genera within Elapidae: taipans (Oxyuranus) and brown snakes (Pseudonaja). The toxins (FXa:FVa) responsible for the exceptional coagulotoxicity of these venoms are a complex of snake venom Factor Xa, and the cofactor Factor Va. These toxins are homologous to mammalian prothrombinase complex– an enzyme complex which is central to the blood coagulation cascade. The efficacy of a newly developed antivenom was found to be surprisingly specific to the genus of snake in the immunising mixture (Oxyuranus) and largely ineffective against coagulotoxins within similarly related species (Pseudonaja). This likely renders the more affordable, new antivenom an unsuitable solution for snakebite by the eastern brown snake (Pseudonaja textilis) in Papua New Guinea. Chapter 3 broadens the scope of the thesis to include an unprecedented sample size of 47 Australian venoms across 42 species and 19 genera, investigating Australian elapid venom effects on human plasma. This timely research uncovered an additional four genera that contain species with previously unknown potent procoagulant venom. Tiger Snake Antivenom was broadly cross-reactive but varied extensively in relative efficacy across venoms. This chapter also examines the molecular evolution of the prominent coagulotoxin within these venoms—Factor Xa—which revealed a clear, stepwise evolution of increased cleavage region and potency via diversifying selection. Chapter 4 investigates the anticoagulant properties of all currently recognised species of black snakes (Pseudechis) and nine localities of mulga snake (P. australis)– the most widely dispersed and medically significant Pseudechis species. Specifically, this chapter determined the mechanism of anticoagulant action of these venoms, as well as their relative response to Black Snake Antivenom. All venoms demonstrated relatively similar potency and were similarly well-neutralised by antivenom, except for P. colletti, which was well neutralised in subsequent tests by Tiger Snake Antivenom (its prescribed antivenom) and extremely well neutralised by a commercially available phospholipase A2 inhibitor drug, called LY315920 (trade name: Varespladib). Geographical variation was observed for P. australis venoms, with the northern and eastern localities significantly more potent than the western, central, and southern localities. The mechanism of anticoagulant action appears conserved across these localities—and all species within Pseudechis. In contrast to previous speculation, the anticoagulant target in these venoms was determined to be multiple factors within the coagulation cascade, not including FXa, but predominantly inhibition of FVa and its formation with the prothrombinase complex. Chapter 5 extends the anthropomorphic focus of this thesis to prized pets—cats and dogs— investigating the relative susceptibility of these two taxa against a carefully selected group of procoagulant snake venoms which accomplish pathological effects through a range of different molecular mechanisms. An unusual phenomenon uncovered in 1998 inspired this test: while 31% of dogs survive eastern brown snake (P. textilis) bites without antivenom, cats survive more than twice as many bites (66%). As expected, dog plasma was more susceptible to procoagulant snake venoms, with quicker venom-induced clotting times than cats (as well as humans). This physiological susceptibility of dog plasma likely is a major factor contributing to the lower survivability of dogs compared to cats in P. textilis envenomations. Further exacerbating the greater susceptibility of dogs to snakebite is, more than likely, predictable behavioural differences pre– and post-bite, which almost certainly further explain the lower survivability in dogs to snakebite compared to cats. Chapter 6 summarises all results and discusses them regarding clinical implications and antivenom production. The research described in this PhD thesis provides clinicians with greater insight into venom effects of medically significant snakes and lesser known species, as well as antivenom efficacy. This knowledge can inform management treatment plans of envenomed patients in the ongoing struggle to reduce the burden of snakebite on society. I conclude that Australian elapid venoms have evolved remarkably successful tricks with which to immobilise their prey, and, despite the catastrophic effects they sometimes produce in human snakebite victims, these venoms may help with the development of haemostatic tools and therapeutic drugs to save lives. Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. Publications included in this thesis Incorporated as Chapter 2, ‘Browns/Taipans’, published Zdenek, C.N., Hay, C., Arbuckle, K., Jackson, T.N.W., Bos, M.H.A., op den Brouw, B., Debono, J., Allen, L., Dunstan, N., Morley, T., Herrera, M., Gutiérrez, J. M., Williams, D. J. Fry, B. G. (2019). Coagulotoxic effects by brown snake (Pseudonaja) and taipan (Oxyuranus) venoms, and the efficacy of a new antivenom. Toxicology in Vitro. 58, 97–109. DOI: 10.1016/j.tiv.2019.03.031. Contributor Statement of contribution Zdenek, C.N. Conception and design (20%) Analysis and interpretation (65%) Performed experiments (100%) Drafting and production (80%) Hay, C. Provided venoms (40%) Edited paper (10%) Arbuckle, K. Analysis and interpretation (5%) Edited paper (10%) Jackson, T.N.W. Analysis and interpretation (5%) Bos, M.H.A. Analysis and interpretation (5%) Brouw, B.O.D. Analysis and interpretation (5%) Debono, J. Edited paper (10%) Allen, L. Provided venoms (30%) Edited paper (5%) Dunstan, N. Provided venoms (30%) Edited paper (5%) Morley, T. Provided antivenoms (25%) Edited paper (10%) Herrera, M. Provided antivenoms (25%) Edited paper (10%) Gutierrez, J.M. Provided antivenoms (25%) Edited paper (10%) Williams, D.J. Provided antivenoms (25%) Edited paper (10%) Fry, B.G. Conception and design (80%) Edited paper (20%) Analysis and interpretation (15%) Drafting and production (20%) Incorporated as Chapter 3, ‘AUS Coag.’, published C.N.Zdenek, B.O.D. Brouw, D. Dashevsky, A. Gloria, N. Youngman, E. Watson, P. Green, C. Hay, N. Dunstan, L. Allen, B.G. Fry. (2019). Clinical implications of convergent procoagulant toxicity and differential antivenom efficacy in Australian elapid snake venoms. Toxicology Letters. 316, 171-182.