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Factor XIIa (FXIIa) is a promising target for developing new drugs that prevent thrombosis without causing bleeding complications. A native cyclotide (MCoTI-II) is gaining interest for engineering FXIIa-targeted anticoagulants as this peptide inhibits FXIIa but not other coagulation proteases. Here, we engineered the native biosynthetic cyclization loop of MCoTI-II (loop 6) to generate improved FXIIa inhibitors. Decreasing the loop length led to gains in potency up to 7.7-fold, with the most potent variant having five residues in loop 6 (

The targeting of specific prey by snake venom toxins is a fascinating aspect of molecular and ecological evolution. Neurotoxic targeting by elapid snakes dominates the literature in this regard; however, recent studies have revealed viper toxins also induce neurotoxic effect. While this effect is thought to primarily be driven by prey selectivity, no study has quantified the taxonomically specific neurotoxicity of the viper clade consisting of Daboia, Macrovipera, Montivipera, and Vipera genera. Here, we tested venom toxin binding from 28 species of vipers from the four genera on the alpha 1 neuronal nicotinic acetylcholine receptors (nAChRs) orthosteric sites of amphibian, avian, lizard, rodent, and human mimotopes (synthetic peptides) using the Octet HTX biolayer interferometry platform. Daboia siamensis and D. russelii had broad binding affinity towards all mimotopes, while D. palestinae had selectivity toward lizard. Macrovipera species, on the other hand, were observed to have a higher affinity for amphibian mimotopes except for M. schweizeri, which inclined more toward lizard mimotopes. All Montivipera and most Vipera species also had higher affinity toward lizard mimotopes. Vipera a. montandoni, V. latastei, V. nikolski, and V. transcaucasina had the least binding to any of the mimotopes of the study. While a wide range of affinity binding towards various mimotopes were observed within the clade, the lowest affinity occurred towards the human target. Daboia siamensis and Macrovipera lebetina exhibited the greatest affinity toward the human mimotope, albeit still the least targeted of the mimotopes within those species. Overlaying this toxin-targeting trait over phylogeny of this clade revealed multiple cases of amplification of this trait and several cases of secondary loss. Overall, our results reveal dynamic variation, amplification, and some secondary loss of the prey targeting trait by alpha-neurotoxins within the venoms of this clade, indicating evolutionary selection pressure shaping the basic biochemistry of these venoms. Our work illustrates the successful use of this biophysical assay to further research snake venom neurotoxins and emphasizes the risk of generalizing venom effects observed on laboratory animals to have similar effects on humans.

Major variations in venom composition can occur between juvenile and adult venomous snakes. However, due to logistical constraints, antivenoms are produced using adult venoms in immunising mixtures, possibly resulting in limited neutralisation of juvenile snake venoms. Daboia russelii is one of the leading causes of snakebite death across South Asia. Its venom is potently procoagulant, causing stroke in prey animals but causing in humans consumptive coagulopathy—a net anticoagulant state—and sometimes death resulting from hemorrhage. In this in vitro study, we compared the venom activity of—and antivenom efficacy against—six 2-week-old D. russelii relative to that of their parents. Using a coagulation analyser, we quantified the relative coagulotoxicity of these venoms in human, avian, and amphibian plasma. The overall potency on human plasma was similar across all adult and neonate venoms, and SII (Serum Institute of India) antivenom was equipotent in neutralising these coagulotoxic effects. In addition, all venoms were also similar in their action upon avian plasma. In contrast, the neonate venoms were more potent on amphibian plasma, suggesting amphibians make up a larger proportion of neonate diet than adult diet. A similar venom potency in human and avian plasmas but varying selectivity for amphibian plasma suggests ontogenetic differences in toxin isoforms within the factor X or factor V activating classes, thereby providing a testable hypothesis for future transcriptomics work. By providing insights into the functional venom differences between adult and neonate D. russelii venoms, we hope to inform clinical treatment of patients envenomated by this deadly species and to shed new light on the natural history of these extremely medically important snakes.

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Cyclotides are plant-derived peptides with complex structures shaped by their head-to-tail cyclic backbone and cystine knot core. These structural features underpin the native bioactivities of cyclotides, as well as their beneficial properties as pharmaceutical leads, including high proteolytic stability and cell permeability. However, their inherent structural complexity presents a challenge for cyclotide engineering, particularly for accessing libraries of sufficient chemical diversity to design potent and selective cyclotide variants. Here, we report a strategy using mRNA display enabling us to select potent cyclotide-based FXIIa inhibitors from a library comprising more than 1012 members based on the cyclotide scaffold of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The most potent and selective inhibitor, cMCoFx1, has a pM inhibitory constant toward FXIIa with greater than three orders of magnitude selectivity over related serine proteases, realizing specific inhibition of the intrinsic coagulation pathway. The cocrystal structure of cMCoFx1 and FXIIa revealed interactions at several positions across the contact interface that conveyed high affinity binding, highlighting that such cyclotides are attractive cystine knot scaffolds for therapeutic development.

The use of venom in predation exerts a corresponding selection pressure for the evolution of venom resistance. One of the mechanisms related to venom resistance in animals (predators or prey of snakes) is the presence of molecules in the blood that can bind venom toxins, and inhibit their pharmacological effects. One such toxin type are venom phospholipase A2s (PLA2s), which have diverse effects including anticoagulant, myotoxic, and neurotoxic activities. BoaγPLI isolated from the blood of Boa constrictor has been previously shown to inhibit venom PLA2s that induced myotoxic and edematogenic activities. Recently, in addition to its previously described and very potent neurotoxic effect, the venoms of American coral snakes (Micrurus species) have been shown to have anticoagulant activity via PLA2 toxins. As coral snakes eat other snakes as a major part of their diet, neonate Boas could be susceptible to predation by this sympatric species. Thus, this work aimed to ascertain if BoaγPLI provided a protective effect against the anticoagulant toxicity of venom from the model species Micrurus laticollaris in addition to its ability shown previously against other toxin types. Using a STA R Max coagulation analyser robot to measure the effect upon clotting time, and TEG5000 thromboelastographers to measure the effect upon clot strength, we evaluated the ability of BoaγPLI to inhibit M. laticollaris venom. Our results indicate that BoaγPLI is efficient at inhibiting the M. laticollaris anticoagulant effect, reducing the time of coagulation (restoring them closer to non-venom control values) and increasing the clot strength (restoring them closer to non-venom control values). These findings demonstrate that endogenous PLA2 inhibitors in the blood of non-venomous snakes are multi-functional and provide broad resistance against a myriad of venom PLA2-driven toxic effects including coagulotoxicity, myotoxicity, and neurotoxicity. This novel form of resistance could be evidence of selective pressures caused by predation from venomous snakes and stresses the need for field-based research aimed to expand our understanding of the evolutionary dynamics of such chemical arms race.

Species within the viperid genus Macrovipera are some of the most dangerous snakes in the Eurasian region, injecting copious amounts of potent venom. Despite their medical importance, the pathophysiological actions of their venoms have been neglected. Particularly poorly known are the coagulotoxic effects and thus the underlying mechanisms of lethal coagulopathy. In order to fill this knowledge gap, we ascertained the effects of venom upon human plasma for Macrovipera lebetina cernovi, M. l. lebetina, M. l. obtusa, M. l. turanica, and M. schweizeri using diverse coagulation analysing protocols. All five were extremely potent in their ability to promote clotting but varied in their relative activation of Factor X, being equipotent in this study to the venom of the better studied, and lethal, species Daboia russelii. The Insoserp European viper antivenom was shown to be highly effective against all the Macrovipera venoms, but performed poorly against the D. russelii venom. Reciprocally, while Daboia antivenoms performed well against D. russelii venom, they failed against Macrovipera venom. Thus despite the two genera sharing a venom phenotype (Factor X activation) driven by the same toxin type (P-IIId snake venom metalloproteases), the surface biochemistries of the toxins differed significantly enough to impede antivenom cross- neutralization. The differences in venom biochemistry were reflected in coagulation co-factor dependence. While both genera were absolutely dependent upon calcium for the activation of Factor X, dependence upon phospholipid varied. The Macrovipera venoms had low levels of dependence upon phospholipid while the Daboia venom was three times more dependent upon phospholipid for the activation of Factor X. This suggests that the sites on the molecular surface responsible for phospholipid dependence, are the same differential sites that prevent inter-genera antivenom cross- neutralization. Due to cold-chain requirements, antivenoms may not be stocked in rural settings where the need is at the greatest. Thus we tested the efficacy of enzyme inhibitor Prinomastat as a field-deployable treatment to stabilise patients while being transported to antivenom stocks, and showed that it was extremely effective in blocking the Factor X activating pathophysiological actions. Marimastat however was less effective. These results thus not only shed light on the coagulopathic mechanisms of Macrovipera venoms, but also provide data critical for evidence-based design of snakebite management strategies.

Snakebite is a neglected tropical disease with a massive global burden of injury and death. The best current treatments, antivenoms, are plagued by a number of logistical issues that limit supply and access in remote or poor regions. We explore the anticoagulant properties of venoms from the genus Micrurus (coral snakes), which have been largely unstudied, as well as the effectiveness of antivenom and a small-molecule phospholipase inhibitor-varespladib-at counteracting these effects. Our in vitro results suggest that these venoms likely interfere with the formation or function of the prothrombinase complex. We find that the anticoagulant potency varies widely across the genus and is especially pronounced in M. laticollaris. This variation does not appear to correspond to previously described patterns regarding the relative expression of the three-finger toxin and phospholipase A2 (PLA2) toxin families within the venoms of this genus. The coral snake antivenom Coralmyn, is largely unable to ameliorate these effects except for M. ibiboboca. Varespladib on the other hand completely abolished the anticoagulant activity of every venom. This is consistent with the growing body of results showing that varespladib may be an effective treatment for a wide range of toxicity caused by PLA2 toxins from many different snake species. Varespladib is a particularly attractive candidate to help alleviate the burden of snakebite because it is an approved drug that possesses several logistical advantages over antivenom including temperature stability and oral availability.

Venoms from Pseudechis species (Australian black snakes) within the Elapidae family are rich in anticoagulant PLA2 toxins, with the exception of one species (P. porphyriacus) that possesses procoagulant mutated forms of the clotting enzyme Factor Xa. Previously the mechanism of action of the PLA2 toxins' anticoagulant toxicity was said to be due to inhibition of Factor Xa, but this statement was evidence free. We conducted a series of anticoagulation assays to elucidate the mechanism of anticoagulant action produced by P. australis venom. Our results revealed that, rather than targeting FXa, the PLA2 toxins inhibited the prothrombinase complex, with FVa-alone or as part of the prothrombinase complex-as the primary target; but with significant thrombin inhibition also noted. In contrast, FXa, and other factors inhibited only to a lesser degree were minor targets. We quantified coagulotoxic effects upon human plasma caused by all nine anticoagulant Pseudechis species, including nine localities of P. australis across Australia, and found similar anticoagulant potency across all Pseudechis species, with greater potency in P. australis and the undescribed Pseudechis species in the NT. In addition, the northern localities and eastern of P. australis were significantly more potent than the central, western, and southern localities. All anticoagulant venoms responded well to Black Snake Antivenom, except P. colletti which was poorly neutralised by Black Snake Antivenom and also Tiger Snake Antivenom (the prescribed antivenom for this species). However, we found LY315920 (trade name: Varespladib), a small molecule inhibitor of PLA2 proteins, exhibited strong potency against P. colletti venom. Thus, Varespladib may be a clinically viable treatment for anticoagulant toxicity exerted by this species that is not neutralised by available antivenoms. Our results provide insights into coagulotoxic venom function, and suggest future in vivo work be conducted to progress the development of a cheaper, first-line treatment option to treat PLA2-rich snake venoms globally.

Ecological variability among closely related species provides an opportunity for evolutionary comparative studies. Therefore, to investigate the origin and evolution of neurotoxicity in Asian viperid snakes, we tested the venoms of Azemiops feae, Calloselasma rhodostoma, Deinagkistrodon acutus, Tropidolaeums subannulatus, and T. wagleri for their relative specificity and potency upon the amphibian, lizard, bird, rodent, and human α-1 (neuromuscular) nicotinic acetylcholine receptors. We utilised a biolayer interferometry assay to test the binding affinity of these pit viper venoms to orthosteric mimotopes of nicotinic acetylcholine receptors binding region from a diversity of potential prey types. The Tropidolaemus venoms were much more potent than the other species tested, which is consistent with the greater prey escape potential in arboreal niches. Intriguingly, the venom of C. rhodostoma showed neurotoxic binding to the α-1 mimotopes, a feature not known previously for this species. The lack of prior knowledge of neurotoxicity in this species is consistent with our results due to the bias in rodent studies and human bite reports, whilst this venom had a greater binding affinity toward amphibian and diapsid α-1 targets. The other large terrestrial species, D. acutus, did not display any meaningful levels of neurotoxicity. These results demonstrate that whilst small peptide neurotoxins are a basal trait of these snakes, it has been independently amplified on two separate occasions, once in Azemiops and again in Tropidolaemus, and with Calloselasma representing a third possible amplification of this trait. These results also point to broader sources of novel neuroactive peptides with the potential for use as lead compounds in drug design and discovery.

Knottins are topologically complex peptides that are stabilised by a cystine knot and have exceptionally diverse functions, including protease inhibition. However, approaches for tuning their activity in situ are limited. Here, we demonstrate separate approaches for tuning the activity of knottin protease inhibitors using light or streptavidin. We show that the inhibitory activity and selectivity of an engineered knottin can be controlled with light by activating a second mode of action that switches the inhibitor ON against new targets. Guided by a knottin library screen, we also identify a position in the inhibitor's binding loop that permits insertion of a biotin tag without impairing activity. Using streptavidin, biotinylated knottins with nanomolar affinity can be switched OFF in activity assays, and the anticoagulant activity of a factor XIIa inhibitor can be rapidly switched OFF in human plasma. Our findings expand the scope of engineered knottins for precisely controlling protein function.

Evidence suggests that snakes can hear, but how snakes naturally respond to sound is still unclear. We conducted 304 controlled experiment trials on 19 snakes across five genera in a sound-proof room (4.9 x 4.9 m) at 27ºC, observing the effects of three sounds on individual snake behavior, compared to controls. We quantified eight snake behaviors (body movement, body freezing, head-flicks, tongue-flicks, hissing, periscoping, head fixation, lower jaw drop) in response to three sounds, which were filtered pink-noise within the following frequency ranges: 0–150Hz (sound 1, which produced ground vibrations, as measured by an accelerometer), 150–300Hz (sound 2, which did not produced ground vibrations), 300–450Hz (sound 3, which did not produced ground vibrations). All snake responses were strongly genus dependent. Only one genus (Aspidites, Woma Pythons) significantly increased their probability of movement in response to sound, but three other genera (Acanthophis (Death Adders), Oxyuranus (Taipans), and Pseudonaja (Brown Snakes)) were more likely to move away from sound, signaling potential avoidance behavior. Taipans significantly increased their likelihood of displaying defensive and cautious behaviors in response to sound, but three of the five genera exhibited significantly different types of behaviors in sound trials compared to the control. Our results highlight potential heritable behavioral responses of snakes to sound, clustered within genera. Our study illustrates the behavioral variability among different snake genera, and across sound frequencies, which contributes to our limited understanding of hearing and behavior in snakes.

Bites from helodermatid lizards can cause pain, paresthesia, paralysis, and tachycardia, as well as other symptoms consistent with neurotoxicity. Furthermore, in vitro studies have shown that Heloderma horridum venom inhibits ion flux and blocks the electrical stimulation of skeletal muscles. Helodermatids have long been considered the only venomous lizards, but a large body of robust evidence has demonstrated venom to be a basal trait of Anguimorpha. This clade includes varanid lizards, whose bites have been reported to cause anticoagulation, pain, and occasionally paralysis and tachycardia. Despite the evolutionary novelty of these lizard venoms, their neuromuscular targets have yet to be identified, even for the iconic helodermatid lizards. Therefore, to fill this knowledge gap, the venoms of three Heloderma species (H. exasperatum, H. horridum and H. suspectum) and two Varanus species (V. salvadorii and V. varius) were investigated using Gallus gallus chick biventer cervicis nerve–muscle preparations and biolayer interferometry assays for binding to mammalian ion channels. Incubation with Heloderma venoms caused the reduction in nerve-mediated muscle twitches post initial response of avian skeletal muscle tissue preparation assays suggesting voltage-gated sodium (NaV) channel binding. Congruent with the flaccid paralysis inducing blockage of electrical stimulation in the skeletal muscle preparations, the biolayer interferometry tests with Heloderma suspectum venom revealed binding to the S3–S4 loop within voltage-sensing domain IV of the skeletal muscle channel subtype, NaV1.4. Consistent with tachycardia reported in clinical cases, the venom also bound to voltage-sensing domain IV of the cardiac smooth muscle calcium channel, CaV1.2. While Varanus varius venom did not have discernable effects in the avian tissue preparation assay at the concentration tested, in the biointerferometry assay both V. varius and V. salvadorii bound to voltage-sensing domain IV of both NaV1.4 and CaV1.2, similar to H. suspectum venom. The ability of varanid venoms to bind to mammalian ion channels but not to the avian tissue preparation suggests prey-selective actions, as did the differential potency within the Heloderma venoms for avian versus mammalian pathophysiological targets. This study thus presents the detailed characterization of Heloderma venom ion channel neurotoxicity and offers the first evidence of varanid lizard venom neurotoxicity. In addition, the data not only provide information useful to understanding the clinical effects produced by envenomations, but also reveal their utility as physiological probes, and underscore the potential utility of neglected venomous lineages in the drug design and development pipeline.

Envenomations by venomous snakes have major public health implications on a global scale. Despite its medical importance, snakebite has long been a neglected tropical disease by both governments and medical science. Many aspects of the resulting pathophysiology have been largely under-investigated. Most research on snake venom has focused on the neurological effects, with coagulotoxicity being relatively neglected, especially for venoms in the Elapidae snake family. In order to fill the knowledge gap regarding the coagulotoxic effects of elapid snake venoms, we performed functional activity tests to determine the fibrinogenolytic activity of 29 African and Asian elapid venoms across eight genera. The results of this study revealed that destructive (non-clotting) fibrinogenolytic activity is widespread across the African and Asian elapids. This trait evolved independently twice: once in the Hemachatus/Naja last common ancestor and again in Ophiophagus. Further, within Naja this trait was amplified on several independent occasions and possibly explains some of the clinical symptoms produced by these species. Species within the Hemachatus/Naja with fibrinogenolytic activity only cleaved the Aα-chain of fibrinogen, whereas Ophiophagus venoms degraded both the Aα- and the Bβ-chain of fibrinogen. All other lineages tested in this study lacked significant fibrinogenolytic effects. Our systematic research across Afro-Asian elapid snake venoms helps shed light on the various molecular mechanisms that are involved in coagulotoxicity within Elapidae.

Snakebite is a neglected tropical disease that disproportionately affects the poor. Antivenom is the only specific and effective treatment for snakebite, but its distribution is severely limited by several factors, including the prohibitive cost of some products. Papua New Guinea (PNG) is a snakebite hotspot but the high costs of Australian antivenoms (thousands of dollars per treatment) makes it unaffordable in PNG. A more economical taipan antivenom has recently been developed at the Instituto Clodomiro Picado (ICP) in Costa Rica for PNG and is currently undergoing clinical trials for the treatment of envenomations by coastal taipans (Oxyuranus scutellatus). In addition to potentially having the capacity to neutralise the effects of envenomations of non-PNG taipans, this antivenom may have the capacity to neutralise coagulotoxins in venom from closely related brown snakes (Pseudonaja spp.) also found in PNG. Consequently, we investigated the cross-reactivity of taipan antivenom across the venoms of all Oxyuranus and Pseudonaja species. In addition, to ascertain differences in venom biochemistry that influence variation in antivenom efficacy, we tested for relative cofactor dependence. We found that the new ICP taipan antivenom exhibited high selectivity for Oxyuranus venoms and only low to moderate cross-reactivity with any Pseudonaja venoms. Consistent with this genus level distinction in antivenom efficacy were fundamental differences in the venom biochemistry. Not only were the Pseudonaja venoms significantly more procoagulant, but they were also much less dependent upon the cofactors calcium and phospholipid. There was a strong correlation between antivenom efficacy, clotting time and cofactor dependence. This study sheds light on the structure-function relationships of the procoagulant toxins within these venoms and may have important clinical implications including for the design of next-generation antivenoms.

What factors influence the evolution of a heavily selected functional trait in a diverse clade? This study adopts rattlesnakes as a model group to investigate the evolutionary history of venom coagulotoxicity in the wider context of phylogenetics, natural history, and biology. Venom-induced clotting of human plasma and fibrinogen was determined and mapped onto the rattlesnake phylogenetic tree to reconstruct the evolution of coagulotoxicity across the group. Our results indicate that venom phenotype is often independent of phylogenetic relationships in rattlesnakes, suggesting the importance of diet and/or other environmental variables in driving venom evolution. Moreover, the striking inter- and intraspecific variability in venom activity on human blood highlights the considerable variability faced by physicians treating envenomation. This study is the most comprehensive effort to date to describe and characterize the evolutionary and biological aspects of coagulotoxins in rattlesnake venom. Further research at finer taxonomic levels is recommended to elucidate patterns of variation within species and lineages.

Rattlesnakes are a diverse clade of pit vipers (snake family Viperidae, subfamily Crotalinae) that consists of numerous medically significant species. We used validatedin vitroassays measuring venom-induced clotting time and strength of any clots formed in human plasma and fibrinogen to assess the coagulotoxic activity of the four medically relevant Mexican rattlesnake speciesCrotalus culminatus, C. mictlantecuhtli, C. molossus, andC. tzabcan. We report the first evidence of true procoagulant activity by Neotropical rattlesnake venom inCrotalus culminatus. This species presented a strong ontogenetic coagulotoxicity dichotomy: neonates were strongly procoagulantviaFactor X activation, whereas adults were pseudo-procoagulant in that they converted fibrinogen into weak, unstable fibrin clots that rapidly broke down, thereby likely contributing to net anticoagulation through fibrinogen depletion. The other species did not activate clotting factors or display an ontogenetic dichotomy, but depleted fibrinogen levels by cleaving fibrinogen either in a destructive (non-clotting) manner orviaa pseudo-procoagulant mechanism. We also assessed the neutralization of these venoms by available antivenom and enzyme-inhibitors to provide knowledge for the design of evidence-based treatment strategies for envenomated patients. One of the most frequently used Mexican antivenoms (Bioclon Antivipmyn®) failed to neutralize the potent procoagulant toxic action of neonateC. culminatusvenom, highlighting limitations in snakebite treatment for this species. However, the metalloprotease inhibitor Prinomastat substantially thwarted the procoagulant venom activity, while 2,3-dimercapto-1-propanesulfonic acid (DMPS) was much less effective. These results confirm that venom-induced Factor X activation (a procoagulant action) is driven by metalloproteases, while also suggesting Prinomastat as a more promising potential adjunct treatment than DMPS for this species (with the caveat thatin vivostudies are necessary to confirm this potential clinical use). Conversely, the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) inhibited the direct fibrinogen cleaving actions ofC. mictlantecuhtlivenom, thereby revealing that the pseudo-procoagulant action is driven by kallikrein-type serine proteases. Thus, this differential ontogenetic variation in coagulotoxicity patterns poses intriguing questions. Our results underscore the need for further research into Mexican rattlesnake venom activity, and also highlights potential limitations of current antivenom treatments.

Is snake venom activity influenced by size? This is a long-standing question that can have important consequences for the treatment of snake envenomation. Ontogenetic shifts in venom composition are a well-documented characteristic of numerous snake species. Although snake venoms can cause a range of pathophysiological disturbances, establishing the coagulotoxic profiles related to such shifts is a justified approach because coagulotoxicity can be deadly, and its neutralisation is a challenge for current antivenom therapy. Thus, we aimed to assess the coagulotoxicity patterns on plasma and fibrinogen produced by B othrops jararacussu venoms from individuals of different sizes and sex, and the neutralisation potential of SAB (anti bothropic serum produced by Butantan Institute). The use of a metalloproteinase inhibitor (Prinomastat) and a serine proteinase inhibitor (AEBSF) enabled us to determine the toxin class responsible for the observed coagulopathy: activity on plasma was found to be metalloprotease driven, while the activity on fibrinogen is serine protease driven. To further explore differences in venom activity, the activation of Factor X and prothrombin as a function of snake size was also evaluated. All the venoms exhibited a potent procoagulant effect upon plasma and were less potent in their pseudo-procoagulant clotting effect upon fibrinogen. On human plasma, the venoms from smaller snakes produced more rapid clotting than the larger ones. In contrast, the venom activity on fibrinogen had no relation with size or sex. The difference in procoagulant potency was correlated with the bigger snakes being proportionally better neutralized by antivenom due to the lower levels of procoagulant toxins, than the smaller. Thus, while the antivenom ultimately neutralized the venoms, proportionally more would be needed for an equal mass of venom from a small snake than a large one. Similarly, the neutralisation by SAB of the pseudo-procoagulant clotting effects was also correlated with relative potency, with the smaller and bigger snakes being neutralized proportional to potency, but with no correlation to size. Thromboelastography (TEG) tests on human and toad plasma revealed that small snakes' venoms acted quicker than large snakes' venom on both plasmas, with the action upon amphibian plasma consistent with smaller snakes taking a larger proportion of anuran prey than adults. Altogether, the ontogenetic differences regarding coagulotoxic potency and corresponding impact upon relative antivenom neutralisation of snakes with different sizes were shown, underscoring the medical importance of investigating ontogenetic changes in order to provide data crucial for evidence-based design of clinical management strategies.

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. nChapter 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.nChapter 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.nI 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.n

Bothrops (lance-head pit vipers) venoms are rich in weaponised metalloprotease enzymes (SVMP). These toxic enzymes are structurally diverse and functionally versatile. Potent coagulotoxicity is particularly important for prey capture (via stroke-induction) and relevant to human clinical cases (due to consumption of clotting factors including the critical depletion of fibrinogen). In this study, three distinct isoforms of P-III class SVMPs (IC, IIB and IIC), isolated from Bothrops neuwiedi venom, were evaluated for their differential capacities to affect hemostasis of prey and human plasma. Furthermore, we tested the relative antivenom neutralisation of effects upon human plasma. The toxic enzymes displayed differential procoagulant potency between plasma types, and clinically relevant antivenom efficacy variations were observed. Of particular importance was the confirmation the antivenom performed better against prothrombin activating toxins than Factor X activating toxins, which is likely due to the greater prevalence of the former in the immunising venoms used for antivenom production. This is clinically relevant as the enzymes displayed differential potency in this regard, with one (IC) in particular being extremely potent in activating Factor X and thus was correspondingly poorly neutralised. This study broadens the current understanding about the adaptive role of the SVMPs, as well as highlights how the functional diversity of SVMP isoforms can influence clinical outcomes. Key Contribution: Our findings shed light upon the hemorrhagic and coagulotoxic effects of three SVMPs of the P-III class, as well as the coagulotoxic effects of SVMPs on human, avian and amphibian plasmas. Antivenom neutralised prothrombin-activating isoforms better than Factor X activating isoforms.

Prey-selective venoms and toxins have been documented across only a few species of snakes. The lack of research in this area has been due to the absence of suitably flexible testing platforms. In order to test more species for prey specificity of their venom, we used an innovative taxonomically flexible, high-throughput biolayer interferometry approach to ascertain the relative binding of 29 &alpha, neurotoxic venoms from African and Asian elapid representatives (26 Naja spp., Aspidelaps scutatus, Elapsoidea boulengeri, and four locales of Ophiophagus hannah) to the alpha-1 nicotinic acetylcholine receptor orthosteric (active) site for amphibian, lizard, snake, bird, and rodent targets. Our results detected prey-selective, intraspecific, and geographical differences of &alpha, neurotoxic binding. The results also suggest that crude venom that shows prey selectivity is likely driven by the proportions of prey-specific &alpha, neurotoxins with differential selectivity within the crude venom. Our results also suggest that since the &alpha, neurotoxic prey targeting does not always account for the full dietary breadth of a species, other toxin classes with a different pathophysiological function likely play an equally important role in prey immobilisation of the crude venom depending on the prey type envenomated. The use of this innovative and taxonomically flexible diverse assay in functional venom testing can be key in attempting to understanding the evolution and ecology of &alpha, neurotoxic snake venoms, as well as opening up biochemical and pharmacological avenues to explore other venom effects.

Snakebite is a common occurrence for pet cats and dogs worldwide and can be fatal. In Australia the eastern brown snake (Pseudonaja textilis) is responsible for an estimated 76% of reported snakebite cases to domestic pets nationally each year, with the primary pathology being venom-induced consumptive coagulopathy. While only 31% of dogs survive P. textilis bites without antivenom, cats are twice as likely to survive bites (66%). Even with antivenom treatment, cats have a significantly higher survival rate. The reason behind this disparity is unclear. Using a coagulation analyser (Stago STA R Max), we tested the relative procoagulant effects of P. textilis venom-as well as 10 additional procoagulant venoms found around the world-on cat and dog plasma in vitro, as well as on human plasma for comparison. All venoms acted faster upon dog plasma than cat or human, indicating that dogs would likely enter coagulopathic states sooner, and are thus more vulnerable to procoagulant snake venoms. The spontaneous clotting time (recalcified plasma with no venom added) was also substantially faster in dogs than in cats, suggesting that the naturally faster clotting blood of dogs predisposes them to being more vulnerable to procoagulant snake venoms. This is consistent with clinical records showing more rapid onset of symptoms and lethal effects in dogs than cats. Several behavioural differences between cats and dogs are also highly likely to disproportionately negatively affect prognosis in dogs. Thus, compared to cats, dogs require earlier snakebite first-aid and antivenom to prevent the onset of lethal venom effects.

Does the venom of Trimeresurus albolabris (white-lipped green tree pit viper) differ between neonate and adults? This species is responsible for most snakebites within south and southeast Asia, yet it is unknown whether ontogenetic variation in venom composition occurs in this species, or how this might affect antivenom efficacy. Using a coagulation analyser robot, we examined the anticoagulant activity of T. albolabris venom from eight individuals across multiple age classes. We then compared the efficacy of Thai Red Cross Green Tree Pit Viper Antivenom across these age classes. Venoms from all age classes were equally potent in their pseudo-procoagulant, fibrinogenolytic activity, in that fibrinogen was cleaved to form weak, unstable fibrin clots that rapidly broke down, thus resulting in a net anticoagulant state. Similarly, this coagulotoxic activity was well neutralised by antivenom across all venoms. Given that coagulotoxicity is the primary serious pathology in T. albolabris envenomations, we conclude that Thai Red Cross Green Tree Pit Viper Antivenom is a valid treatment for envenomations by this species, regardless of age or sex of the offending snake. These results are relevant for clinical treatment of envenomations by T. albolabris.

Night adders (Causus species within the Viperidae family) are amphibian specialists and a common source of snakebite in Africa. Some species are unique in that they have the longest venom glands of any viper, extending approximately 10% of the body length. Despite their potential medical importance and evolutionary novelty, their venom has received almost no research attention. In this study, venoms from a short-glanded species (C. lichtensteinii) and from a long-glanded species (C. rhombeatus) were compared using a series of proteomic and bioactivity testing techniques to investigate and compare the toxin composition and functioning of the venoms of these two species. Both C. rhombeatus and C. lichtensteinii were similar in overall venom composition and inhibition of blood coagulation through non-clotting proteolytic cleavage of fibrinogen. While the 1D gel profiles were very similar to each other in the toxin types present, 2D gel analyses revealed isoformic differences within each toxin classes. This variation was congruent with differential efficacy of South African Institute for Medical Research snake polyvalent antivenom, with C. lichtensteinii unaffected at the dose tested while C. rhombeatus was moderately but significantly neutralized. Despite the variation within toxin classes, the similarity in overall venom biochemistry suggests that the selection pressure for the evolution of long glands served to increase venom yield in order to subjugate proportionally large anurans as a unique form of niche partitioning, and is not linked to significant changes in venom function. These results not only contribute to the body of venom evolution knowledge but also highlight the limited clinical management outcomes for Causus envenomations.

Envenomations are complex medical emergencies that can have a range of symptoms and sequelae. The only specific, scientifically-validated treatment for envenomation is antivenom administration, which is designed to alleviate venom effects. A paucity of efficacy testing exists for numerous antivenoms worldwide, and understanding venom effects and venom potency can help identify antivenom improvement options. Some spider venoms can produce debilitating injuries or even death, yet have been largely neglected in venom and antivenom studies because of the low venom yields. Coagulation disturbances have been particularly under studied due to difficulties in working with blood and the coagulation cascade. These circumstances have resulted in suboptimal spider bite treatment for medically significant spider genera such as Loxosceles and Sicarius. This study identifies and quantifies the anticoagulant effects produced by venoms of three Loxoscles species (L. reclusa, L. boneti, and L. laeta) and that of Sicarius terrosus. We showed that the venoms of all studied species are able to cleave the fibrinogen Aα-chain with varying degrees of potency, with L. reclusa and S. terrosus venom cleaving the Aα-chain most rapidly. Thromboelastography analysis revealed that only L. reclusa venom is able to reduce clot strength, thereby presumably causing anticoagulant effects in the patient. Using the same thromboelastography assays, antivenom efficacy tests revealed that the commonly used Loxoscles-specific SMase D recombinant based antivenom failed to neutralize the anticoagulant effects produced by Loxosceles venom. This study demonstrates the fibrinogenolytic activity of Loxosceles and Sicarius venom and the neutralization failure of Loxosceles antivenom, thus providing impetus for antivenom improvement.

A paradigm of venom research is adaptive evolution of toxins as part of a predator-prey chemical arms race. This study examined differential co-factor dependence, variations relative to dietary preference, and the impact upon relative neutralisation by antivenom of the procoagulant toxins in the venoms of a clade of Australian snakes. All genera were characterised by venoms rich in factor Xa which act upon endogenous prothrombin. Examination of toxin sequences revealed an extraordinary level of conservation, which indicates that adaptive evolution is not a feature of this toxin type. Consistent with this, the venoms did not display differences on the plasma of different taxa. Examination of the prothrombin target revealed endogenous blood proteins are under extreme negative selection pressure for diversification, this in turn puts a strong negative selection pressure upon the toxins as sequence diversification could result in a drift away from the target. Thus this study reveals that adaptive evolution is not a consistent feature in toxin evolution in cases where the target is under negative selection pressure for diversification. Consistent with this high level of toxin conservation, the antivenom showed extremely high-levels of cross-reactivity. There was however a strong statistical correlation between relative degree of phospholipid-dependence and clotting time, with the least dependent venoms producing faster clotting times than the other venoms even in the presence of phospholipid. The results of this study are not only of interest to evolutionary and ecological disciplines, but also have implications for clinical toxinology.

The ability to identify individuals within a population is often essential for a detailed understanding of the ecology and conservation of a species. However, some species, including large parrots, are notoriously difficult to catch and mark for individual identification. Palm cockatoos (Probosciger aterrimus) are a large, poorly understood species of parrot which are likely in severe decline within the eastern part – and possibly the western part – of their range on Cape York Peninsula, Australia. Here, we investigated whether three different palm cockatoo call types are sufficiently individually distinctive to function as a non-invasive “marker” for identifying individuals over time. Using Discriminant Function Analysis, overall identification accuracy among 12 putative individuals for all call types was 81% (i.e. 148 out of 183 calls were assigned to the correct individual) on the basis of multiple temporal, energy (amplitude) and frequency measurements on the spectrogram. For three different c...

Papuan Frogmouths Podargus papuensis are large nocturnal birds about which relatively little is known. A prolonged aggressive interaction between two Papuan Frogmouths that involved interlocking beaks was filmed in the Lockhart River region on Cape York Peninsula, Queensland, and is described here. Two additional Papuan Frogmouths were present during the event, one of which gave a call-type ( OomWoom ) previously unrecorded for this species. Difficulties associated with detecting such agonistic behaviour mean that the prevalence of these contests and their behavioural significance are currently unknown and require further research.

Australian elapid snakes are some of the most venomous snakes in the world and are unique among venomous snakes in having mutated forms of the blood clotting factor X in an activated form (FXa) as a key venom component. In human bite victims, an overdose of this activated clotting enzyme results in the systemic consumption of fibrinogen due to the large amounts of endogenous thrombin generated by the conversion of prothrombin to thrombin by venom FXa. Within Australian elapids, such procoagulant venom is currently known from the tiger snake clade (Hoplocephalus, Notechis, Paroplocephalus, and Tropidechis species), brown/taipan (Oxyuranus and Pseudonaja species) clade, and the red-bellied black snake Pseudechis porphyriacus. We used a STA-R Max coagulation analyser and TEG5000 thromboelastographers to test 47 Australian elapid venoms from 19 genera against human plasma in vitro. In addition to activity being confirmed in the two clades above, FXa-driven potent procoagulant activity was found in four additional genera (Cryptophis, Demansia, Hemiaspis, and Suta). Ontogenetic changes in procoagulant function was also identified as a feature of Suta punctata venom. Phylogenetic analysis of FX sequences confirmed that snake venom FXa toxins evolved only once, that the potency of these toxins against human plasma has increased in a stepwise fashion, and that multiple convergent amplifications of procoagulant activity within Australian elapid snakes have occurred. Cofactor dependence tests revealed all procoagulant venoms in our study, except those of the tiger snake clade, to be highly calcium-dependent, whereas phospholipid dependence was less of a feature but still displayed significant variation between venoms. Antivenom testing using CSL Tiger Snake Antivenom showed broad but differential cross-reactivity against procoagulant venoms, with P. porphyriacus and S. punctata extremely well neutralised but with Cryptophis, Demansia, and Hemiaspis less well-neutralised. The relative variation was not in accordance to genetic relatedness of the species used in antivenom production (Notechis scutatus), which underscores a fundamental principle that the rapid evolution characteristic of venoms results in organismal phylogeny being a poor predictor of antivenom efficacy. Our results have direct and immediate implications for the design of clinical management plans in the event of snakebite by such lesser known Australian elapid snake species that have been revealed in this study to be as potent as the better studied, and proven lethal, species.

Snakebite is a global tropical disease that has long had huge implications for human health and well-being. Despite its long-standing medical importance, it has been the most neglected of tropical diseases. Reflective of this is that many aspects of the pathology have been underinvestigated. Snakebite by species in the Elapidae family is typically characterised by neurotoxic effects that result in flaccid paralysis. Thus, while clinically significant disturbances to the coagulation cascade have been reported, the bulk of the research to date has focused upon neurotoxins. In order to fill the knowledge gap regarding the coagulotoxic effects of elapid snake venoms, we screened 30 African and Asian venoms across eight genera using in vitro anticoagulant assays to determine the relative inhibition of the coagulation function of thrombin and the inhibition of the formation of the prothrombinase complex through competitive binding to a nonenzymatic site on Factor Xa (FXa), thereby preventing FXa from binding to Factor Va (FVa). It was revealed that African spitting cobras were the only species that were potent inhibitors of either clotting factor, but with Factor Xa inhibited at 12 times the levels of thrombin inhibition. This is consistent with at least one death on record due to hemorrhage following African spitting cobra envenomation. To determine the efficacy of antivenom in neutralising the anticoagulant venom effects, for the African spitting cobras we repeated the same 8-point dilution series with the addition of antivenom and observed the shift in the area under the curve, which revealed that the antivenom performed extremely poorly against the coagulotoxic venom effects of all species. However, additional tests with the phospholipase A2 inhibitor LY315920 (trade name: varespladib) demonstrated a powerful neutralisation action against the coagulotoxic actions of the African spitting cobra venoms. Our research has important implications for the clinical treatment of cobra snakebites and also sheds light on the molecular mechanisms involved in coagulotoxicity within Naja. As the most coagulotoxic species are also those that produce characteristic extreme local tissue damage, future research should investigate potential synergistic actions between anticoagulant toxins and cytotoxins.

Bitis are well known for being some of the most commonly encountered and medically important snake species in all of Africa. While the majority of species possess potently anticoagulant venom, only B. worthingtoni is known to possess procoagulant venom. Although known to be the basal species within the genus, B. worthingtoni is an almost completely unstudied species with even basic dietary information lacking. This study investigated various aspects of the unique procoagulant effects of B. worthingtoni venom. Coagulation assays determined the primary procoagulant effect to be driven by Factor X activating snake venom metalloprotease toxins. In addition to acting upon the mammalian blood clotting cascade, B. worthingtoni venom was also shown to clot amphibian plasma. As previous studies have shown differences in clotting factors between amphibian and mammalian plasmas, individual enzymes in snake venoms acting on plasma clotting factors can be taxon-selective. As venoms evolve under purifying selection pressures, this suggests that the procoagulant snake venom metalloprotease toxins present in B. worthingtoni have likely been retained from a recent common ancestor shared with the related amphibian-feeding Proatheris superciliaris, and that both amphibians and mammals represent a substantial proportion of B. worthingtoni current diet. Thus, taxon-specific actions of venoms may have utility in inferring dietary composition for rare or difficult to study species. An important caveat is that to validate this hypothesis field studies investigating the dietary ecology of B. worthingtoni must be conducted, as well as further investigations of its venom composition to reconstruct the molecular evolutionary history of the toxins present.