Prey size influences venom delivery. These data, obtained from medium and large Northern Pacific Rattlesnakes Crotalus oreganus striking at small and large mice, support the interpretation that snakes can control, or meter, how much venom they inject.
Source: Hayes et al. Defensive bites Bite movies cool stuff! Predatory bites. More recently, we have begun to examine defensive strikes, which are particularly important to the problem of human snakebite Hayes et al. To our surprise, we learned that rattlesnakes appear to inject more venom into models of human limbs warm, human-scented, saline-filled gloves than into mice.
However, roughly 10 percent of the bites are dry, which is much more frequent than that observed for predatory bites. We also found that, contrary to popular belief, large rattlesnakes inject far more venom than small rattlesnakes when biting defensively. The larger rattlesnakes have much more venom available and experimental evidence from predatory strikes suggests that even smaller snakes can control, or meter, their venom. We have discovered some interesting differences between species.
When delivering three defensive bites at model human limbs within a relatively brief time, southern Pacific rattlesnakes Crotalus helleri inject slightly declining amounts with each successive bite, whereas cottonmouths Agkistrodon piscivorus inject less in the first bite and more in subsequent bites. Also, rattlesnakes inject similar amounts when striking at gloves and when grasped and forced to bite a membrane-covered beaker i.
More exciting, we have found that articles of clothing such as blue jeans may interfere with venom delivery. A yet-to-be-published experiment revealed that small and large southern Pacific rattlesnakes inject approximately two-thirds less venom into model human limbs when they are covered by denim! Obviously, millions of Americans should be interested to learn that jeans can provide significant protection when in rattlesnake habitat!
By the way, Levi, Inc. Our research on defensive bites continues at a steady pace, and we look forward to more interesting discoveries. However, you may wish to check out our research on Severity and Treatment of Snakebite with photographs.
Some of our conclusions find support in our analyses of factors associated with snakebite severity. Venom extraction. Figure 1. Structure of PLA2s from snake venoms. Green: N-terminal region critical for enzymatic and neurotoxic properties, C-terminal region essential for enzymatic activity and central Histidine in the catalytic site Rouault et al. D,E Highlighted in blue are the amino acids positions involved in the enzymatic, toxic and pharmacological properties of Crotoxin B Soares et al.
The central histidine in the catalytic site of Crotoxin B is highlighted in red. A number of PLA2s exert strong myotoxic effects which often lead to severe necrosis Harris and Maltin, ; Gutierrez and Ownby, , and many of these toxins also promote inflammation, including edema formation, cytokine production and leukocyte recruitment, pain by inducing thermal allodynia and mechanical hyperalgesia, paralysis through block of neuromuscular transmission and intensify hemorrhage by inhibiting coagulation Table 1 Camara et al.
Neurotoxic effects caused by these toxins, as well as some of their proinflammatory effects, occurs via the modulation of pre-synaptic terminals as well as sensory nerve-endings Camara et al. Overall, these pre-synaptic effects induce robust exocytosis of the neurotransmitters vesicles reserves which consequently lead to the depletion of neurotransmitter release in the neuromuscular junction to promote muscle paralysis Harris et al.
Table 1. Snake toxins and their multifunctional roles in the toxicity induced by snakebites. The inflammation induced by PLA2s has non-neurogenic and neurogenic substance-P dependent components Camara et al. The non-neurogenic component is mostly caused by the hydrolysis of membrane lipids that generate potent pro-inflammatory lipid mediators Costa et al. Additional non-neurogenic and neurogenic inflammations induced by PLA2s use more complex mechanisms still not fully understood.
For example, leukocyte recruitment De Castro et al. Furthermore, substance-P mediated neurogenic inflammation has been described to be induced by PLA2s from Crotalus durissus cascavella Camara et al. Interestingly, the C-terminal of Myotoxin-II a LysPLA2 isolated from Bothrops asper was able to activate macrophages, showing this region maybe be crucial for the observed enzymatic-independent inflammation Giannotti et al.
The pain induced by PLA2s is driven by inflammatory processes and sensory neuronal activation. Bradykinin is an important mediator of the inflammatory pain induced by PLA2s Moreira et al.
This suggests that PLA2s contribute to an increase in arachidonic acid release from cell membranes and its availability to be processed by cyclooxygenase resulting in prostaglandin production Verri et al.
Direct activation of sensory neurons was demonstrated by MitTx from Micrurus tener tener , a heteromeric complex between a PLA2 and a kunitz peptide Bohlen et al. This agonistic effect induces robust pain behavior in mice via activation of ASIC1 channels on capsaicin-sensitive nerve fibers Bohlen et al. BomoTx also activated a cohort of sensory neurons to induce ATP release followed by activation of purinergic receptors Zhang et al.
Unfortunately, the primary target of this neuronal activation is still unknown. The multifunctionality of PLA2s is evidenced by their myotoxic, neurotoxic and enzymatic functions, as well as by their inflammatory properties.
There is evidence that separate domains and regions of the PLA2s structure participate in these various activities Figures 1A,B. For example, for the LysPLA2 from Bothrops asper and Agkistrodon piscivorus piscivorus , the C-terminal region of these toxins residues — were identified as the active sites responsible for their myotoxic effects Lomonte et al. Interestingly, the same C-terminal region in BpirPLA2-I isolated from Bothrops pirajai had anticoagulant activity through inhibition of platelet aggregation Teixeira et al.
Crotoxin B, an AspPLA2, and a major component of the venom of Crotalus durissus terrificus , has toxic active sites fully independent of its enzymatic activity Soares et al. A detailed mutational study using the PLA2 OS2 from the Australian Taipan snake Oxyuranus scutellatus scutellatus revealed that a fold loss in enzymatic activity had only a minor effect on its neurotoxicity Rouault et al.
Furthermore, the enzymatic activity of OS2 was dependent of the N- and C-terminal regions, and the N-terminal region had a major role in the central nervous system neurotoxicity. In this study, the mutant ArgAla lost both nociceptive and edematogenic properties, LysAla and LysAla lost the nociceptive effects without interfering with the edema formation and LysAla lost the nociceptive properties and had weak inflammatory effects Figure 1E.
Similarly, an independent study showed the LysAla substitution led to reduced membrane damaging and myotoxic activities Ward et al. This C-terminal region is characterized by the presence of basic and hydrophobic residues which have been strongly associated with the ability of PLA2s to interact and penetrate the lipid bilayer Delatorre et al.
Many snake venom toxins are known to be encoded by multi-locus gene families Casewell et al. The process of gene duplication and loss underpins the evolution of many snake venom toxin families, including the PLA 2 s Lynch, ; Vonk et al. Indeed, studies have demonstrated that extremely divergent venom phenotypes e. It remains unclear as to the specific processes that underpin such diversity, although natural selection driven by environmental factors and hybridization events have both been proposed Dowell et al.
These toxins are major components of viper venoms and play a key role in the toxicity of these snake venoms Table 1 ; Tasoulis and Isbister, The final class, P-I SVMPs which consist only of the metalloproteinase domain, appeared to have evolved on multiple independent occasions in specific lineages as a result of loss of the P-II disintegrin-encoding domain Casewell et al. Throughout this diverse evolutionary history, SVMPs show evidence of extensive gene duplication events, coupled with bursts of accelerated molecular evolution Casewell et al.
These abundance differences likely underpin the distinct pathologies observed following envenomings by snakes found in these families. SVMPs contribute extensively to the hemorrhagic and coagulopathic venom activities following bites by viperid snakes, and the diversity of SVMPs isoforms often present in their venom likely facilitate synergistic effects, such as simultaneous action on multiple steps of the blood clotting cascade Kini and Koh, ; Slagboom et al.
However, it is relatively uncommon for elapid snakebites to cause systemic hemotoxicity Slagboom et al. Figure 2. Structure of metalloproteinaises from snake venoms. Cysteines are colored in red, the disintegrin-like domain is highlighted in green and the cysteine-rich domain is highlighted in blue.
The metalloproteinase domain is colored in orange, the disintegrin-like domain D-like is colored in green and the cysteine-rich domain Cys-rich is colored in blue. The disulphide bridges are colored in yellow. Research has revealed that the effects of SVMP-induced hemorrhage relies on a mechanism that occurs in two steps Gutierrez et al.
First, SVMPs cleave the basement membrane and adhesion proteins of endothelial cells-matrix complex to weaken the capillary vessels. During the second stage, the endothelial cells detach from the basement membrane and become extremely thin, resulting in disruption of the capillary walls and effusion of blood from the fragile capillary walls.
In addition to the proteinase activity, SVMPs impact on homeostasis by altering coagulation, which contributes to their toxic hemorrhagic effects Markland, ; Takeda et al. This occurs through modulation of factors such as fibrinogenase and fibrolase that mediate the coagulation cascade, depletion of pro-coagulation factors through consumption processes e.
Some SVMPs also induce inflammation, including edema, and pain by triggering hyperalgesia Dale et al. Neurogenic inflammation was also implicated in the local hemorrhage induced by Bothrops jararaca which was shown to be dependent on serotonin and other neuronal factors Goncalves and Mariano, The mechanisms on how neurogenic inflammation is triggered by the snake venom components and how it participates in the hemorrhagic process are still not understood.
Pain induced by SVMPs is characterized by hyperalgesia and inflammatory pain, which is dependent on the production of cytokines, nitric oxide, prostaglandins, histamine, leukotrienes, and migration of leukocytes, mast cell degranulation and NFkB activation Fernandes et al.
However, the mechanisms underlying SVMP-induced pain are still poorly understood, with neurogenic inflammation and neuronal excitatory properties still underexplored. The multifunctional properties of SMVPs are also well-described. These observations suggest that these domains are involved in the inflammatory hyperalgesia induced by SVMPs.
Furthermore, the pronounced hemorrhagic and necrotic activities are strongly dependent on biological effects driven by the disintegrin-like and cysteine-rich domains, as observed for BJ-PI2 da Silva et al. The hemorrhagic activity of Bothrops jararaca venom was also shown dependent on neurogenic inflammation Goncalves and Mariano, These venom toxins have evolved from kallikrein-like serine proteases and, following their recruitment for use in the venom gland, have undergone gene duplication events giving rise to multiple isoforms Fry et al.
SVSPs catalyze the cleavage of polypeptide chains on the C-terminal side of positively charged or hydrophobic amino acid residues Page and Di Cera, ; Serrano, Whilst the SVMPs are well-known for their ability to rupture capillary vessels, SVSPs execute their primary toxicity by altering the hemostatic system of their victims, and by inducing edema and hyperalgesia through mechanisms still poorly understood Table 1.
Hemotoxic effects caused by SVSPs include perturbations of blood coagulation pro-coagulant or anti-coagulant , fibrinolysis, platelet aggregation and blood pressure, with potential deadly consequences for snakebite victims Murakami and Arni, ; Kang et al. Figure 3. Structure of Serine proteinases from snake venoms.
For example, the activation of prothrombin produces thrombin which in turn produces fibrin polymers that are cross-linked. Thrombin also activates aggregation of platelets which, together with the formation of fibrin clots, results in coagulation Murakami and Arni, In addition, platelet-aggregating SVSPs will activate the platelet-receptors to promote binding to fibrinogen and clot formation Yip et al.
These procoagulant and platelet-aggregating activities will lead to the rapid consumption of key factors in the coagulation cascade and clot formation. Furthermore, fibrinolytic SVSPs play an important role in the elimination of blood clots by acting as thrombin-like enzymes or plasminogen activators, which eliminates the fibrin in the clots and contributes significantly to the establishment of the coagulopathy Kang et al.
Little is known about inflammatory responses and hyperalgesia induced by SVSPs. SVSPs in the venoms of Bothrops jararaca and Bothrops pirajai induce inflammation through edema formation, leucocyte migration mainly neutrophils and mild mechanical hyperalgesia, however, the mediators involved in these effects are still unknown Zychar et al.
Three-fingers toxins 3FTXs are non-enzymatic neurotoxins ranging from 58 to 81 residues that contain a three-finger fold structure stabilized by disulfide bridges Osipov and Utki, ; Kessler et al. They are present mostly in the venoms of elapid and colubrid snakes, and exert their neurotoxic effects by binding postsynaptically at the neuromuscular junctions to induce flaccid paralysis in snakebite victims Barber et al. Furthermore, they can exist as monomers and as covalent or non-covalent homo or heterodimers.
The diversity of 3FTX isoforms described above are a direct result of a diverse evolutionary history, whereby ancestral 3FTXs have diversified by frequent gene duplication and accelerated rates of molecular evolution. These processes, which are broadly similar to those underpinning the evolution of the other toxin families described above, are particularly associated with the evolution of a high-pressure hollow-fanged venom delivery system observed in elapid snakes Sunagar et al.
For example, gene duplication events have resulted in the expansion of 3FTX loci from one in non-venomous snakes like pythons, to 19 in the elapid Ophiophagus hannah king cobra Vonk et al. The consequences of this evolutionary history are the differential production of numerous 3FTX isoforms that often exhibit considerable structural differences and distinct biological functions Figures 4B—E.
Although many elapid snakes exhibit broad diversity of these functionally varied toxins in their venom e. Figure 4. Structure of three-finger toxins from snake venoms. K Neurotoxin II from N. L Neurotoxin b NTb from O. Despite the shared three-finger fold, the 3FTXs have diverse targets and biological activities.
Their toxic biological effects include flaccid or spastic paralysis due to the inhibition of AChE and ACh receptors Grant and Chiappinelli, ; Changeux, ; Marchot et al. 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.
Furthermore, 3FTXs are relatively small compared to the other snake toxins discussed herein, and do not exhibit multiple domains to produce their multiple toxic functions.
Nevertheless, the number of receptors, ion channels, and enzymes targeted by snake 3FTXs highlights the unique capacity of this fold to modulate diverse biological functions and the arsenal of toxic effects that are induced by 3FTXs. The unique multifunctionality of the 3FTX scafold occurs because of their resistance to degradation and tolerance to mutations and large deletions Kini and Doley, Therefore, the structure-activity relationship of the 3FTXs is complex and yet to be fully understood.
Their functional sites are located on various segments of the molecule surface. Conserved regions determine structural integrity and correct folding of 3FTXs to form the three loops, including eight conserved cysteine residues found in the core region.
Additional disulfide bonds can be observed either in the loop I or loop II which can potentially change the activity of the 3FTX in some cases. Specific amino acid residues in critical segments of the 3FTXs have been identified to be important for binding to their targets. For example, the interactions between fasciculin and AChE enzyme has been studied. The first loop or finger of fasciculin reaches down the outer surface of the enzyme, while the second loop inserts into the active site and exhibit hydrogen bonds and hydrophobic interaction Harel et al.
Several basic residues in fasciculin make key contacts with AChE. From docking studies, hydrogen bonds, and hydrophobic interactions where shown to establish receptor-toxin assembly. Hydrophobic interactions are also observed between eight amino acid residues Lys32, Cys59, Val34, Leu48, Ser26, Gly36, Thr15, Asn20 from fasciculin and the enzyme active site Waqar and Batool, These interactions involve charged residues but lacks intermolecular salt linkages.
Muscarinic toxins from mamba venoms, such as MT1 and MT7 Figures 4G,H , act as highly potent and selective antagonists of M1 receptor subtype through allosteric interactions with the M1 receptor. Fruchart-Gaillard et al. In this study, substitution within loop 1 and loop 3 weaken the toxin interactions with the M1 receptor, resulting in a 2-fold decrease in affinity Figures 4I,J. Furthermore, modifications in loop 2 of the MT1 and MT7 significantly reduce the affinity for the M1 receptor.
These two residues were not located at the tip of the toxin loop, however, they played a critical role in the interactions with their molecular targets Bourne et al. The insertion of the loop II into the binding pocket of a nAChR induces the neurotoxin activity and significantly determines the toxin-receptor interactions, while loop I and III contact the receptor residues by their tips only and determine the immunogenicity of the short neurotoxins.
The structure of neurotoxin b NTb , a long neurotoxin from Ophiophagus hannah , has been elucidated Peng et al. Conserved residues in loop II also play an important role in the toxicity of the long neurotoxins by making ionic interactions between toxin and receptor. Positively charged residues Trp27, Lys24 and Asp28 are highly conserved residues in the long neurotoxins.
Furthermore, a modification of the Trp27 in the long neurotoxin analog of NTb from king cobra venom led to a significant loss in neurotoxicity. The additional disulphide bridge in loop II of long neurotoxins does not affect the toxin activity. Nevertheless, cleavage of the additional disulphide bridge in loop II can disrupt the positively charged cluster at the tip of loop II. Changes in loop II conformation will affect the binding of the long neurotoxin to the target receptor resulting the loss of neurotoxicity Peng et al.
Long and short neurotoxins show sequence homology and similar structure. Previous studies show that many residues located at the tip of loop II are conserved in both short and long neurotoxins.
However, significant differences between long-chain neurotoxin and short chain neurotoxin are indicated by the immunological reactivity. Many of the residues involved in the antibody-long neurotoxins binding are located in loop II, loop III, and in the C-terminal, while in short neurotoxins the antibody's epitope makes interactions with the loop I and loop II Engmark et al.
Animal-derived antivenoms are considered the only specific therapy available for treating snakebite envenoming Maduwage and Isbister, ; Slagboom et al. These consist of polyclonal immunoglobulins, such as intact IgGs or F ab' 2 , or Fab fragments Ouyang et al. Antivenoms can be classified as monovalent or polyvalent depending on the immunogen used during production.
Monovalent antivenoms are produced by immunizing animals with venom from a single snake species, whereas polyvalent antivenoms contain antibodies produced from a cocktail of venoms of several medically relevant snakes from a particular geographical region. Polyvalent antivenoms are therefore designed to address the limited paraspecific cross-reactivity of monovalent antivenoms by stimulating the production of antibodies against diverse venom toxins found in different snake species, and to avoid issues relating to the wrong antivenom being given due to a lack of existing snakebite diagnostic tools O'leary and Isbister, ; Abubakar et al.
However, polyvalent therapies come with disadvantages—larger therapeutic dose are required to effect cure, potentially resulting in an increased risk of adverse reactions, and in turn increasing cost to impoverished snakebite victims Hoogenboom, ; O'leary and Isbister, ; Deshpande et al. Variation in venom constituents therefore causes a great challenge for the development of broadly effective snakebite therapeutics.
The diversity of toxins found in the venom of any one species represents considerable complexity, which is further enhanced when trying to neutralize the venom of multiple species, particularly given variations in the immunogenicity of the multi-functional toxins described in this review. Antivenom efficacy is therefore, typically limited to those species whose venoms were used as immunogens and, in a number of cases, closely-related snake species that share sufficient toxin overlap for the generated antibodies to recognize and neutralize the key toxic components Casewell et al.
Because variation in venom composition is ubiquitous at every level of snake taxonomy e. Such studies have revealed surprising cross-reactivity of antivenoms against distinct, non-targeted, snake species, such as: i the potential utility of Asian antivenoms developed against terrestrial elapid snakes at neutralizing the venom toxicity of potent sea snake venoms Tan et al.
The later of these studies demonstrated cross-neutralization between distinct snake lineages e. Thus, detailed knowledge of venom composition can greatly inform studies assessing the geographical utility of antivenoms.
Such studies have stimulated much research into the development of novel therapeutic approaches to tackle snakebite. These include the use of monoclonal antibody technologies to target key pathogenic toxins found in certain snake species Laustsen et al. It is anticipated that in the future these new therapeutics may offer superior specificities, neutralizing capabilities, affordability and safety over conventional antivenoms.
However, the translation of their early research promise into the mainstay of future snakebite treatments will ultimately rely on further research on the toxins that they are designed to neutralize.
Specifically, the selection, testing and optimization of new tools to combat snake envenoming is reliant upon the characterization of key pathogenic, and often multifunctional, toxins found in the venom of a diverse array of medically important snake species.
The first drug derived from animal venoms approved by the FDA is captopril, a potent inhibitor of the angiotensin converting enzyme sACE used to treat hypertension and congestive heart failure Cushman et al. Captopril was derived from proline-rich oligopeptides from the venom of the Brazilian snake Bothrops jararaca Ferreira et al.
This milestone in translational science in the late 70's revealed the exceptional potential of snake venoms, and possibly other animal venoms such as from spider and cone snails, as an exquisite source of bioactive molecules with applications in drug development. More recently, an anti-platelet drug derived from the venom of the southeastern pygmy rattlesnake Sistrurus miliarius barbouri was commercialized as Integrillin by Millenium Pharmaceuticals, and is used to prevent acute cardiac ischemia Lauer et al.
The resulting product is now commercialized as Syn-AKE. Snake toxins have been applied with great success in diagnostics. Snake toxins also have the potential to become novel painkillers. These findings, alongside current research into venom toxins, suggest an exciting future for the use of snake venoms in the field of drug discovery. Snake venoms are amongst the most fascinating animal venoms regarding their complexity, evolution, and therapeutic applicability.
They also offer one of the most challenging drugs targets due to the variable toxin compositions injected following snakebite. The multifunctional approach adopted by the major components of their venoms, by using multidomain proteins and peptides with promiscuous folds e. Gaining a better understanding of the evolution, structure-activity relationships and pathological mechanisms of these toxins is essential to develop better snakebite therapies and novel drugs.
Recent developments in genomics, proteomics and bioactivity assays, as well as in the understanding of human physiology in health and disease, are enhancing the quality and speed of research into snake venoms. We hope to improve the therapies used to neutralize the toxic effects of PLA2s, SVMPs, SVSPs and 3FTXs, and to develop drugs as new antidotes for a broad-spectrum of snake venoms that could also be effective in preventing the described inflammatory reactions and pain induced by snakebite.
Finally, a diversity of biological functions in snake venoms is yet to be explored, including their inflammatory properties and their intriguing interactions with sensory neurons and other compartments of the nervous system, which will certainly lead to the elucidation of new biological functions and the development of useful research tools, diagnostics and therapeutics. FC provided theme, scope, and guidance. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Abubakar, I. Randomised controlled double-blind non-inferiority trial of two antivenoms for saw-scaled or carpet viper Echis ocellatus envenoming in Nigeria. PLoS Negl. Ainsworth, S. Different people may consider a bee sting to be a minor nuisance or unbearable, but everyone agrees that it hurts immediately.
The survey asked people who had collectively received bites from different snake species. By far the most common experience involved relatively low pain levels after a bite within the first five minutes, when the pain might deter a predator in time for the snake to escape injury or death. More severe pain often followed later. We also investigated the presence of venoms that caused early-onset pain throughout an evolutionary tree of snake species.
We found that venoms which cause early pain evolved on several occasions, but were usually quickly lost again during the course of snake evolution. There are likely exceptions though. For instance, some coral snakes and pit vipers have specifically pain-inducing toxins in their venoms.
Spitting cobras have unique behavioural adaptations for defensive venom use, and their venoms cause intense pain upon contact with eyes. Did you know that you are nine times more likely to die from being struck by lightning than to die of venomous snakebite?
The graph below compares deaths from venomous snakebites to some leading causes of death, lightning strikes and other animal related deaths. Poisons are substances that are toxic cause harm if swallowed or inhaled.
Venoms are generally not toxic if swallowed, and must be injected under the skin by snakes, spiders, etc. However, we do NOT recommend drinking venom! The venom gland is a modified salivary gland, and is located just behind and below the eye. The size of the venom gland depends on the size of the snake - this image shows the approximate size of the venom gland in relation to the skull of this Timber Rattlesnake Crotalus horridus.
In a study comparing snake venoms, researchers milked the largest amount of venom from an Eastern Diamondback Rattlesnake Crotalus adamanteus --more than from any other species they studied. A comparative study found that the snake venom that is most toxic to mice of the species tested is that of the Inland Taipan Oxyuranus microlepidotus , found in Australia. The most toxic venom of U. It is important to note that these venoms were only tested on mice.
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