ADVANCES IN CLINICAL CHEMISTRY, VOL.

57

BIOCHEMISTRY OF ENVENOMATION
Prameet Kaur,* Vibha Ghariwala,* Kun Song Yeo,*
Hui Zhing Tan,* Jian Chye Sam Tan,* Arunmozhiarasi
Armugam,* Peter N. Strong,† and Kandiah Jeyaseelan*,1

*Department of Biochemistry, Yong Loo Lin School of Medicine,

National University Health System, National University of
Singapore, Singapore
†
Biomedical Research Centre, Biosciences Division,
Sheffield Hallam University, Sheffield, United Kingdom

1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
2. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
3. Animal Venoms, Their Action, and Clinical Manifestations . . . . . . . . . . . . . . . . . . . . . 190
3.1. Neurotoxins Acting at the Neuromuscular Junction . . . . . . . . . . . . . . . . . . . . . . . . 191
3.2. Ion Channel Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
3.3. Cardiotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
3.4. Hemotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
4. Biochemical Basis of Venoms in Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
4.1. Neuroprotective Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
4.2. Cardioprotective Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
4.3. Ion Channel Toxins and Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
4.4. Hemostasis and Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
4.5. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Abbreviations

ASIC acid-sensing ion channel

CTX cardiotoxin
GLP-1 glucagon-like peptide I
m.e.p.p. miniature endplate potential

1
Corresponding author: Kandiah Jeyaseelan, e-mail: bchjeya@nus.edu.sg

187

0065-2423/12 $35.00 Copyright 2012, Elsevier Inc.

DOI: 10.1016/B978-0-12-394384-2.00007-3 All rights reserved.
188 KAUR ET AL.

mAChR muscarinic acetylcholine receptor

nAChR nicotinic acetylcholine receptor
PLA2 phospholipase A2
SVMP snake venom metalloproteinase
a-LTX a-latrotoxin

1. Abstract

Venoms and toxins are of significant interest due to their ability to cause a
wide range of pathophysiological conditions that can potentially result in death.
Despite their wide distribution among plants and animals, the biochemical
pathways associated with these pathogenic agents remain largely unexplored.
Impoverished and underdeveloped regions appear especially susceptible to
increased incidence and severity due to poor socioeconomic conditions and
lack of appropriate medical treatment infrastructure. To facilitate better man-
agement and treatment of envenomation victims, it is essential that the bio-
chemical mechanisms of their action be elucidated. This review aims to
characterize downstream envenomation mechanisms by addressing the major
neuro-, cardio-, and hemotoxins as well as ion-channel toxins. Because of their
use in folk and traditional medicine, the biochemistry behind venom therapy
and possible implications on conventional medicine will also be addressed.

2. Introduction

Estimates of annual worldwide snakebite vary widely between 400,000

(20,000 fatalities) and 1.8 million (40,000 fatalities), primarily in the countries
of Southern and Southeast Asia and sub-Saharan Africa [1]. The incidence of
snakebite envenomation is likely underestimated since a substantial number
of victims seek treatment outside traditional hospital settings, thus minimiz-
ing the impact to public health statistics. This nontraditional practice is more
likely in impoverished regions, thus contributing to additional socioeconomic
hardship [2].
Venoms are often confused with poisons; venom consists of a cocktail of toxic
molecules, contained in an isolated, specialized gland within the venomous
animal and directly or indirectly injected into the host. On the other hand, poison
is a broad term for any introduced substance causing illness, injury, or death via
molecular targets and can be physical, chemical, or biological [3]. Venom is
produced endogenously and secreted by animals for hunting and defense. For
example, snake venom functions primarily as a prey immobilization mechanism
and digestive aid [4]. Injection of venom is typically accomplished via a
 ENVENOMATION 189

specialized apparatus that can penetrate the dermis thus bringing the venom into
immediate contact with proximal tissues and subsequent distal organs via the
circulation. In addition to bites and stings, envenomation can occur by a variety
of other methods. For example, the Gila monster propels venom from glands to
the teeth via chewing and capillary action [5].
Exposure to the toxic components in the venom produces a wide range of
clinical symptoms via dysregulation of normal physiological function. Be-
cause envenomation is a dynamic process, the full extent of symptoms may
not manifest immediately. However, in the absence of symptoms after 6–8h,
the patient may be considered to be medically cleared [6]. Clinical effects
depend on the venomous species involved and toxicity and mechanisms of
the venom. For instance, envenomation by North American elapids proceeds
via local, early systemic, and late systemic effects. Local effects can manifest
as edema, pain, or discoloration but is an unreliable indicator of the severity.
Once the venom enters the blood stream or lymphatic system, it may spread to
other parts of the body and depending on the specificity of action of the toxins
present, may affect different organs or systems. Early systemic effects may
include lethargy, weakness, nausea, vomiting, salivation, ptosis, thrombocy-
topenia, and abnormal reflexes. Late systemic effects include respiratory
failure, usually due to paralysis, and cardiovascular and neurological dys-
function, possibly leading to death [7]. Envenomation by species lower down
in the evolutionary tree, such as insects, arthropods, and marine animals, may
have different effects following the entry of the venom and the severity may
vary. The unpredictable nature of such envenomations and the multitude of
mechanisms conferred by the different toxins make the assessment and man-
agement of clinical outcomes more difficult, as treatment against a single
toxin will prove to be ineffective. Table 1 provides a representative list of
venomous species and the predominant mode of action of the major toxins.
Toxins found in venom are often of small size, displaying a range of
functional and structural diversity and able to exert multiple effect on a
variety of molecular targets, including enzymes and ion channels [8]. This
review elaborates on how the major systems in our body can be dysregulated
by animal venoms and highlights examples of recent advances in venom
toxicology. It emphasizes the effects of envenomation on major bodily func-
tions from a biochemical and molecular point of view. The review discusses
general mechanisms with reference to these examples but is not intended to
be exhaustive, given the considerable literature in this field.
Toxins and venoms have also been used in traditional and folk medicine for
centuries. The range and biodiversity of venoms and toxins provide a range of
compounds from which therapeutic agents may be developed [9], and with
recent developments in science and both laboratory based and clinical studies,
they have made their mark in therapeutic medicine. The review also provides
190 KAUR ET AL.

TABLE 1
VARIOUS TYPES OF VENOMOUS AND POISONOUS ANIMALS AND EXAMPLES OF THEIR
RESPECTIVE TOXINS

Animals Representative components of venoms/toxins

Box jellyfish
Hemolysin (Hemotoxin) [340]

Myotoxins (T1 and T2) [341]
Sea anemone
Ion channel toxins
○ Sea anemone toxin [115], APETx2 [172]
Cone snail
Ion channel toxins
○ a-Conotoxin [85], o-conotoxin [161,162]
Stingray
Local tissue damage
○ Hyaluronidase [342]
Scorpionfish
Local tissue damage
○ Plumieribetin (a1b1 integrin inhibitory activity) [343]

Hemotoxin
○ Scorpaena plumieri Cytolytic Toxin (Sp-CTx) [344]
Frogs
Neurotoxins
○ Batrachotoxin [345]
Bee
Hemotoxin
○ Bee venom serine protease [248]

Ion channel toxin
○ Apamin [145]
Scorpion
Ion channel Toxin
○ Scorpion a-toxin [128], Noxiustoxin [157]
Spider
Neurotoxins
○ o-Agatoxins [162], m-agatoxins [346], Hanatoxin 1 (HaTxl) [347],
Hanatoxin 2 (HaTx2) [347], a-latrotoxin [16]
Snakes
Neurotoxins
○ b-Neurotoxins (presynaptic)—crotoxin [21], a-bungarotoxin [13],
anticholinesterase (synaptic cleft)—fasciculin [14], nAChR
(postsynaptic)—cobrotoxin [92]

Hemotoxin
○ Ancrod [238], Batroxobin [240], Ecarin [246]

Cardiotoxin
○ Cardiotoxin III [332]

an insight into the pharmacopoeia of various venoms in the treatment of

common diseases and the biochemical basis of therapy conferred by venoms.

3. Animal Venoms, Their Action, and Clinical Manifestations

There are possibly more than 100 different toxic and nontoxic compounds
(proteins and peptides, as well as nontoxic carbohydrates, lipids, and amines)
within the venom of any species [10]. Venom can be organ specific or affect
 ENVENOMATION 191

entire systems, depending on the toxic components present. Envenomation

can manifest itself in several pathological conditions, including muscle and
dermal necrosis, hypotension, muscle paralysis, myocardial effects, hemor-
rhage, and respiratory arrest. This section elucidates several biochemical
aspects of envenomation with respect to major physiological systems. The
different venoms are classified under neurotoxins that act at the neuromus-
cular junction of the peripheral nervous system, ion channel toxins, cardio-
toxins (CTXs), and hemotoxins. Since the toxins acting on ion channels
affect the neuromuscular junction as well as other organs, we have discussed
these in a separate section.

3.1. NEUROTOXINS ACTING AT THE NEUROMUSCULAR JUNCTION

Neurotoxins from snake venoms act on different sites at the neuromuscu-
lar junction via excitatory or inhibitory mechanisms, so as to immobilize prey
either by causing respiratory failure or by inducing flaccid paralysis of the
voluntary muscles, resulting in death due to suffocation [10,11]. Most
venoms have several different, simultaneous modes of action to immobilize
prey as effectively as possible [3]. Toxins acting at the neuromuscular junc-
tion are found mainly in elapids, hydrophids, and some species of vipers,
with representative examples such as Mojave toxin from Crotalus scutulatus
scutulatus (Mojave rattlesnake) [12], a-bungarotoxin from Bungarus multi-
cinctus [13], and fasciculin from the green mamba, Dendroaspis angusticeps
[14]. Neurotoxins either cause continuous blockade of neuromuscular trans-
mission [15] or increase neurotransmitter levels [16]. High-affinity binding is
common for both pre- and postsynaptically acting toxins, which undermines
the efficacy of treatment with antivenom, although particular improvements
are evident in treating snakebite due to the effects of postsynaptic toxins [17].
Anticholinesterases acting in the synaptic cleft lengthen the timecourse of
acetylcholine action and can therefore be used to counter the effects of
postsynaptic neurotoxicity [18,19].
Neurotoxins acting at the neuromuscular junction can be classified into
various groups: (1) presynaptic neurotoxins with phospholipase A2 (PLA2)
activity, (2) presynaptic toxins which cause massive release of neurotransmit-
ter, (3) synaptic cleft toxins with anticholinesterase activity, and (4) postsyn-
aptic toxins acting on neurotransmitter receptors.

3.1.1. Presynaptic Toxins: PLA2 Enzymes

The largest and most widely studied groups of neurotoxins display presyn-
aptic neurotoxicity. They are also known as b-neurotoxins, which are unable
to cross the blood–brain barrier and therefore do not reach the central
nervous system [20]. Crotoxin from the South American rattlesnake,
192 KAUR ET AL.

Crotalus durissus terrificus, was the first neurotoxin isolated in crystalline

form by Slotta and Fraenkel-Conrat [21] which blocks neuromuscular trans-
mission, resulting in peripheral respiratory paralysis and eventually death
[22]. Interestingly, different experimental animals show a different progres-
sion of clinical symptoms; chicks and kittens demonstrate flaccid paralysis of
the extremities followed by respiratory depression, whereas the order is
reversed in the mice and rats. Upon death, which ensued 2–8h after enven-
omation, all animals had strongly beating hearts and were still responsive to
nociception [22]. Edelson et al. [23] looked at the response of the lungs to
intratracheal administration of PLA2 from Naja naja venom in adult male
rats. Animals displayed acute signs of inflammation (e.g., the accumulation
of inflammatory cells, interstitial and alveolar edema, as well as alveolar wall
thickening) [23]. By analogy, envenomed patients displayed signs of ptosis
and mydriasis, followed by bulbar and respiratory paralysis, before total
flaccid paralysis ensued [10].
Most PLA2 toxins have a similar enzymatic mechanism of action and
induce similar consequential physiological changes, although there are slight
differences in toxin binding sites, many of which are thought to be over-
lapping. Although PLA2 toxins belong to a broad structurally homologous
family (40–99% amino acid sequence identity) [24], there is no correlation
between enzymatic activity and toxicity [25]. PLA2 toxins exist variously as
monomers, dimers, and multimeric proteins. Notexin from Notechis scutatus
scutatus is a single-chain PLA2 (Fig. 1) [26], b-bungarotoxin from B. multi-
cinctus consists of two covalently linked subunits of which one is a PLA2
enzyme [27], taipoxin from Oxyuranus scutellatus scutellatus comprises of a
trimer in which one subunit is a PLA2 enzyme [28], while textilotoxin from
Australian elapid Pseudonaja textilis consists of five PLA2 subunits [29].
Among these toxins, the subunits which are devoid of PLA2 activity act as
chaperones and increase the specificity of binding to individual target sites
[30]. Crotoxin is an example of a noncovalently linked dimer: a weakly toxic,
basic PLA2 subunit and an acidic nontoxic subunit which is devoid of
enzymatic activity (Fig. 1) [31,32]. Crotoxin comprises a mixture of several
isoforms with similar polypeptide sequences but dramatically different enzy-
matic and pharmacological activities. On its own, the basic phospholipase
subunit binds nonspecifically to diverse low-affinity binding sites; in the
presence of the acidic subunit, however, there is an increase in both binding
affinity and specificity of the basic subunit to high-affinity presynaptic bind-
ing sites (comprising of negatively charged mono- and diphosphoinositide
phosphates), which boosts the pharmacological efficacy of the phospholipase
[32]. Interestingly, the acidic subunit is subsequently released from the dimer
upon binding to the membrane [32,33]. There is disagreement as to whether
the effects of crotoxin are solely presynaptic. Binding studies carried out
 ENVENOMATION 193

Presynaptic toxins (b-neurotoxins)

Notexin (1AE7)[360] Crotoxin (2QOG)[361]

Synaptic cleft toxins (anticholinesterase)

Fasciculin 2 (1FSC)[362]
Postsynaptic toxins

a-Conotoxin (2GCZ)[363] Erabutoxin (1QKD)[364] Cobratoxin (2CTX)[365]

Short-chain neurotoxin Long-chain neurotoxin
Ion channel toxins

d-Conotoxin (1FU3)[366] w-Conotoxin (1V4Q)[367]

FIG. 1. Three-dimensional (3D) structures of a- and b-neurotoxins, synaptic cleft neurotoxins,

and ion channel toxins.
194 KAUR ET AL.

in vitro, using postsynaptic Electrophorus electroplaque preparations, showed

blocking effects at 10 lower concentrations as those reportedly required for
presynaptic blockage, supporting a postsynaptic effect. However, experiments
carried out on mammalian diaphragm preparations failed to show any de-
crease in miniature endplate potential (m.e.p.p) amplitude, a necessary criteri-
on for postsynaptic activity [34]. The exact mechanism of crotoxin action
therefore appears to depend on the preparation studied.
Several studies have demonstrated that some PLA2 toxins show high
binding specificity, for example, b-bungarotoxin, which is one of the most
specific presynaptic neurotoxins known [35]. Daboiatoxin, another PLA2
toxin from Daboia russelli siamensis venom, also binds with high specificity
and affinity to rat cerebrocortical synaptosomes and synaptic membrane
fragments [36]; this study demonstrated that evolution has given rise to
high-specificity tissue binding by these PLA2 toxins as the PLA2 subunits
target unique specific proteins or membranes structures [30]. Pražnikar et al.
[37] recently found that after the initial binding step, ammodytoxin, a PLA2
toxin from Vipera ammodytes ammodytes, was translocated into the cytosol
of a motoneuron-like cell by clathrin-mediated endocytosis, subsequently
binding to calmodulin and 14-3-3 proteins. Ammodytoxin has also been
detected in the nucleus, as well as the cytosol, of rat hippocampal neurons
[38]. This internalization process allowed the PLA2 activity of the toxin to act
on intracellular organelles like mitochondria, accelerating processes that lead
to synaptic vesicle recycling and cell degeneration [37], events which are
crucial for the second and third phase of PLA2 toxin action (discussed
below). The discovery of this internalization process could be an important
step in deciphering the enigmatic and complex mechanism of action of PLA2
toxins.
Napias and Heilbronn [39] investigated the binding of notexin and Naja
nigricollis basic PLA2 and found that these toxins preferentially bind to
synaptosomal membranes as well as mitochondria. They suggested that
synaptosomal phosphatidylcholine could be the specific target of these
PLA2 toxins. It can therefore be postulated that once PLA2 toxins are
internalized as shown by Pražnikar et al. [37], these toxins would target the
mitochondrial membrane, resulting in mitochondrial damage as evidenced
by the rounding of mitochondrial structures and the appearance of a less
defined interior.
Following an initial specific binding step, presynaptic PLA2 toxins cause
failure of neuromuscular transmission by a triphasic mechanism, comprising
(i) an early inhibition of neurotransmitter release, followed by (ii) a long
phase of facilitated neurotransmitter release, and (iii) an irreversible decline
in neurotransmitter release [40,41]. This is evidenced by an immediate initial
decrease in m.e.p.p. frequency, followed by a temporary increase in m.e.p.p.
 ENVENOMATION 195

frequency as well as quantal content [13] due to the release of bursts of

acetylcholine, and finally a decrease in m.e.p.p. frequency until m.e.p.p.
s can no longer be detected. Taken together, these observations provide
cellular mechanistic support for the observed systemic paralytic symptoms
when animals are injected with PLA2 toxins [42].
The first phase of PLA2 toxin action has been shown to be independent of
PLA2 activity. In contrast, the second and third phases are enzyme depen-
dent [42]. It has been hypothesized that phase changes in presynaptic termi-
nal membrane structure after incorporation of PLA2 toxins into the
membrane give rise to the first phase of toxin action [41], though the exact
mechanism is still unclear. The second and third phases require the activity of
PLA2, which catalyzes the Ca2þ-dependent hydrolysis of membrane glycer-
ophospholipids, to yield lysophospholipids and fatty acids [42,43]. It has
been proposed that this initially causes a change in the conformation of the
presynaptic plasma membrane, facilitating fusion of synaptic vesicles with
the presynaptic membrane and an increase in exocytotic neurotransmitter
release. At the same time, the toxins inhibit vesicle recycling, which eventu-
ally leads to a depletion of vesicles in the terminal and a cessation of
neurotransmitter release [44]. The second phase has also been speculated to
be due to a rise in cytosolic Ca2þ in the nerve terminals to encourage vesicle
release [45]. Toxins like b-bungarotoxin have also been shown to block
potassium channels, thus providing a second, indirect way of stimulating
neurotransmitter release [46].
Rather than inhibiting recycling [44], the picture is confused by the sugges-
tion that PLA2 toxins might actually enter nerve endings by the vesicle
recycling process [37,38,47]. Internalization results in the uncoupling of
mitochondria [48], a loss of Ca2þ homeostasis and the inhibition of ATP
synthesis, the latter giving rise to an inhibition of protein phosphorylation,
degradation of synaptic vesicles, inhibition of endocytosis, and the disinte-
gration of the cytoskeleton. Leakiness of the presynaptic plasma membrane
increases cytosolic calcium ion concentrations, which in turn increases PLA2
toxin activity and culminates in the total degeneration of the nerve terminal
and consequent neuromuscular blockade [47].
The third and last phase of PLA2 toxin action is morphologically char-
acterized by swollen and enlarged axon terminals, void of synaptic vesicles
and multiple clathin-coated o-shaped invaginations of the plasma mem-
brane, indicative of incomplete synaptic vesicle retrieval [42,49–51]. These
events are followed by the presence of vacuoles and inflamed mitochondria
within the terminal [49,50]. The suggestion of faulty vesicle retrieval was
supported by immunocytochemical data demonstrating the presence of syn-
aptic vesicle markers such as synaptophysin I (SypI) and vesicle-attached
membrane protein 2 (VAMP2) on neurite bulges, and the presence of the
196 KAUR ET AL.

luminal domain of synaptotagmin on the cell surface. These morphological

changes were accompanied by glutamate release from neuronal cultures,
mimicking the release of acetylcholine from neuromuscular junctions [52].
The swelling of neurites could be attributed to the enzymatic production of
lysophospholipids and fatty acids which increases the membrane permeability
to ions, resulting in increased osmotic pressure [52,53]. This phospholipid
hydrolysis mechanism, coupled with the action of a PLA2 toxin such as
taipoxin, to disrupt the synaptic F-actin barrier to release reserved secretory
vesicles [54] gives rise to neurotransmitter release but is not supported by
synaptic vesicle replenishment [52]. Disorganization of the endplate, engulf-
ment of the axon terminal by Schwann cells, and structural modifications to
the preterminal motor nerve, as well as eventual myonecrosis, can be seen
after intramuscular injection of crotoxin [51]. Fletcher and Middlebrook [55]
have shown that b-bungarotoxin and N. naja atra PLA2 toxin both stimulate
acetylcholine release from synaptosomes but at the same time block choline
uptake (which is required to synthesize acetylcholine, thereby inhibiting
acetylcholine release) from synaptosomes. This indirectly results in complete
inhibition of neurotransmitter release by depleting existent neurotransmitter
vesicles as well as preventing the formation of new ones.
Human envenomation from snake venoms containing large quantities of
b-neurotoxins results in profound neuromuscular weakness, thus broadly
supporting the mechanisms proposed from biochemical, cellular, and physi-
ological studies. Unfortunately, treatment with antivenoms, anticholines-
terases, or diaminopyridines has little effect on neuromuscular blockade,
due to the high-affinity binding of PLA2 toxins [56]. Harris and colleagues
[56] showed that injection of notexin and taipoxin into the hind limb of a rat
showed irreversible damage to the presynaptic nerve terminal within 1h.
Seventy percent of the muscle fibers were denervated in 24h. Likewise,
exposure of frog sartorius muscle and rat diaphragm muscle to b-bungaro-
toxin led to disappearance of the motor axon from the muscle endplate, while
Schwann cells established extensive synaptic contact with the muscle fiber.
After only half an hour of b-bungarotoxin treatment, some endplates
attained a stage of denervation similar to that seen several days after trans-
ecting the motor nerve [42]. These cellular level observations explain why
clinical envenomation with b-neurotoxins results in a severe and prolonged
paralysis which lasts several weeks. They also rationalize the need for pro-
longed assisted mechanical ventilation of the victim, due to weakness of
respiratory muscles [57]; recovery depends on regeneration of the terminal
axons [58].
Neuromuscular blockade induced by PLA2 toxins accounts for the weak-
ness of respiratory muscles but cannot explain the pulmonary inflammation
and edema which also play a crucial role in respiratory paralysis [23].
 ENVENOMATION 197

Cher et al. [59] therefore investigated the effect of the PLA2 from the venom
of Naja sputatrix on gene expression in a rat model. This PLA2 is implicated
in peripheral respiratory paralysis, resulting from inflamed and edematous
lungs. Inflammatory genes (e.g., cytokines, interleukins, and tumor necrosis
factor) showed increased levels of expression, whereas in contrast there
was decreased expression of the gene encoding Naþ/Kþ ATPase, which is
required for the clearance of edema fluid and thus giving rise to fluid
accumulation [59].
Some PLA2 toxins overlap in the mechanisms by which they confer toxicity.
For instance, the highly toxic Mojave toxin and a basic PLA2 from
N. nigricollis venom have been shown to have lethal direct hemolytic action,
whereas caudoxin and notexin show indirect hemolytic activity [35]. The
hemolytic action of PLA2 toxins is discussed in Section 3.4.4.2.

3.1.2. Presynaptic Toxins: Excitatory Neurotoxins

Another group of presynaptic neurotoxins are those that excite peripheral
and central nervous system synapses to cause massive release of neurotrans-
mitter. The most widely known is a-latrotoxin (a-LTX) from the black
widow spider (Latrodectus sp.). a-LTX binding dysregulates the function of
cation channels, leading to a flood of Ca2þ into the cytosol [16,60]. This
results in an uncontrolled release of the reserve pool of synaptic vesicles
containing neurotransmitters like acetylcholine and norepinephrine [61]
without inhibition of synaptic vesicle recycling or internalization of the
toxin into the presynaptic cell [15]. Activation of sympathomimetic and
cholinergic synapses thus occurs and the neuromuscular junction is hyper-
stimulated [62].
Schaivo et al. [15] have proposed a possible mechanism of action of a-LTX
and similar molecules centered on the existence of a large macromolecular
structure at the presynaptic membrane which is responsible for neuroexocy-
tosis. The structure comprises a Ca2þ channel/syntaxin complex bound to
neurexin Ia and the Ca2þ-independent LTX receptor, via a Ga protein.
Neurexin could be connected to the nerve terminal active zones via interac-
tions with synaptotagmin, and the LTX receptor could function to ensure
proper assembly of the neuroexocytosis machinery. a-LTX binding would
lead to a conformational change in its receptor that would signal to induce
neuroexocytosis. These signals are likely to be detected by nerve terminal Ca2þ
channels which subsequently open, facilitating influx of Ca2þ into the terminal
and adding to the a-LTX induced neurotransmitter release. Neuroexocytosis is
also amplified by the presence of calcium permeable a-LTX channels, which
assemble when a-LTX inserts into the presynaptic terminal membrane after
toxin binding [15].
198 KAUR ET AL.

Administration of a sublethal dose of black widow spider venom into the

calf muscles of mice resulted in swollen nerve terminals devoid of synaptic
vesicles in just half an hour. After 6h, terminals were disrupted and engulfed
by Schwann cells. Finally, after 24h, every endplate was denervated with
concomitant signs of degeneration. At this stage, neurotransmission was lost
(absence of m.e.p.p.s and endplate potentials) for 3days postadministration
[63]. Recovery occurred in stages, with reinnervation, the reestablishment of
axon terminals at the majority of endplates, and a subsequent return to nor-
mal morphology being observed at 2, 3, and 8days, respectively. Analogous
to the presynaptically acting snake neurotoxins discussed above, endplate
reinnervation facilitated recovery and similarities in morphological changes
reinforce the notion that increased Ca2þ levels in the nerve terminal are the
main cause of synapse degeneration initiated by both a-LTX and PLA2
neurotoxins [20].
Clinically, a person bitten by a black widow spider first experiences a
painless prick sensation at the site of envenomation which slowly progresses
to a small erythema. Muscle cramps develop over time, either at the site of
envenomation or sometimes involving all systemic muscles, which can be-
come severe and last for several days [64–66]. Critical cases may show signs of
abdominal rigidity, nausea and headache, hypertension, tachycardia, facial
edema, and renal failure [62,67].

3.1.3. Synaptic Cleft Toxins

Acetylcholinesterase is present at neuromuscular junctions and cholinergic
synapses of the central nervous system. The enzyme terminates synaptic
transmission by hydrolyzing acetylcholine into choline and acetic acid. Cho-
line is recycled into the presynaptic cell and is converted back to acetylcholine
by the enzyme choline acetyltransferase [68]. Inhibitors of acetylcholinester-
ase prevent this degradation, resulting in prolonged excitation due to exces-
sive acetylcholine in the synaptic cleft [14,69].
Fasciculins are toxins with anticholinesterase activity that are found in the
venoms of the green mamba, D. angusticeps [14] and the black mamba,
Dendroaspis polylepis [70]. Intraperitoneal injection of fasciculin into mice
gives rise to continuous, uncontrolled muscle movements, clinically known as
fasciculations and thus accounting for the toxin’s name. Fasciculations are
followed by elevated secretions from the respiratory tract as well as lacrimal
and salivary glands, which leads to neuromuscular paralysis and eventually
death by asphyxiation [14,69]. The skeletal muscles of mice injected with
fasciculin showed a vast reduction in acetylcholinesterase activity at neuro-
muscular junctions [71]. At the cellular level, this was manifested by elevated
twitch responses of the mouse phrenic nerve-hemidiaphragm preparation
and enhanced levels of neuromuscular transmission, evidenced by increasing
 ENVENOMATION 199

the duration and amplitude of end plate potentials. The actions of fasciculin
could not be reversed by washing [72], similar to PLA2 toxins.
There are two isoforms of fasciculin (fasciculins 1 and 2), each having 61
amino acids with four disulfide bridges. They display a ‘‘three-finger’’ struc-
ture (Fig. 1) [71], similar to a-neurotoxins. A large proportion of the posi-
tively charged residues of fasciculin are hidden in the center of the toxin,
whereas the negatively charged residues are found at the C-terminus. The
toxin binds to a negatively charged site on the surface of acetylcholinesterase,
inducing a conformational change at the catalytic active site and inhibiting
enzyme activity [69–71]. Fasciculins noncompetitively inhibit both muscle
and brain acetylcholinesterases [14,69], although the effects of fasciculin on
synapses in brain in vivo have not been reported.
Formation of the fasciculin 2/acetylcholinesterase complex is very favor-
able as the conformational changes induced on complex formation give
rise to a very low energy state as compared to the individual proteins [73].
Eastman et al. [74] have reported that the acetylcholinesterase active site
comprises a gorge filled with aromatic residues, with a serine residue near the
base. This is the location of an acyl enzyme intermediate, formed during
hydrolysis of the substrate and known as the acylation site. Residues near the
entrance to the gorge consist of a peripheral site where fasciculin 2 binds. The
toxin blocks catalysis by inducing a conformational change of the active site,
resulting in inhibition of proton transfer during acylation. Toxin binding
to the peripheral site is believed to provide steric hindrance for enzyme
substrates at the opening to the narrower gorge [75]. In addition, there are
significantly higher dynamic movements of parts of the enzyme once the
complex is formed which disrupt the catalytic active site [73,75,76].
Using fluorescently tagged fasciculin 2, it has been shown that dissociation
of toxin/ enzyme complexes at synaptic junctions is extremely slow. Removal
of complexes from the synaptic cleft takes place in two stages; the first stage
has a half-life of approximately 3days after which the half-life decreases to
approximately 12days. These results have been demonstrated in vivo as well
as in vitro [77,78]. If acetylcholinesterase is completely removed, there is also
substantial elimination of postsynaptic nicotinic acetylcholine receptors
(nAChRs) [77].

3.1.4. Postsynaptic Toxins

3.1.4.1. Nicotinic Acetylcholine Receptor Toxins. Toxins that bind to and
inhibit nAChRs on postsynaptic membranes are present as a-neurotoxins in
Elapidae and Hydrophidae snakes and as a-conotoxins in marine Conus
snails [79,80]. In the clinical setting, these toxins give rise to a block of
neuromuscular transmission eventually leading to death by asphyxiation
[80]. Administration of a sublethal dose of Naja siamensis venom into the
200 KAUR ET AL.

soleus muscle of a mouse prevented neuromuscular transmission for 2–3days

and the muscle responded by increasing sensitivity to acetylcholine and
innervating with another nerve [81]. Typically in vitro, toxin II from the venom
of the South African ringhals cobra (Hemachatus haemachatus) produced
a decline in twitch tension of toad sciatic nerve-gastrocnemius preparations
[82].
a-Conotoxins and a-neurotoxins have three-dimensional structural simila-
rities and bind specifically to similar sites on the nAChR [83–85], even though
they have low sequence homologies (Fig. 1) [86,87]. a-Conotoxins are the
most well characterized; examples include a-conotoxin M1 from Conus geo-
graphus [88] which targets the nAChR from the Torpedo electric ray and
the mammalian muscle AChRs, both of which have similar structures.
a-Conotoxin ImI from Conus imperialis targets the a7 subunit of neuronal
AChRs [89]. Hydrophobic residues of a-conotoxin M1 are vital for its tar-
geted binding to muscle AChRs [88], whereas using pairwise mutations, it was
reported that recognition of a7 AChR by a-conotoxin ImI is governed by a
hydrophobic interaction of toxin Arg7 with the Tyr195 on the receptor [89].
Within the a-neurotoxin family, there is a large degree of conservation and
sequence similarity between different toxins, all of which form a three-finger-
loop structure (Fig. 1) [90]. These toxins are categorized into two groups:
short-chain toxins of 60–62 amino acid residues, cross-linked by four disul-
fide bridges, or long-chain toxins of 71–74 amino acid residues, cross-linked
by five disulfide bridges [91]. Short-chain toxins are found in N. naja atra
venom (cobrotoxin), Naja oxiana venom (neurotoxin II), and Laticauda
semifasciata (erabutoxin b; Fig. 1). Examples of long-chain toxins include
cobratoxin from N. naja kaouthia (Fig. 1), neurotoxin I from N. oxiana, and
a-bungarotoxin from B. multicinctus [92].
There are various isoforms of the same neurotoxin found in the same
species. Afifiyan et al. [93] have characterized three genes which give rise to
the respective isoforms of the a-neurotoxin from the venom of the spitting
cobra, Naja naja sputatrix. The group has also elucidated their promoters,
transcription factor binding sites, 50 UTR, 30 UTR, and their structures.
These toxins are specific, competitive inhibitors of acetylcholine at
nAChRs, multisubunit ligand-gated ion channels on the postsynaptic mem-
brane. The toxins decrease the ability of acetylcholine to depolarize the
postsynaptic cell [94]. a-Neurotoxins bind to the highly conserved a-subunit
of the nAChR, preventing opening of the nAChR-gated Naþ channel and
giving rise to blockage of electrical transmission [95]. Postsynaptic toxins like
a-bungarotoxin bind irreversibly [96], whereas venom from the Australian
tiger snake binds reversibly. The paralytic effects of tiger snake venom can be
quickly reversed by treatment with tiger snake antivenom, which initiates
dissociation of the venom a-neurotoxin from the nAChR. This is in stark
 ENVENOMATION 201

contrast to the PLA2-dependent presynaptic effects of tiger snake venom

which are difficult to reverse, most probably due to the structural changes
that the presynaptic membrane undergoes [97].
Crotoxin, a well-studied presynaptic neurotoxin, also displays postsynap-
tic characteristics, though only on the electric organs of the eel, Electrophorus
electricus and the ray, Torpedo marmorata. Crotoxin blocks the increase of
22
Naþ efflux caused by the acetylcholine agonist, carbamylcholine from
excitable Torpedo microsacs. Crotoxin binds to a desensitized form of the
electric organ nAChR, characterized by its high agonist affinity, and as a
result blocks neuromuscular transmission. Binding is saturable [32]. In con-
trast, the effects of crotoxin on postsynaptic mammalian membranes have
been inconclusive [22,34].

3.1.4.2. Muscarinic Acetylcholine Receptor Toxins. Muscarinic acetyl-

choline receptors (mAChRs) belong to the G-protein-coupled receptor family.
They are found throughout the body with five subtypes, M1–M5. mAChRs
have important roles in neurotransmitter release and cognitive function, as well
as smooth and cardiac muscles [98]. Several muscarinic toxins are found in
mamba snake venom (D. augusticeps). Some toxins act as irreversible, subtype-
specific mAChR antagonists, while others act as agonists [99,100]. M1, M2, and
M4 subtype-specific toxins have been identified [101–103]. Muscarinic toxins
are homologous to a-neurotoxins and have a three-finger structure similar to
fasciculins and CTXs [98].
Jerusalinsky and colleagues [104] first showed that muscarinic toxins in-
hibit mAChR in an allosteric fashion [98]. Toxins MT1 and MT2 also have
affinity for some a-adrenoceptors [105]. Physiological antimuscarinic effects
can be observed after treatment with whole Dendroaspis venoms. Dendroas-
pis jamesoni venom causes hypotension and bradycardia in cats by acting on
the floor of the fourth ventricle via sympathetic efferents in the vasomotor
center [106]. D. angusticeps venom induces bradycardia and hypotension due
to activation of central muscarinic sites [107].
Muscarinic antagonists from Dendroaspis venom therefore increase or
decrease blood pressure by inhibiting vascular and spinal M1, M2, and M4
receptors. The mamba paralyzes its victims by flooding neuromuscular
synapses with acetylcholine. Free acetylcholine would result in vasodilation
in smooth muscle and glandular tissues by activation of M3 receptors, while
antagonism of M1 (glandular tissues), M2 (smooth muscle), and M4 (lungs)
receptors would antagonize vasoconstriction [4,108]. The consequent global
burst of vasodilation would aggravate the hypotension and shock [109]. This
experimental scenario is born out from a clinical report of a human victim of
D. angusticeps envenomation, who faced persistent hypotension for more
than 5days after envenomation [110].
202 KAUR ET AL.

The biochemical and cellular pathways involved in synaptic communica-

tion, with specific reference to the mechanism of action of pre- and postsyn-
aptically acting neurotoxins and those toxins acting in the synaptic cleft are
shown (Fig. 2).

3.2. ION CHANNEL TOXINS

Ion channels are transmembrane protein complexes that span across the
lipid bilayer forming a central pore that regulate the flow of ions across the
membranes and form major components responsible for cellular regulation
and signal transduction. Channels can be classified either by how their open-
ing and closing is regulated (‘‘gating’’) or by the nature of the ion that flows
through the channel. Here, the focus will be mainly on toxins that target
voltage-gated and ligand-gated ion channels (see Fig. 3 for a summary).
Voltage-gated ion channels open and close in response to changes in
transmembrane voltage [111]. Voltage differences between the interior and
the exterior and of a cell arise as a result of different ion concentrations,
leading to the movement of gating charges across the membrane thereby
driving conformational changes that open and close the pore [112]. With
ligand-gated ion channels, the pore opens when a specific ligand (e.g., a
neurotransmitter) binds to an allosteric site on the extracellular face of the
channel and closes when the ligand dissociates.
Most animal toxins acting on ion channels are short peptides that bind
selectively and with high affinity to specific ion channels. These toxins variously
cause membrane destabilization and interference with neuronal signaling path-
ways, both in the central and in the peripheral nervous systems. As a conse-
quence, this generally leads to immobilization and paralysis in envenomed
animals [113,114]. These toxins can act as gating modifiers or channel blockers.
Toxins that modify voltage gating (e.g., Naþ channel toxins from scorpions
and sea anemones) employ a voltage-sensor trapping mechanism [115,116]. In
contrast, channel-blocking toxins bind to the transmembrane pore, resulting in
occlusion of the pore and the inhibition of ion conductance [117].

3.2.1. Voltage-Gated Sodium Channel Toxins

Naþ channels are involved in action potential conduction, synaptic trans-
mission, excitation–contraction coupling as well as the maintenance of
extracellular fluid volume and blood pressure, in skeletal, nerve, and cardiac
cells [118,119]. The Naþ channel is made up of a pore-forming a-subunit
associated with one or two smaller b-subunits [118]. The association with
b-subunits modifies channel function and mediates protein interactions. The
a-subunit has four repeating domains (I–IV) containing six transmembrane
segments (Fig. 4). Many different toxins from diverse species have both high
 ENVENOMATION 203

Presynaptic
cell
Action
potential

Membrane
damage Clathrin-mediated
endocytosis
Mitochondrial
multi uncoupling
+
a-LTX component ACoA
uptake +
Choline Choline b-Neurotoxin

lip
Hy to fa
Phase (ii)

ids
dro tty
ACh

lys ac
is
Ca2+ Phase (i)

of ds
i
2+
Ca
sio –
K+
fu le
n
e sic
an e
br te v

Ca2+ Phase (iii)

em ita
m cil

1 2
Fa

Reuptake
mAChR
Choline + acetic acid
3
AChE
Synaptic
Anticholinesterase cleft
a-Neurotoxin
mAChR toxins
4 Na+

nAChR
mAChR

Postsynaptic Depolarization
cell

Vasoconstriction

FIG. 2. Mechanism of action in a normal physiological state (blue arrows) [(1)–(4) illustrate the
mechanism by which synaptic transmission takes place in physiological conditions.] and when
neurotoxins like a-neurotoxins, b-neurotoxins, anticholinesterases, nAChR inhibitors, and
mAChR inhibitors act on the different structures in the synapse to result in a block in neurotransmis-
sion (red lines). Activation of pathways is denoted by (!) and inhibition by (a).

specificities and affinities for Naþ channels and binding studies have revealed
a number of distinct toxin binding sites (Table 2).
Binding site 1 is situated in the pore loop between S5 and S6 segments of
the channel [120]. This is the target for the hydrophilic guanidinium moieties
204 KAUR ET AL.

Tetrodotoxin, saxitoxin, m-conotoxin

Batrachotoxin

Scorpion a-toxin
Sodium
channel
Scorpion b-toxin

d-Conotoxin

Crotamine

Voltage
Dendrotoxin
gating
Potassium
channel
Noxiustoxin

w-Conotoxin

Taicotoxin
Ion channel Calcium
channel
Atrotoxin

Grammotoxin

Psalmotoxin 1
Ligand
Acid sensing
gating
APETx2

FIG. 3. An overview of the ion channels and the toxins that target them.

of the small organic molecules tetrodotoxin (puffer fish) and saxitoxin

(dinoflagellates), as well as the peptidic m-conotoxins (cone snail). The toxins
bind to the extracellular face of the pore and inhibit Naþ ion conduction
[121–125]. Binding site 2 is positioned on the S6 segment of domains I and IV
and is the target of batrachotoxin, a lipid-soluble toxin isolated from the skin
of South American arrow frogs [126]. Batrachotoxin inhibits channel inacti-
vation by holding the transmembrane segment in domain IV in an inward
position resulting in a prolonged open state of the channels during depolari-
zation [115,116,127].
 ENVENOMATION 205

I II III IV
Scorpion b-toxin
Scorpion a-toxins
d-conotoxins

S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6

Ciguatoxin
brevetoxin

Voltage sensor

Receptor site1 (tetrodotoxin, saxitoxin)

Receptor site1 (m-conotoxin)
Receptor site2
Receptor site 3
Receptor site 4
Receptor site 5

FIG. 4. Schematic diagram of the a-subunit of sodium channel and the five toxin receptor sites.
a-Subunit composes of four repeating domains (I–IV), each domain has six transmembrane
segments (S1–S6) and connected with internal and external loops [117]. The voltage sensors are
located on S4 in each domain within the channel.

TABLE 2
NEUROTOXINS THAT BIND TO THE CORRESPONDING RECEPTOR SITE ON THE SODIUM CHANNEL AND
THEIR MODE OF ACTION [348]

Receptor site Neurotoxin Mode of action

Site 1 Tetrodotoxin Block sodium conductance

Saxitoxin
m-Conotoxin
Site 2 Batrachotoxin Persistent activation and block of activation
Site 3 Scorpion a-toxin Delay inactivation
Sea anemone toxin
d-Atracotoxins
Site 4 Scorpion b-toxin Prolonged channel activation
Site 5 Brevetoxin Prolonged channel activation
Ciguatoxin
Site 6 d-Conotoxin Delay inactivation

Binding site 3 was first identified on the mammalian Naþ channel using
scorpion a-toxins [128] which contain 60–70 amino acid residues cross-linked
by four disulfide bonds [129]. The localization of site 3 was first investigated
by photoaffinity labeling and mapping studies with sequence-specific
206 KAUR ET AL.

antibodies [130,131]. Site 3 is found on the extracellular linker of domain IV

between S3 and S4 segments. Other toxins such as d-atracotoxins and sea
anemone polypeptides bind to this site as well.
Binding site 4 is the target for b-toxins from New World scorpion venoms.
The binding site includes the S3–S4 extracellular loop in domain II. The
mechanism action of b-toxins can be described by a three-step process
involving toxin binding, depolarization-dependent activation of the voltage
sensor, and rapid trapping of the activated voltage sensor in the open state
[116]. This leads to continuous, prolonged, and repetitive firing of somatic,
sympathetic, and parasympathetic neurons. The spider toxin magitoxin 5
(Macrothele gigas) has also been demonstrated to bind to site 4, through
competitive binding experiments using rat brain synaptosomes [132].
Ciguatoxin and the family of brevetoxins, all lipid soluble, small organic
molecules isolated from dinoflagellates, target binding site 5 which was
identified on S5 and S6 segments by photoaffinity-labeling experiments
[133]. Toxin binding results in persistent activation of the channel [134].
Exposure of mice to ciguatoxin resulted in both central responses (initial
lowering of body temperature, a delayed fever-like response, and more
persistent effects on spinal heat antinociception) and reduced motor activity
[135]. Brevetoxin alters cell proliferation, cytokine production and causes
apoptotic DNA damage in immune cell lines (e.g., Jurkat) via activation of
voltage-gated Naþ channels [136].
Binding site 6 is the target of the d-conotoxin cone snail family (Fig. 1).
d-Conotoxins specifically inhibit Naþ current inactivation [137]. Despite
having the same physiological effects as the toxins binding to site 3,
d-conotoxins differ in that their binding is not voltage dependent [138].
Exposure to d-conotoxins leads to massive electrical hyperexcitation [139].
Crotamine, a myotoxin isolated from the venom of South American
rattlesnake (C. durissus terrificus), is a 42 amino acid toxin cross-linked by
three disulfide bridges. The toxin acts on skeletal muscle Naþ channels [140]
and is able to induce depolarization-dependent muscle contractions in cats,
rats, and mice by increasing the Naþ ion permeability [141–143], resulting in
spasmodic seizures and convulsions [144].

3.2.2. Voltage-Gated Potassium Channel Toxins

Kþ channels are important in various cellular processes in both excitable
and nonexcitable cells, for example, nerve conduction and muscle contrac-
tion, cell growth and differentiation, blood pressure regulation, immunity,
and hormone secretion. There are four main classes of Kþ channels—those
with six transmembrane channels (voltage-gated and Ca2þ-activated) are the
main focus in this review, as they provide the primary targets of most toxins.
 ENVENOMATION 207

Toxins affecting neuronal Kþ channels include apamin from bee (Apis

mellifera) venom [145], dendrotoxins from mamba snakes (Dendroaspis)
[146], and charybdotoxin from the Old World scorpion Leiurus quinquestria-
tus [147]. Apamin is specific for low-conductance Ca2þ-activated Kþ chan-
nels, which results in the inhibition of delayed hyperpolarization that follows
a neuronal action potential. Dendrotoxins, on the other hand, act on a subset
of voltage-dependent Kþ channels. Charybdotoxin blocks both voltage-
dependent Kþ channels and large-conductance forms of Ca2þ-activated Kþ
channels. Some toxins act exclusively on neurons (e.g., dendrotoxins), where-
as others (e.g., charybdotoxin) act on many different cells, including neurons,
smooth muscle, and lymphocytes [148,149].
Kþ channel toxins have between 22 and 60 amino acid residues and are
cross-linked by two to four disulfide bridges [150]. Formation of disulfide
bridges confers stability and rigidity to the toxins, allowing key residues to be
positioned for optimal interactions with the ion channel. A distinct motif
identified in the toxins is the inhibitory cysteine knot [151].
Dendrotoxins found in mamba snake venom [152] contain 57–60 amino
acid residues cross-linked by three disulfide bridges and act on neuronal Kþ
channels [153]. Kv1.1 and Kv1.2, cloned Kþ channels expressed in Xenopus
oocytes, were shown to be blocked by a-dendrotoxin from D. augusticeps
[154]. Novel Kþ channel toxins (e.g., from sea anemone toxins) have been
initially identified through their ability to inhibit binding of radiolabeled
a-dendrotoxin [153]. Dendrotoxins promote neurotransmitter release as well
as induce repetitive neuronal firing [152] which gives rise to excessive muscular
activity, trembling, and fasciculations [155]. The intracerebroventricular injec-
tion of a-dendrotoxin induces epileptiform seizures and brain damage in rats
[156]. Noxiustoxin from the Mexican scorpion Centruroides noxius [157]
blocks Ca2þ-activated Kþ channel in skeletal muscle; specific interactions
with the channel were revealed through site-directed mutagenesis [158].

3.2.3. Voltage-Gated Calcium Channel Toxins

Ca2þ channels can be found in the plasma membrane of excitable cells such
as neurons and myocytes [159]. They open in response to cell depolarization,
and the influx of Ca2þ ions into the cell can trigger a variety of intracellular
events, including neurotransmitter release, secretion of hormones such as
insulin, gene expression, and muscle contraction. Ca2þ channels can be gated
by either voltage or ligands, and it is common to find several different types
of Ca2þ channels on most cells. Voltage-gated channels can be classified into
five distinct types as shown in Table 3; the five types of channels differ in the
strength of depolarization required for channel activation.
One of the largest groups of toxins [160] that block Ca2þ channels are
members of the o-conotoxin family (Fig. 1), first isolated from C. geographus.
208 KAUR ET AL.

TABLE 3
CLASSIFICATION OF VOLTAGE-GATED CALCIUM CHANNELS BASED ON THEIR
ELECTROPHYSIOLOGICAL AND PHARMACOLOGICAL PROPERTIES [349]

Type Location Function

L type Skeletal muscle, cardiac, smooth muscle, Muscle contraction, hormone release,
endocrine cells regulation of gene expression
N type Neuroendocrine cells Neurotransmitter release
P/Q type Neuroendocrine cells Neurotransmitter release
R type Cerebellar granule neurons Repetitive firing
T type Cardiac, smooth muscle myoctes, neurons Regulating pacemaking

These toxins cause immediate paralysis and death in fishes. They physically
occlude the Ca2þ channel pore and selectively block N- and L-type channels
[161,162].
Taicatoxin isolated from the venom of Australian taipan snake O. scutel-
latus was found to specifically block L-type Ca2þ channel in selected tissues,
including cardiac muscle and chromaffin cells [163,164]. In contrast, atro-
toxin from the venom of rattlesnake Crotalus atrox prolongs the Ca2þ
channel in open state but the mechanism remains inconclusive. Atrotoxin
causes necrosis and edema at the bitten area, as well as hemorrhage and
hypotension [165].
Grammotoxin, a 36 amino acid toxin found in the venom of the tarantula
Grammostola spatulata, inhibits closed N- and P/Q-type Ca2þ channels in rat
cerebellar purkinje neurons. As a result, a larger membrane depolarization is
required to activate these channels [166]. Grammotoxin also binds to Kþ
channels but with a lower affinity [167].

3.2.4. Acid-Sensing Ion Channel Toxins

Proton-gated Naþ channels belong to a class of acid-sensing ion channels
(ASICs) that respond to extracellular pH, expressed in both the peripheral
and the central nervous systems. ASICs are made up of four proteins forming
functional channel subunits (ASIC1a, ASIC1b, ASIC2a, and ASIC3) and
two proteins (ASIC2b and ASIC4) without any known activators [168]. They
are associated with nociception, taste transduction, and perception of extra-
cellular pH fluctuations in the brain [169].
Psalmotoxin 1, a 40-amino acid residue toxin isolated from tarantula
Psalmopoeus cambridgei venom, was the first specific blocker of an ASIC to
be characterized [169]. The toxin has a specific affinity for ASIC1a, by
binding to the cysteine-rich extracellular loop structure, and can distinguish
between ASCI1a and ASC11b splice variants. The toxin activates an
 ENVENOMATION 209

endogenous enkephalin pathway [170]. Although the role of psalmotoxin in

prey capture has yet to be understood, ASIC channels are implicated in
nociception and neuronal death during ischemia, and understanding toxin–
channel interactions could provide insights into future therapeutic treat-
ments for stroke [171].
APETx2, a 42 amino acid residue toxin from sea anemone (Anthopleura
elegantissima) venom [168], selectively interacts with ASIC3 channels.
APETx2 serves as a useful tool to study the physiological involvement of
ASIC3 channels in neuronal excitability and pain coding [172,173].

3.3. CARDIOTOXINS
CTXs disrupt the membranes of neuronal and skeletal and cardiac muscle
cells [174,175]. CTXs are speculated to act on cell membranes to form pores,
which results in depolarization and an influx of Ca2þ [176], causing muscle
contraction, cell lysis, and cardiac arrest. Ventricular tissue is very suscepti-
ble to the actions of CTXs; the toxins cause loss of the fast phase of the action
potential when injected into perfused rat heart [177]. CTX-induced myone-
crosis in skeletal muscle cells results in rapid lysis of the sarcolemma, myofi-
bril clumping, and hypercontraction of sarcomeres [178]. b-CTX from
Ophiophagus hannah (king cobra) venom [179] has a unique action, binding
to b-adrenergic receptors with high affinity and lowering heart rate; in
contrast, other CTXs increase heart rate. CTX-4b from cobra (N. sputatrix)
venom causes neuronal cell death in mouse primary cortical neurons by
elevating levels of reactive oxygen species as well as activating calpains.
These effects can be countered by antioxidants [180].
It has been speculated that CTXs depolarize cell membranes by acting on
specific phospholipid structures associated with Naþ and Ca2þ channels
[181]. In model studies, CTXs have been shown to perturb the structure of
phospholipid bilayers by inducing aggregation, fusion, and altering the
permeability of a variety of phospholipid liposomes [182,183]. In the pres-
ence of cardiolipin, inverted micellar structures are formed [184] and with the
addition to fusion of sphingomyelin, there is widespread leakage of vesicular
contents [185]. There are two types of CTXs (P- and S-type) which differ in
their phospholipid binding capacity; both types interact with anionic phos-
pholipids but only P-type CTXs bind to zwitterionic lipids [186]. Several
groups have reported specific and targeted binding of CTXs to phospholipid
membranes, and they propose that the structural changes induced in these
membranes account for the cytotoxic effects of CTXs [187,188].
Five CTX isoforms (CTX I–V) have been isolated from the venom of the
Taiwan cobra Naja atra, showing varying degrees of potency with CTX-I
and -V being twice as potent as the remaining three [189,190]. They share
210 KAUR ET AL.

similar structural features of triple- and double-stranded antiparallel

b-sheets. An X-ray crystallographic study of CTX-VII4 from Naja mossambica
mossambica has revealed a dimeric structure with six-stranded antiparallel
b-sheets [191].
The CTX-II gene from N. sputatrix is expressed in many different tissues
(e.g., liver, heart, and muscle) as well as venom glands. Different transcript
variants are expressed in different tissues at dissimilar levels, and this has
been attributed to regulation by different promoters [192]. CTX isoforms
found many cobras, as well as rattlesnake (C. scutulatus scutulatus) venom,
comprising 60–62 amino acid residues with four disulfide bridges similar to
a-neurotoxins acting on the nAChRs [193,194]. Further structural studies of
CTX have revealed a three-finger motif with three loops projecting from a
globular head [195], which form trimeric structures in synaptosomal mem-
branes. These trimers are asymmetric and possess a hydrophobic exterior
and a hydrophilic center pore, suggesting that these structures function as ion
channels [196,197]. Unlike a-neurotoxins, CTXs contain a large amount of
positively charged residues; when positively charged Lys residues on CTX
were neutralized or converted to negative charges, the toxin lost its cytolytic
activity [30]. Variation in amino acid sequences between CTX isoforms from
Naja species occurs mostly at positions 7–11, 27–32, and 45–47, which are
located at or near the tip of the loop structure and are responsible for the
biological activity of the toxin [193]. Transfection into Chinese hamster
ovary cells of cDNAs coding for CTX molecules from N. sputatrix and
chimeric toxins in which the loops had been interchanged gave rise to cell
lysis with the first two loops being the most important for lytic action [198].
A cationic site flanked by a hydrophobic site is a common structural
feature that is predicted to give rise to the lytic activity of a large group of
cytolysins [199]. CTXs inhibit membrane-bound acetylcholinesterase; the
presence of an exposed arginine on loop I of CTX that interacts with Trp-
86 of acetylcholinesterase is essential for enzyme inhibition. Different CTXs
from the venom of the Taiwan cobra (N. atra) also bind to the surface of the
enzyme via loop II of the CTX [200]. This has led to the general conclusion
that CTXs appear to stabilize the functional conformation of acetylcholines-
terase, in comparison to fasciculins which stabilize the nonfunctional con-
formation of enzyme.
CTX from N. naja kaouthia at low concentrations had no effect on 3H-
deoxyglucose-6-phosphate or hemoglobin release [201] from erythrocytes.
However, higher concentrations significantly enhanced release, signifying
that there is complete lysis of a subpopulation of cells.
CTX-I from N. naja atra was also tested for its ability to cause skeletal
muscle necrosis. Light and electron microscopic examination showed that
the toxin caused necrosis of mouse skeletal muscle and rupture of the
 ENVENOMATION 211

sarcolemma [202]. Myofibrils were condensed into dense clumps, alternating

with clear areas containing elements of the sarcotubular system. Eventually
cells contained only remnants of myofibrils and swollen mitochondria.
Bieber et al. [12] showed that intoxication of anesthetized rabbits with
Mojave toxin resulted in cardiovascular collapse while the diaphragm was
still responsive to phrenic nerve stimulation, indicative of a direct cardiovas-
cular action. This is in contrast to other studies suggesting that the cardio-
toxic effects of Mojave toxin are secondary to its neurotoxic action [203].
Envenomation by Vipera aspis has an effect on coagulation and cardiovas-
cular failure which is probably due to the paralysis of muscle fibers by a
similar triphasic mechanism as shown by PLA2 toxins [204]. The CTX of
D. jamesoni has also been shown to give rise to muscle fiber necrosis and
neuronal damage [205]. CTXs probably increase capillary membrane perme-
ability and cause hypotension, although the presence of histamine in many
venoms may also contribute [206]. Cher et al. [207] carried out a gene-
expression analysis of mice envenomed by a fatal dose of CTX from
N. sputatrix. Largest changes in gene expression were observed in the heart,
affecting genes with roles in inflammation, apoptosis, energy metabolism,
and ion transport. Deteriorating heart function and myocardial damage
suggested that cardiotoxic effects signal the first stages of envenomation by
this cobra.

3.4. HEMOTOXINS
The hemostasis system is a complex process involving interactions of
multiple proteins to sense hemorrhage and trigger platelet aggregation, as
well as a cascade of clotting factors to initiate blood clotting. Hemostasis
requires the interplay of two different events: the formation of the platelet
plug and activation of the coagulation cascade [208]. Even though the event
of platelet plug formation is more prominent at the beginning, the two events
occur along side each other and they share several common protein compo-
nents in their pathways such as von Willebrand factor (vWF) and fibrinogen.
Both are initiated by the exposure of subendothelial proteins in the extracel-
lular matrix (ECM) such as basement membrane protein and other tissue
factors [208] when the endothelium is damaged.
Animal venoms targeting the hemostasis system have evolved a set of
hemostatically active components to mimic or inhibit the functions of vari-
ous endogenous protein regulators involved in hemostasis. Generally, these
venom components are proteins with characteristics of metalloproteinases
and serine proteases, such as thrombin-like enzymes (TLEs) and hemorrha-
gins, but there are also nonenzymatic proteins such as disintegrins and other
binding proteins. Historically, viper bites are well known for their local
212 KAUR ET AL.

hemorrhagic effects [209] which may explain the greater amount of the
literature dealing with hemotoxins in viper venoms. However, other animals
such as the caterpillar of the moth, Lonomia spp., also have potent hemor-
rhagic venoms, most notably containing clotting factor activators [210].
Studies of hemotoxins from these more unusual species might yield great
benefits for the pharmaceutical industry and medical research.
Symptoms of envenomation that variously affect the hemostatic system
result in a marked decline in blood coagulability, giving rise to a higher
propensity to bleed and a damage to blood vessels. Systemic effects include
intracerebral hemorrhage, hypovolemic shock, and renal damage, as well as
the development of pathological thromboses, such as pulmonary embolism
[155].
The classification of hemotoxins in this section is based on Markland’s
review [211]. The first section deals with toxins affecting the endothelium
(hemorrhagins). This is followed by a section on toxins inhibiting the plate-
let-activation pathway (platelet-aggregation inhibitors) and finally toxins
affecting the coagulation cascade (procoagulants, anticoagulants, and fibri-
nolytic enzymes). The molecular targets of the various toxins in the hemosta-
sis pathway are shown in Fig. 5.

3.4.1. Hemorrhagins
In addition to mechanical injury inflicted during envenomation, hemor-
rhagins damage the endothelium by digesting the basement membrane sup-
porting the endothelium. Hemorrhagins are zinc-dependent snake venom
metalloproteinases (SVMPs) that mediate hemorrhage by targeting consti-
tuents of the basement membrane underneath endothelial cells, such as
fibronectin, laminin, and type IV collagen [212]. Without a substratum, the
endothelial cells disperse and the vascular wall integrity is compromised
resulting in a perforated vasculature and subsequent symptoms of edema
[212]. However, it should be noted that SVMPs may have additional non-
hemotoxic functions; Stejnihagin from Trimeresurus stejnegeri venom inhi-
bits L-type Ca2þ channel and blocks Kþ-induced blood vessel contraction
[213].
Bjarnason and Fox [214] have categorized SVMPs based on their domain
composition. All four groups (PI–PIV) share homologous signal, pro- and
metalloproteinase domains. The PI group (20–30kDa) consists of a single
metalloproteinase domain, the PII group (30–60kDa) has an additional
disintegrin-like domain at the C-terminal, and the PIII group (60–100kDa)
has an additional cysteine-rich domain following the disintegrin-like domain
and contains the most potent hemorrhagins [215,216]. Last, the PIV group
adds an additional lectin-like domain at the C-terminal.
 Intrinsic pathway F IX/X BP
Extrinsic pathway Main coagulation cascade
Damaged endothelium—proteins exposed Normal endothelium–VWF and basement membrane
Common pathway Platelet aggregation under endothelium

PK K

XII XIIa Tissue Hemorrhagin

factor
2+
Ca
XI XIa
2+
VIIa VII
Ca
F IX/X IX IXa FX
BP activator
VIII VIIIa
Disintegrin
X Xa

Protein C Activated 2+
Ca Platelet activation
protein C
F IX/X PL
Fibrinogen
BP PLA2
V Va production

Protein C Fibrinogen
Prothrombin Thrombin receptor activation
activator
FV XIIIa XIII
Prothrombin Fibrinogen
activator activator TI

Fibrinogen Fibrin Activated fibrinogen

Cross-linked receptor
tPA TLE
PA fibrin
inducer Fibrinogenase
Plasminogen Plasmin
FDP

FIG. 5. The hemostasis pathway. Activation, (!); cofactor activation, (–!); inhibition, (a); FDP, fibrin-degradation product; PK, prekallikrein;
PLA2, phospholipase A2; K, kallikrein; TI, thrombin inhibitor; TLE, thrombin-like enzyme; tPA, tissue plasminogen activator; vWF, von Willebrand
factor. The actions of toxins (green boxes) are also indicated.
214 KAUR ET AL.

Experimental studies assaying the systemic hemorrhagic potential of

SVMPs have examined the development of hemorrhage in lungs introduced
via intravenous SVMP injection. The minimum dosage required to induce
one hemorrhagic spot and the number/total area of hemorrhagic spots at
fixed doses were determined [217]. From a study on viper SVMPs, the
superior potency of the PIII group was explained by the ability of a combi-
nation of the disintegrin-like and cysteine-rich domains to recognize and
bind ECM protein targets with high specificity and affinity [216,218]. The
resistance of PIII-SVMPs to inhibition by the endogenous protease inhibitor,
a2-macroglobulin, which allows them to travel through the circulatory sys-
tem without loss of activity [219], also enhances their potency, a characteris-
tic lacking in PI-SVMPs [220].
Studies of jararhagin-C, a PIII-SVMP isolated from Bothrops jararaca
venom, have shown that in addition to being able to degrade basement
membranes, the toxin can also degrade platelet collagen receptors and vWF
[221], both of which are necessary for platelet aggregation. Sugiki et al. [222]
discovered that jarahagin promotes fibrinolysis by dissociating plasminogen
activator inhibitor-1 (PAI-1) from tissue plasminogen activator (tPA).
Hemorrhagins therefore act at the site of envenomation and induce local
as well as systemic hemorrhage. The substantial loss of red blood cells alone
can be a cause of death; however, even in nonlethal doses, pathophysiological
conditions resulting from a hindrance in the proper blood supply to the distal
parts of the body can be devastating for the envenomed patient [212].

3.4.2. Platelet-Aggregation Inhibitors

Platelet activation is essential for the early phase of hemostasis, and it is
characterized by the rearrangement of the platelet cytoskeleton and the release
of its granular contents, leading to aggregation and vascular plug formation in
blood vessels [223]. Normally, platelet activation is initiated by thrombin
activity and binding of platelet integrin receptors to proteins found in the
ECM. Such conditions are usually met at sites of vascular damage where the
endothelium is displaced to expose vWF, collagen and tissue factor in the ECM
[223]. Tissue factor is involved in the extrinsic pathway of the coagulation
cascade, to generate thrombin which acts upon protease-activated receptors
and integrin aIIbb3 receptors [224]. The binding of vWF and collagen to
glycoprotein Ib (GPIb) receptors trigger signal transduction within the platelet
[225]. Both pathways will alter the conformation of the aIIbb3 receptor and
prevent fibrinogen and ECM proteins such as fibronectin and vWF from
binding. Fibrinogen subsequently cross-links platelet aIIbb3 receptors to
begin platelet aggregation on the exposed ECM; this stimulates the release of
secondary agonists such as ADP, thromboxane A2, and serotonin, in addition
to more vWF, to recruit more platelets to grow the platelet plug [225].
 ENVENOMATION 215

3.4.2.1. Disintegrins. Disintegrins are a family of small (40–100 amino

acids), nonenzymatic, cysteine-rich polypeptides that are potent antagonists
of integrin receptors. The first to be described was trigramin, isolated from
the venom of Trimeresurus gramineus [226]. They are generated from the
proteolytic cleavage of disintegrin domains from metalloproteinase precur-
sors (PII- and PIII-SVMPs). PIII-disintegrins are wide spread among five
families of snakes (Colubroidea, Viperidae, Elapidae, Atractaspididae, and
Colubridae), whereas PII-disintegrins are only found in the venom of vipers
and rattlesnakes (Viperidae). This suggests an evolutionary pathway for the
disintegrins as demonstrated phylogenetically by Juárez et al. [227]. Disin-
tegrins have also been categorized by their number of disulfide bonds [227] as
well as by the motifs recognized by the companion integrin [228]. The most
prominent example would be the RGD (Arg-Gly-Asp) motif which enables
disintegrins to compete with fibrinogen for the aIIbb3 integrin receptor,
thereby inhibiting platelet aggregation [227]. Other motifs include slight
variants such as KGD, MVD, MGD, and WGD, all of which inhibit
RGD-recognizing integrins; KTS selectively binds to a1b1 integrin receptors
specific for Type IV collagen [228].
These disintegrin motifs are located at the tip of a highly flexible and
protruding loop. From the NMR structure of AaHIV, a PIII-SVMP from
Agkistrodon acutus venom which releases the disintegrin, acucetin, it has been
proposed that the disintegrin motif of acucetin plays a target recognition role
for subsequent proteolysis by the metalloproteinase domain [229].
Disintegrins therefore inhibit the formation of the platelet plug and inhibit the
aggregation of platelets required for hemostasis. This in turn results in continued
bleeding which can worsen with the other damaging effects of the venom.

3.4.3. Procoagulants
The final common pathway of the coagulation cascade is dependent on
Factor Xa, an endopeptidase which converts prothrombin to thrombin.
Thrombin is a key protease which plays three major roles. It converts
fibrinogen to fibrin monomers, activates Factor XIII which is responsible
for cross-linking fibrin polymer chains to an insoluble mesh-like complex,
thereby forming the building blocks of a blood clot [230], and finally activates
Factors V and XI increase thrombin activation in a positive-feedback loop.
Procoagulant toxins therefore induce blood clotting by various mechanisms
which can be subcategorized into TLEs, prothrombin activators, Factor
X activators, Factor V activators, and other activators, based on which
components of the cascade they act upon (Fig. 5).
3.4.3.1. Thrombin-Like Enzymes. Fibrinogen is a hexamer consisting of
2 Aa, Bb, and g chains, and its activation by thrombin requires cleavage of
fibrinopeptides A (FPA) and B (FPB) at the N-terminal of the Aa and Bb
216 KAUR ET AL.

chains [231]. Once cleaved, the activated fibrin molecules can polymerize with
other monomers to form a weakly stable complex which is stabilized by
cross-links formed by thrombin-activated Factor XIIIa in the presence of
Ca2þ [232].
TLEs from snake venoms share almost the same function as thrombin
[233] and are classified by their selectivity toward cleaving FPA, FPB, or both
[234]. Gabonase from Bitis gabonica venom [235] represents this class of
enzymes. The venombin AB group of TLEs resembles thrombin the most
and is able to cleave FPA, FPB, and activate Factor XIII. Venombin A and
venombin B partially cleave fibrinogen to release either FPA or FPB, respec-
tively [236]. In addition, they do not activate Factor XIII, resulting in clots
which are only supported by noncovalent intra-fibril interactions and thus
are highly susceptible to plasmin activity [237].
Ancrod [238,239] and batroxobin [240,241] are TLEs (venombin A from
Calloselasma rhodostoma and Bothrops atrox, respectively). They have been
shown to induce plasminogen activator (PA) release from endothelial cells
and boost the plasma plasmin concentration, thereby increasing fibrinolytic
activities. They are reduced to soluble nonfunctional fibrin degradation
products and removed from the plasma [237]. Ancrod is presently in phase
III clinical trials for the treatment of acute ischemic stroke. Batroxibin is used
in clinical laboratories to test the contractile properties of platelets, as a
heparin-insensitive alternative to the determination of thrombin time. In
comparison, venzyme, a venombin B isolated from venom of Agkistrodon
contortrix [242], clots blood only after prolonged incubation.
While a coagulant venom, by definition, induces some degree of clotting, in
most cases this is accompanied by active fibrinolysis, resulting in a net loss of
clotting capacity [243]. When the fibrinogen store is exhausted by TLEs, the
victims’ blood is unable to coagulate effectively and individual is said to suffer
from venom-induced consumption coagulopathy (VICC). The risk of severe
hemorrhage increases greatly when TLEs are coupled with vascular damage
caused by hemorrhagins or wounds incurred during envenomation [234].
3.4.3.2. Prothrombin Activators. Prothrombin is the zymogen form of
thrombin and exists as a single-chain glycoprotein. Its conversion to active
thrombin is mediated by cleavage at the C-terminal side of Arg271 and
Arg320 residues to generate a two-chain molecule. This process is mediated
by prothrombinase and a complex of factor Xa and nonenzymatic cofactors:
factor Va, Ca2þ ions, and membrane phospholipids [244].
Prothrombin activators from snake venoms are endopeptidases that func-
tion similar to Factor Xa, which provides the catalytic site for prothrombin
cleavage. However, there are distinct groups of prothrombin activators classi-
fied by their requirement for cofactors [245]. Group A activators are cofactor-
independent metalloproteinases which cleave at the Arg320 site to form
 ENVENOMATION 217

meizothrombin, an active prothrombin–thrombin intermediate which converts

autocatalytically to thrombin. Ecarin isolated from the venom of Echis carina-
tus [246] is an example of a group A activator. Group B activators are
also metalloproteinases, but they require Ca2þ for activity. Group C and
D activators are serine proteases, commonly found in Australian elapid snake
venoms [247]. Group C activators possess a Factor Va-like subunit which is
likely to be resistant to activated protein C degradation, as found in the
prothrombin activator from P. textilis venom. They can function independent-
ly of Factor Va. Group D activators require the presence of all the cofactors, in
a similar manner to mammalian Factor Xa. Prothrombin activators may also
cause anticoagulant effects, such as bumblebee (Bombus ignitus) venom serine
protease (Bi-VSP) which has a plasmin-like (fibrinolytic) function [248].
3.4.3.3. Factor X Activators. Factor X is synthesized as a single-chain
glycoprotein and matures as a two-chain protein circulating in the plasma. It
can be activated by Factor VIIa during the initiation phase of coagulation via
the extrinsic pathway, by cleavage of a single peptide bond that removes the
first 52 amino-terminal residues of the heavy chain which carries the glyco-
sylated glutamic acid residue. Upon its removal, the active site becomes fully
exposed and capable of activating prothrombin [249].
Tans and Rosing [249] have concluded that the presence of snake venom
Factor X activators is rather widespread, especially in viperid, crotalid, and a
few elapid venoms. They are either metalloproteinases or serine proteases.
Russell’s viper (D. russellii formerly Vipera russellii) venom contains a potent
activator of human Factor X (RVV-X) [209]. RVV-X is a disulfide-linked
two chain serine metalloproteinase composed of a heavy chain of 427 resi-
dues and a light chain of 123 residues [250]. Purified RVV-X activates Factor
X by cleaving the Arg194–Ile195 bond and is also able to activate Factor IX
and protein C by specific cleavage of Arg–Ile and Arg–Val bonds [251]. It was
proposed that the C-type lectin-like domain in the light chain serves as an
exosite by which RVV-X recognizes and binds to the glycosylated domain of
Factor X, since nonglycosylated Factor X is less susceptible to RVV-X
degradation [249].
3.4.3.4. Factor V Activators. Factor V circulates in plasma as a single-
chain glycoprotein and needs to be first cleaved and activated by thrombin or
Factor Xa to express procoagulant activity [252]. Factor Va is a cofactor for
Factor Xa-mediated prothrombin activation and enhances the rate of throm-
bin formation more than 1000-fold and activation proceeds via specific
cleavage of peptide bonds at Arg709, Arg1018, and Arg1545 [253]. Kinetic
analysis has shown that Factor Va accelerates prothrombin activation by
acting as a receptor that promotes the binding of Factor Xa and prothrom-
bin to membrane phospholipids, thereby enhancing the catalytic activity of
Factor Xa [254,255].
218 KAUR ET AL.

Factor V activators are proteases that cleave Factor V. The most studied
member of this family is a 236 amino acid, single-chain serine protease from
Russell’s viper venom, Factor V activator (RVV-V) [256,257]. RVV-V acti-
vates Factor V by a specific cleavage of a single peptide bond at Arg 1545,
which distinguishes it from thrombin which additionally cleaves Factor V at
Arg709 and Arg1018 [258]. RVV-V-activated Factor V showed no- differ-
ences in procoagulant activity from thrombin-activated Factor V [259].
When RVV-V and Factor V activators from two other members of the
Viperidae family Macrovipera lebetina (formerly V. lebetina) and Vipera
ursinii were assessed for differences in Factor V activation kinetics, there
were no significant differences [257], although it took 40-fold higher concen-
trations of snake activators to follow the same kinetic profile as thrombin.

3.4.4. Anticoagulants
Anticoagulant proteins cause coagulopathy via a different mechanism
from VICC as caused by some procoagulants. Rather than depleting the
fibrinogen store for clotting, they prevent clotting by degrading or inhibiting
the clotting factors’ activity.
3.4.4.1. Protein C Activator. Protein C is a zymogen that is activated by
thrombin on the endothelial surface in the presence of thrombomodulin
[260]. Activated protein C degrades Factors Va and VIIIa. Cleavage at
Arg306, Arg506, and Arg679 of Factor Va results in A2 domain dissociation,
as well as loss of factor Xa binding and cofactor function [261]. This cripples
the formation of the prothrombinase complex and prevents continuation of
the common pathway. One commercialized protein C activator is ProtacÒ
which is isolated from the venom of A. contortrix [262].
3.4.4.2. Factor IX/X-Binding Protein. The Factor IX/X-binding proteins
are nonenzymatic proteins which bind to Factors IX and X and inhibit their
activities in a Ca2þ-dependent manner. The binding proteins interact with
clotting factors at their N-terminal g-carboxy glutamic acid residues [263]
which bind Ca2þ ions. In the case of Factor Xa, binding proteins also hinder
its incorporation into the prothrombinase complex [264]. Factor IX/X-binding
proteins have been isolated from the venoms of A. acutus [264], Trimeresurus
flavoviridis [265], and Echis carinatus leucogaster [266]. A possible lectin-like
mechanism of action has been ruled out by observing that there was no decrease
in binding affinity of the binding protein to deglycosylated Factor IX [266].
PLA2 enzymes also have anticoagulant properties in addition to the afore-
mentioned neurotoxic effects (Section 3.1.1), by hydrolyzing phospholipid
cofactors, for example, phosphatidylserine, as shown in Vipera berus venom
[267]. However, enzyme independent binding of PLA2 to phospholipids has
also been found to prevent formation of the prothrombin complex, as shown
in N. nigricollis, H. haemachatus, and N. atra [268]. Alkylation of His48 at the
 ENVENOMATION 219

active site of CM-IV (one of three anticoagulant PLA2 enzymes from

N. nigricollis venom) inactivates phospholipase activity, but inhibition of
the prothrombinase complex is largely unaffected. Inhibition of prothrombin
cleavage is not affected even after complete phospholipid digestion by CM-I
and CM-II. It is proposed that, rather than competing for the active site of the
prothrombinase complex or binding to prothrombin itself, CM enzymes
compete with Factor Va [30,269].
3.4.4.3. Thrombin Inhibitor. Bothrojaracin is a thrombin inhibitor, pur-
ified from B. jararaca [270]. It competitively inhibits heparin binding to
thrombin via the enzyme’s exosite 2 [270,271]. Bothrojaracin also binds to
the fibrinogen-specific exosite 1 of thrombin, rather than its active site, to slow
down the formation of the fibrin mesh [272]. It also inhibits thrombin-induced
platelet activation and aggregation [271]. Bothrojaracin was shown to reduce
thrombus size by 95% and at the same dosage, protect 100% of mice from
death in a thrombin-induced pulmonary thromboembolism model [270].

3.4.5. Fibrinolytic Enzymes

Plasmin, a serine protease made up of 810 amino acids (Mr ¼92kDa) [273],
is woven into the fibrin clot in its zymogen form, plasminogen. Once acti-
vated, it begins to break down the fibrin mesh into soluble fibrin degradation
products which are removed from the plasma, in a process known as fibrino-
lysis [274]. Plasminogen is physiologically activated by tPA and urokinase
plasminogen activator (uPA) which are released by endothelial cells and are
inhibited by serpins, PAI-1 and PA inhibitor-2 [275].
3.4.5.1. Fibrinogenase. This class of enzymes targets the a and b chains
of fibrin and dissolves the fibrin clots in the blood stream. To date, there are
no reports of a g chain-specific fibrinogenase. Since fibrinogen carries a and b
chains in the form of Aa and Bb, they are prone to the enzymes’ fibrinogen-
olysis, and the enzymes are aptly named a-chain and b-chain fibrinogenase
[276]. Their fibrinogenolytic and fibrinolytic properties can prevent throm-
bosis by dissolving clots and depleting the fibrinogen stores in a fashion
similar to TLE-induced defibrinogenation. Markland [234] has reviewed
the structure and function of venom fibrinogenases, all of which act upon
fibrins and fibrinogens directly without enlisting plasmin activity. Most are
metalloproteinases with higher specificity for the a chain than the b chain.
3.4.5.2. Plasminogen Activator Inducer and Plasminogen Activator-Like
Enzyme. C. atrox and Crotalus adamanteus venoms are potent inducers of
PAs [277]. TLEs, such as batroxobin from Bothrops moojeni and habutoxin from
T. flavoviridis venoms [241,278], have also been reported to induce tPA and uPA
in porcine and bovine endothelial cells, respectively. Another PA-like serine
protease, TSV-PA, was extracted from the venom of T. stejnegeri, which was
insensitive to PAI-1 and was able to function without fibrin stimulation [279].
220 KAUR ET AL.

Snake venom, therefore, contains a multitude of different proteins that act

via various different mechanisms to disable the complex hemostasis system
of the body after envenomation. Inhibition or activation of coagulant fac-
tors, damage to the vascular endothelium, and inhibition or induction of
platelet aggregation all act in synergy (Fig. 5) to result in consumption
coagulopathy, anticoagulant activity, a reduction in platelet numbers, and
excessive bleeding, caused by damage to the vasculature [10].
Individual toxins act simultaneously when injected together as a complex
mixture in the form of venom. The action of toxins on the various biochemi-
cal processes discussed above account for the symptoms seen in the debili-
tated victim. Figure 6 summarizes the target molecules of these toxin groups
and the clinical manifestations they give rise to.

4. Biochemical Basis of Venoms in Therapy

Traditional medicine encompasses systems such as Indian Ayurveda, Arabic

Unani, and traditional Chinese medicine. For hundreds of years, traditional
medicine has seen the use of remedies made from leaves, herbs, tree bark,
animals, mineral substances, and other materials found in nature [280].
Although venoms and toxins deliver varying levels of toxicity to our physiolog-
ical systems, advancements in biotechnology have allowed for discovery of
their therapeutic potentials. Folk and traditional communities have used ani-
mal venoms and toxins as tools for the therapeutic treatment of pathological
conditions. They have provided stepping stones for the development of drugs
from natural sources. The biodiversity of venoms and toxins provide a range of
molecules from which therapeutic agents may be developed [9]. Snake venom,
for example, contains many constituents that can also be developed for the
treatment of thrombosis, arthritis, cancer, and many other diseases [281].
Traditional medication such as huachansu, a Chinese medicine derived from
dried toad venom from the skin glands of Bufo gargarizans or Bufo melanos-
tictus, has been used in various cancer treatments for several years [282]. Other
venom therapies include bee venom acupuncture (apitherapy) for providing
relief from pain for patients with knee osteoarthritis [283].
The previous sections have addressed the biochemistry of envenomation
from the viewpoint of the cardiotoxic, neurotoxic, and hemostatic properties
of venom toxins, as well as their effects on ion channels. While invariably toxic,
often because of their synergistic and simultaneous actions when injected in the
form of venom, many of these individual peptides and proteins have therapeutic
potential either in their own right or as models to develop small molecule drugs.
This section focuses on the applications of venoms in biomedicine and the
biochemical mechanisms that provide a rationale for their use.
 ENVENOMATION 221

Neurotoxins (neuromuscular junction)

• Clinical manifestations of enzymatic presynaptic NTXs (PLA2)—Ptosis,
mydriasis, paralysis of other muscles, respiratory paralysis, bulbar paralysis, flaccid
paralysis, pulmonary inflammation, and edema.
• Clinical manifestations of presynaptic excitatory NTXs—Cramping of muscles at
site of envenomation or of all systemic muscles, abdominal rigidity, nausea,
headache, palpitations, hypertension, tachycardia, facial edema, renal failure, and
anxiety.
• Clinical manifestations of synaptic cleft NTXs—Faciculations, neuromuscular
paralysis, and eventually death by asphyxiation.
• Clinical manifestations of postsynaptic NTXs (nAChR)—Paralysis, death by
asphyxiation.
• Clinical manifestations of postsynaptic NTXs (mAChR)—Hypotension, bradycardia.

Ion channel toxins

Cardiotoxins
• Clinical manifestations: cardiac
arrest, low blood pressure,
severe shock, myonecrosis.
• Effector molecules: cardiotoxins
like CTX I, II, III, IV, V.
• Clinical manifestations:
hyperexcitability, convulsion,
paralysis and death.
Envenomation
• Targets molecules: voltage-gated
Na+ channels, voltage-gated K+
channels, voltage-gated Ca2+
channels, acid-sensing ion
channels (ASICs).

Hemotoxins
• Clinical manifestations: decline in coagulability of blood, bleeding, systemic
effects like intracerebral hemorrhage, hypovolemic shock, or renal damage,
development of pathological thrombosis especially pulmonary embolism.
• Effector molecules: hemorrhagin, disintegrin, thrombin-like enzymes,
prothrombin activators, Factor X activators, Factor V activators, protein C
activator, Factor IX/X binding protein, thrombin inhibitor, fibrinogenase,
plasminogen activator (PA) inducer, and PA-like enzyme.

FIG. 6. An overview of the major toxin groups, their associated clinical symptoms, and their
target molecules.

4.1. NEUROPROTECTIVE EFFECTS

The elevated intracellular PLA2 activities found in neurological disorders
are closely associated with inflammation and oxidative stress. PLA2 inhibi-
tors, such as the large protein (Mr 100kDa) isolated from the serum of the
Habu snake (T. flavoviridis) [284], have been shown to inhibit the hemorrhag-
ic and lethal activity of Habu venom [285] by forming specific complexes with
PLA2 enzymes. Habu PLA2 inhibitor has been shown to play a role in
reducing inflammatory reactions associated with trauma and brain tumors,
222 KAUR ET AL.

as well as various neurodegenerative diseases such as Alzheimer’s and

Parkinson’s diseases [286].
Thwin et al. [287] isolated a PLA2 inhibitor from the serum of the nonven-
omous snake Python reticulatus that was termed phospholipase inhibitor
from python (PIP). A cDNA clone obtained from python liver RNA encoded
PIP (182 amino acids) as a 603-bp open-reading frame with a 19-residue
signal sequence. PIP was able to neutralize the toxic effects of several snake
venoms and toxins, including the formation of edema in mice. The evolution-
ary origins of most PLA2 inhibitors can be traced back to the sera of two
major families of venomous snakes, namely the Elapidae and Viperidae [287].
Recent studies have seen the use of PLA2 antisense oligonucleotides to
inhibit enzyme activity and to suppress PLA2 expression [286]. An extension
of this would be to address the efficient in vivo delivery of such oligonucleo-
tides to specific regions of the brain for neuroprotection. Armugam and
colleagues [288] have shown that secretory PLA2 enzymes isolated from
N. sputatrix (Malayan spitting cobra) reduced the infarct volume in middle
cerebral artery occluded models for stroke. A study conducted by the same
group found that administration of N. sputatrix PLA2 reduced the extent
of neuronal damage in hippocampal slices subjected to oxygen-glucose
deprivation, possibly due to a reduction in the extent of apoptosis.
Exendin-4, a 39 amino acid peptide isolated from the saliva or venom of the
gila monster lizard (Heloderma suspectum), has been shown to be almost
pharmacodynamically identical to glucagon-like peptide 1 (GLP-1) [289].
GLP-1 was first described and cloned in 1985 [290]. It acts on a specific
G-protein-linked receptor, stimulating adenylate cyclase activity, increasing
levels of cAMP, and affecting downstream gene expression [289,290]. Since
exendin-4 has a similar pharmacological profile to GLP-1, it is thought to follow
the same mechanism of action and increase the expression of antiapoptotic
genes Bc1-2 and Bcl-xl [291], as a result of nuclear factor kappa B (NF-kB)-
dependent transcription of Bc1-2 and inhibitor of apoptosis protein (IAP)-2
[291,292]. This implies that exendin-4 could have therapeutic potential for use
in poststroke conditions as well as neurodegenerative disorders. The beneficial
effects of exendin-4 serving as a neuroprotectant by increasing neurogenesis
have been demonstrated in laboratory models of Parkinson’s disease [293].

4.2. CARDIOPROTECTIVE EFFECTS

4.2.1. Antihypertensive Agents
A snake bite may cause hypotension due to various activities such as
altering blood vessel permeability and causing hypovolemia from extravasa-
tion of plasma andoxins, which act directly or indirectly on cardiac muscle,
 ENVENOMATION 223

vascular smooth muscle, and other tissues [10]. Individual venom compo-
nents may also indirectly act to lower blood pressure. Murayama et al. [294]
cloned and sequenced cDNA isolated from B. jararaca venom glands, and it
was found to encode two distinct classes of bioactive peptides, bradykinin-
potentiating oligopeptide and a C-type natriuretic peptide. These two pep-
tides act in synergy to lower blood pressure. Bradykinin-potentiating activity
of the venom prolongs activity of bradykinin by inactivating peptidyl dipep-
tidase which would otherwise destroy bradykinin. Inhibition of this peptidyl
dipeptidase also prevents the conversion of angiotensin I to the vasoconstric-
tor peptide angiotensin II. These observations caught the interest of Nobel
Prize winner Sir John Vane, who promoted the use of viper venom as an
angiotensin-converting enzyme (ACE) inhibitor and brought it to the atten-
tion of the pharmaceutical industry, resulting in the invention of captopril
(CapotenÒ), the first commercial antihypertensive ACE inhibitor [295].
Bradykinin-potentiating peptides and ACE inhibitor peptides have now
been isolated from a variety of crotaline and viperine venoms [10].

4.2.2. Natriuretic Peptides

Natriuretic peptides also have the ability to reduce blood pressure by
several mechanisms. All natriuretic peptides have a 17-residue ring structure
with a highly conserved internal sequence and exert their biological effects by
binding to membrane-bound receptors that have guanylyl cyclase activity
[296]. For instance, Dendroaspis natriuretic peptide, which was isolated from
the venom glands of D. angusticeps, is both a potent natriuretic and diuretic
peptide, which, like endogenous atrial natriuretic peptide (ANP) secreted by
heart muscle cells, is associated with an increase in urinary and plasma
cGMP [296]. This allows for its use as a biomarker for congestive heart
failure. ANP has also been found to be beneficial to patients with congestive
heart failure, by regulating pulmonary capillary pressure, systemic blood
pressure, vascular resistance, fatigue, and dyspnea, as well as increasing
stroke volume and cardiac output [297].

4.2.3. Endothelins
Other venoms such as that of Israeli burrowing asp (Atractaspis engadden-
sis) contain sarafotoxins, which are homologous to mammalian endothelins
[298]. The cDNAs encoding sarafotoxins were found to be arranged in a
unique ‘‘rosary-type’’ manner that differs from cDNA-encoding endothelins
[298]. Both endothelins and sarafotoxins can vasoconstrict arteries and delay
atrioventricular conduction [299]. Eight naturally occurring peptides of the
endothelin/sarafotoxin family, each with a high degree of sequence homology,
are now known. They possess four cysteinyl resides and about 60–70% of their
21 amino acid residues are identical [300]. Recent studies have shown that
224 KAUR ET AL.

sarafotoxin 6c has therapeutic potential due to its ability to reduce the infarct
size in ischemic and reperfused rats [301]. Pretreatment with sarafotoxin 6c
before coronary occlusion protected the intact rabbit heart against infarction
and arrhythmias, postischemia, and reperfusion [302].
Another example of venoms that can deliver cardioprotective effects is
maxadilan, a potent 61 amino acid vasodilator peptide isolated from the
salivary gland lysates of the sand fly Lutzomyia longipalpis [303]. It has been
found to act on the type I receptor for pituitary adenylate cyclase-activating
peptide, leading to the accumulation of cAMP [303].

4.3. ION CHANNEL TOXINS AND THERAPY

Management of acute and chronic pain (nociception) has been of interest
to modern medicine for many years. Ion channels have been attractive
targets for the development of novel analgesics [304]. ASICs, or proton-
gated Naþ channels, are expressed throughout the central and the peripheral
nervous systems [304]. ASIC3 is predominantly implicated in pain sensations
in dorsal root ganglion neurons [305], while ASIC1a, ASIC2a, and ASIC2b
are abundant in brain and spinal cord neurons [306]. Psalmotoxin 1 (see
Section 3.2.4) has been found to possess potent analgesic properties against
many various kinds of pain, including thermal, mechanical, neuropathic, and
inflammatory [307]. Psalmotoxin 1 inhibits ASIC1a channels, leading to
activation of the endogenous enkephalin pathway [307]. Enkephalin is a
pentapeptide involved in regulating nociception in the body. Psalmotoxin
has been found to be nonlethal, even at high concentrations, making it a
suitable therapeutic candidate for nociception management.
Certain species of marine snail from the Conus family contain a group of
Cys-rich, paralytic polypeptidic molecules known as o-conotoxins (o-GVIA,
o-MVIIA, o-MVIIC, and o-CVID) which bind with high affinity to Ca2þ
channels to block Ca2þ flux [308]. Many conotoxins also show several
posttranslational modifications, which further increase their structural diver-
sity [309]. Ziconotide (PRIALTÒ) is a synthetic version of an o-conotoxin
and is administered intrathecally for chronic pain management [310]. N-type
and T-type Ca2þ channels are particularly attractive molecular targets for the
development of novel analgesic drugs [311] and ziconotide functions by
selectively blocking presynaptic N-type Ca2þ channels in the spinal cord,
thereby reducing the release of pronociceptive neurotransmitters in the dorsal
horn and inhibiting the transmission of pain signals [311]. a-Conotoxins are
also found to be efficient in the management of chronic pain in several
preclinical models [312]. They are nAChR antagonists, with specificity for
a3-subunits. A PCR screen of a cDNA library from the fish-eating cone snail
 ENVENOMATION 225

Conus catus has recently identified two novel o-conotoxins, CVIE and CVIF,
which are N-type Ca2þ channel blockers [313].

4.4. HEMOSTASIS AND THERAPY

4.4.1. Disintegrins
Metastasis is often the cause of mortality in cancer [314]. Integrins are
essential for the recognition of tumor receptors on ECM molecules, and this
interaction leads to the invasive growth of cancerous cells into surrounding
tissues, resulting in metastasis [315]. Integrins are heterodimeric glycopro-
teins in which different a subunits combine with specific b subunits, resulting
in specific interactions with a range of ECM proteins [316]. These specific
interactions enable integrins to function as adhesion and cell signaling recep-
tors, influencing the rate of cell proliferation, migration, and survival. They
function to grip the ECM by binding to its components, providing traction
and allowing cells to migrate. One such integrin, avb3, is found on the surface
of cancer cells and is implicated to have a critical role in metastasis [317].
Disintegrins are small, disulfide-rich, Arg-Gly-Asp-containing peptides
that bind to integrins on the surface of both malignant and normal cells
and have been characterized from snake venoms as platelet-aggregation
inhibitors [318]. Disintegrins, which competitively inhibit integrins by bind-
ing to the latter on cell surfaces, disrupt ligand–integrin interactions and
could effectively prevent metastasis [319]. Disintegrins are cysteine-rich,
nonenzymatic polypeptides that have been found in the venoms of four
families of snakes, namely, Atractaspididae, Elapidae, Viperidae, and Colu-
bridae [319]. Crotatroxin 2, a disintegrin isolated from the venom of the
Western diamondback rattlesnake (C. atrox), has been shown to inhibit, not
just platelet aggregation but cancer cell migration and lung tumor coloniza-
tion, via different integrins [315]. Contortrostatin (ProtacÒ), a homodimeric
disintegrin (Mr¼13.5kDa) from the southern copperhead snake, modulates
integrins on tumor cells and angiogenic vascular endothelial cells [320]. Each
of the two chains has an Arg-Gly-Asp motif and binds integrins specific for
fibronectin (a5b1), vitronectin (avb3, avb5), and fibrinogen (aIIbb3), con-
ferring anti-invasive properties to tumor cells as well as anticoagulation
effects. A special liposomal delivery method has been designed to administer
contortrostatin without loss of its biological activity, for potential treatment
of metastatic breast cancer [320].
Disintegrins have also been used as templates to design compounds that
bind with high affinity to endogenous blood clotting factors, such as fibrino-
lytic activators and hemorrhagins, thereby inducing or inhibiting platelet
aggregation [321]. This has led to the development of two drugs, eptifibatide
226 KAUR ET AL.

(IntegrilinÒ), designed from the disintegrin, echistatin, and tirofiban

(AggrastatÒ), which is a reverse-engineered copy of the venom derived
from the South American viper, E. carinatus. AggrastatÒ prevents heart
attacks by acting as an anticlotting agent. Both of these antiplatelet drugs
are used in the therapy of acute coronary ischemic syndrome and during
cardiac interventions such as balloon angioplasty [321], to prevent coagula-
tion. AggrastatÒ is used in combination with heparin and aspirin as the
standard therapy for unstable angina and works by inhibiting glycoprotein
IIb/IIIa receptors found on blood platelets, preventing their ability to adhere
to abnormal surfaces and aggregate [321].

4.4.2. Anticoagulants
The anticoagulant and hemostatic properties of individual proteins and
peptides in some snake venoms are primarily responsible for their increasing
involvement in the development of new therapeutic agents for cardiovascular
disorders. A natural anticoagulant secretory PLA2 from the venom of the
spitting cobra, N. sputatrix, has been found to reduce infarct volume in rats
subjected to focal transient cerebral ischemia [288]. PLA2 enzymes are impli-
cated in a wide range of pharmacological effects ranging from antiplatelet
activity to cardiotoxic activity. Spitting cobra venom contains two neutral
and one acidic PLA2 isozymes [288], of which one of the neutral forms
functions as a potent anticoagulant protein [322]. This binds to all muscarinic
receptors, with highest affinity for the M5 subtype [323]. The anticoagulant
activities of snake venoms arise by a variety of means, for example, inhibition
of the activation of Factor X to Factor Xa by extrinsic tenase complex and
inhibition of the activation of prothrombin to thrombin by the prothrombi-
nase complex [324].
Snake venoms have also been found to have metalloproteinases, serine
proteinases, L-amino acid oxidase, as well as nonenzymatic proteins such as
C-type lectin-related proteins and three-finger toxins (3FTx) [324], all of
which possess anticoagulant properties and can be used for stroke therapy
by reducing the formation of clots. 3FTx are a family of nonenzymatic
polypeptides containing 60–74 amino acid residues [325]. Two 3FTx which
have been found to inhibit platelet aggregation include dendroaspin isolated
from green mamba (D. angusticeps) venom [326] and a 3FTx with Arg-Gly-
Asp recognition sequences from the venom of the Taiwan branded krait
(B. multicinctus) [327]. Dendroaspin has been found to have adhesive function
in several protein structures [325].
Cobra venom factor, processed into a mature three-chain protein, has also
been found to reduce postischemic cerebral infarct volume in adults and post-
hypoxic–ischemic cerebral atrophy in neonates [328]. Ancrod (ViprinexÒ),
derived from the Malayan Pit Viper (C. rhodostoma), is being investigated in
 ENVENOMATION 227

clinical trials worldwide for its defibrinogenating properties. It is a protease

that produces rapid decreases in serum fibrinogen by accelerating cleavage of
the fibrinogen A-a chain [329] and has proven to be effective in reducing
infarct volume in animal models of acute stroke as well as in randomized
clinical trials [330]. Defibrinogenation has proven to be effective in treatment
of stroke by preventing blood coagulation, hence decreasing blood viscosity,
improved blood circulation, and enhancement of clot-specific thrombolysis
by stimulation of endogenous PAs. ViprinexÒ has been used for reperfusion
therapy for a number of clinical conditions such as peripheral vascular
disease and deep vein thrombosis, although it is presently only marketed in
Canada [331].

4.5. OTHERS
4.5.1. Induction of Apoptosis
Several toxins may function by targeting the downstream apoptotic cas-
cade and promoting programmed cell death or apoptosis. For example, CTX
III, a basic polypeptide isolated from N. naja atra venom, has been impli-
cated for its anticancer activity by induction of cell apoptosis [332]. Admin-
istration of CTX III has been correlated with an upregulation of
proapoptotic proteins such as Bax and Bad and a decrease in antiapoptotic
proteins such as Bcl-2 and survivin. Turan blunt-nosed viper (Vipera lebetina
turanica) venom has also been found to induce cell apoptosis in several cell
lines via reactive oxygen species-dependent disruption of the mitochondrial
membrane potential and upregulation of proapoptotic genes [333]. Bee
venom has been used in traditional medicine to treat a range of conditions
such as arthritis, rheumatism, back pain, skin diseases, as well as malignant
tumors [334]. Melittin, an amphipathic 26-amino acid peptide present at 50%
(w/w) in bee venom, is responsible for enhancing the cytotoxic properties of
the venom on cancer cells by stimulating PLA2 activity [335].

4.5.2. Antivenoms
Efforts to generate antivenoms in order to treat patients who have suffered
from snake and scorpion envenomation have been ongoing for many years.
Antivenoms are biological products produced based on the principle of
vaccination. Isolated component(s) of a venom are injected into animal
hosts (e.g., horses or sheep) to generate venom-specific antibodies or anti-
venoms [336]. Some antivenoms are effective against the venom of a single
species, while others are effective against the venoms of a range of species,
giving rise to the classifications of monovalent and polyvalent antivenoms,
228 KAUR ET AL.

respectively. Antivenoms can be whole IgG molecules, F(ab0 )2 fragments, or

Fab fragments [336].
Apart from developing therapeutic agents from venom, venomous animals
can be studied for the presence of endogenous antivenoms that protect
against their own toxins. For example, crotoxin inhibitor extracted from
the serum of Crotalus druissus terrificus snakes protects against b-neurotox-
ins from the Viperidae family [337]. Manufactured antivenoms have been
highly effective in the neutralization of Viperidae toxins which cause hemor-
rhage, coagulopathy, and neurotoxicity as well as other systemic effects [338].
There are many studies on the pharmacological and physiological effects of
antivenoms. For example, the efficacy of antivenom in preventing cardiovas-
cular depression and coagulopathy induced by different species of brown
snake (P. textilis, Pseudonaja affinis) was investigated in mechanically
anesthetized, ventilated dogs [339]. Despite the high efficacy of antivenoms,
there are always concerns of the risks of antivenom-induced hypersensitivity
[336]. Efforts need to be made to improve the efficacy and safety of these
antivenoms as well as to increase their deployment and distribution in rural
regions highly prone to snakebite or scorpion sting.
Table 4 summarizes the different venoms/individual venom constituents
that have discovered use as therapeutic agents, only some of which have been
able to be discussed in this section.

5. Conclusion

This chapter has provided an insight into the biochemistry of envenom-

ation and how venoms and toxins target several major systems in the body.
Envenomation can lead to a myriad of systemic conditions which may affect
the physiological state of being and potentially lead to pathogenesis. The
chapter has tried to shine some light on various types of toxins and provides a
breakdown of the biochemical pathways that follow envenomation account-
ing for their clinical manifestations. This includes neurotoxins which target
several ion channels and neurotransmitter receptors as well as those that
induce specific enzyme activities to dysregulate neurological functions. Other
major toxin groups have been addressed, including CTXs and hemotoxins,
which affect the cardiovascular system and disrupt hemostasis of the whole
body, as well as at the site of envenomation. Besides the biochemical aspects,
this review has also addressed how biotechnological advancements have
allowed us to use venoms in their entirety or purified venom constituents,
to develop agents of therapeutic value.
Venoms possess a concoction of compounds, some of which have already
proved themselves in the treatment of diverse disorders. In order to exploit
 TABLE 4
SUMMARY OF VENOMS USED IN THERAPY

Class/order Family Organism Toxin name Drug name Properties Toxin category Mode of action Therapeutic potential

Mollusca— Conidae Cone snail (Conus o-Conotoxin Ziconotide [310], Contains 25 amino Antinociception N-type voltage-gated Chronic pain
Gastropoda magus) peptide PRIALTÒ (Azur acids, 6 of which are calcium channel blocker
Pharma cysteine residues reduces the release of
International linked in pairs by 3 pronociceptive
Limited 1) disulfide bonds neurotransmitters in the
dorsal horn of the spinal
cord, thereby inhibiting
pain signal transmission
Arachnida— Theraphosidae South American Psalmotoxin [307] – 40-amino acid peptide Antinociception Inhibits acid-sensing Nociception (acute or
Araneae tarantula containing three channel protein 1a chronic pain)
(Psalmopoeus disulfide bridges (involved in pain
cambridgei) sensation)
Insecta— Apidae Honey bee – Found to have several Proapoptotic, Enhances cytotoxic effects Arthritis, rheumatism,
Hymenoptera components, antiarthritic of cancer cells via the back pain, skin
including melittin, a increase in PLA2 activity diseases as well as
26-amino acid [334,335] cancerous tumors
peptide (prostate and
breast cancer)
Insecta—Diptera Psychodidae Sand fly Maxadilan [303] Potent 61 amino-acid Vasodilator Binds to type I receptor for Cardiovascular
(Lutzomyia vasodilator peptide pituitary adenylate diseases (e.g.,
longipalpis) isolated from the cyclase activating hypertension and
salivary gland peptide, leading to stroke)
lysates accumulatin of cAMP
and vasodilation effect
Amphibia— Bufonidae Bufo gargarizans – Huachansu Contains several Anesthetic, Control the cell signal Hepatocellular
Anura or B. (extracted from cardiac glycosides antitumor transduction pathways, carcinoma, non
melanostictus dried toad venom such as bufalin, controlling cell small-cell lung
from skin glands) resibufogenin, and proliferation cancer or
[282,350] cinobufagin pancreatic cancer
(antitumor and anesthetic,
properties) cardiotonic, and
diuretic functions

(continues)
 TABLE 4 (Continued)

Class/order Family Organism Toxin name Drug name Properties Toxin category Mode of action Therapeutic potential

Reptilia— Helodermatidae Gila monster Exendin-4 Exenatide [289,351], 39-Amino-acid peptide Antidiabetic— Acts like endogenously Type II diabetes
Squamata (Heloderma BYETTAÒ was found to display antiapoptotic, expressed GLP-1 which is Neurodegenerative
suspectum) similar property as proneurogenesis an important hormone diseases
glucagon-like that helps to regulate
peptide 1 (GLP-1) insulin and supress
abnormal elevation of
glucagon release
Downregulates
proapoptotic factors via
glucogon-like peptide 1
receptor
Atractaspididae – – Cysteine-rich, Disintegrin Prevents metastasis by Cancer
nonenzymatic inhibition of integrins
polypeptides [319]
Elapidae Naja naja atra Cardiotoxin III – Cysteine-rich, Disintegrin— Upregulation of Cancer
(Chinese cobra) [319,352] nonenzymatic proapoptotic proapoptotic proteins
polypeptides such as Bax and Bad and
(phospholipids and decrease in antiapoptotic
glycospingolipids) proteins such as Bcl-2 and
surviving
Naja Naja Natrahagin – Contain Anticoagulant— Reduce infarct volume Stroke
sputatrix [288,324] metalloproteinases, fibrinogenolysis during cerebral ischemia
(Malayan serine proteinases, Inhibition of activation of
spitting cobra) L-amino acid FX to FXa by extrinsic
oxidase as well as tenase complex as well as
nonenzymatic inhibition of the
proteins such as activation of
C-type lectin-related prothrombin to thrombin
proteins and three- by the prothrombinase
finger toxin complex
Green mamba Dendroaspin – Contains B-type Hypotensive— Regulates pulmonary Hypertension and
(Dendroaspis (muscarinic natriuretic peptide anticoagulant capillary pressure, conditions such as
angusticeps) toxins) [297,326] and three-finger systemic blood pressure, stroke and
toxins vascular resistance, congestive heart
fatigue, and dyspnea as failure
well as increases stroke Hemorrhagic
volume and cardiac conditions
output
Viperidae Western Crotatroxin 2 – Cysteine-rich, Disintegrin— Inhibit platelet aggregation, Cancer
diamondback (Atrocollastatin) nonenzymatic inhibition of metastasis, and lung
rattlesnake [315,319,353] polypeptides platelet tumor colonization via
(Crotalus adhesion different integrins
atrox)
Southern Contortrostatin ProtacÒ [3,320,354] 13.5-kDa homodimeric Binds to Modulates integrins on Cancer (especially
copperhead (Centerchem Inc.) protein integrins— tumor cells and metastatic breast
snake antiangiogenic, angiogenic vascular cancer)
(Agkistrodon antitumor endothelial cells Protein C activator/
contortrix) clinical diagnosis of
hemostatic disorder
Turan blunt- Snake venom – b-Chain and random Proapoptotic Induce cell apoptosis in Cancer (especially
nosed viper toxins (SVT) coil structures several cell lines via the human prostate
(Vipera lebetina [333] predominate in these reactive oxygen species cancer)
turanica) proteins (via disruption of the
mitochrondrial
membrane potential and
upregulation of
proapoptotic genes)
Malayn Pit viper Kistomin Ancord [329–331], Metalloprotease Defribrinogen— Protease that produces rapid Stroke (reduction in
(Calloselasma ViprinexÒ anticoagulant decreases in serum infarct volume),
rhodostoma) (Nordmark) fribrinogen by vascular disease,
accelerating cleavage of deep vein
the fibrinogen A-a chain; thrombosis
stimulates plasminogen
activators (reduces blood
viscosity and improved
blood circulation)
Saw-scaled viper Echistatin Tirofiban [3,355] Contains 49 amino Disintegrin Nonpeptide inhibitor acting Unstable angina
(Echis AggrastatÒ acids and 4 cystine at glycoprotein (GP) IIb/
carinatus) (Medicure bridges, cysteine- IIIa receptors in human
Pharma) rich peptides platelets; prevents
containing the Arg- platelet coagulation
Gly-Asp (RGD)
sequence
Dusky Pygmy Disintegrin Eptifibatide [3,353], Synthetic cyclic Anticoagulant (by Non-peptide inhibitor Unstable angina
rattlesnake IntegrilinÒ hexapeptide that preventing acting at glycoprotein
(Sistrurus (Millennium binds to platelet platelet (GP) IIb/IIIa receptors in
miliarius Pharmaceuticals, receptor conjugation) human platelets
barbouri) Inc.) glycoprotein and
inhibits platelet
aggregation

(continues)
 TABLE 4 (Continued)

Class/order Family Organism Toxin name Drug name Properties Toxin category Mode of action Therapeutic potential

Brazilian jararaca Bradykinin- Captopril [356], 5–13 amino acid Antihypertensive Activates bradykinin, which Hypertension and
(Bothrops potentiating CapotenÒ residues, with agent acts as a hypotensive conditions such as
jararaca) peptide (Bristol-Myers common structural agent hemorrhagic stroke
Squibb), enalapril, features such as high Inactivates peptidyl
and other ACE- proportion of dipeptidase, which is
inhibiting peptides proline residues and involved in the
the same structure destruction of bradykinin
for 3–4 amino acids Prevents conversion of
angiotensin I to II
Jararhagin ReptilaseÒ [357] Zinc-containing Cutaneous and Fibrinogenolysis and Prevention of
(hemorrhagins) (American metalloproteases subcutaneous platelet aggregation that coagulation
Diagnostica, Inc.) characterized by the bleeding facilitate hemorrhage
presence of a
protease domain
Crotalinae and Bradykinin- Captopril [358], 5–13 amino acid Hypotensive agent Activates bradykinin Hypertension and
Ò
Viperinae potentiating Capoten residues, with conditions such as
species peptide (Bristol-Myers common structural hemorrhagic stroke
Squibb), enalapril, features such as high
and other ACE- proportion of
inhibiting peptides proline residues and
the same structure
for 3–4 amino acids
Brazillian Batroxobin DefibraseÒ [359] A serine protease Defibrinogenic Thrombin and prothombin Acute cerebral
lancehead (Pentapharm) inhibitor; cleaves the infarction,
(Bothrops alpha chain of fibrinogen unspecific angina
moojeni) pectoris
Colubridae – Cysteine-rich, Disintegrin [319] Cancer
nonenzymatic
polypeptides
Atractaspididae Atractaspis Sarafotoxins – Homologous to Antihypertensive Vasoconstrict arteries and Hypertension and
engaddensis: [298,299, endogenous delay atrioventricular conditions such as
Atractaspididae 301,302] mammalian conduction stroke
endothelins
 ENVENOMATION 233

these particular molecules as therapeutic agents, understanding the biochem-

istry of envenomation becomes crucial. Folklore and traditional medicine
have seen the use of such venoms for several centuries, and it is only
comparatively recently that some of these remedies have been validated via
laboratory and clinical testing; other remedies continue to intrigue clinicians
and medical scientists in terms of their therapeutic potential. Venoms there-
fore could potentially weave a new direction for conventional medicine and
combination therapy.

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