Chapter I
GENERAL INTRODCTION
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Snakes
Snakes are elongated, limbless, flexible reptiles. Their body shape depends on the
habitat in which they live. Aquatic snakes usually have a flattened body; those living in
trees are long and slender with a prehensile tail while burrowing snakes tend to be compact.
Snakes are found in a huge range of colors, from bright to dull. Brightly colored snakes are
usually venomous, their coloration serving as a warning to predators, while dull colored
snakes use their coloration for camouflage. Some snakes mimic the color and pattern of
venomous snakes.
Snakes are highly evolved reptiles belonging to the phylum: Chordata, order:
Squamata and sub order: Serpentes. They are widely distributed throughout the world
except in Arctic, New Zealand and Ireland (Deoras, 1965). They are common in tropical
and subtropical regions, but are rarely found at high altitudes while their number increases
in humid regions (Russell, 1980). Nearly 3500 species of snakes have been identified
allover the world, among them, 400 species of snakes are known to be venomous (Russell
and Brodie, 1974; Philip, 1994). Based on their morphological characteristics like
arrangement of scales, dentition, osteology and sensory organs, these snakes are classified
into different families. As per the recent update of classification, venomous snakes have
been grouped into four families under the order: Serpents (Wuster 1996, 1997).
Viperidae: folding fang snakes including typical vipers such as European adders
(vipera berus), Gaboon vipers (Bitis gabonica); saw-scaled or carpet vipers (Echis);
Rusell’s vipers (Daboia russelii); and pit vipers such as rattle snakes (Crotalus),
bushmasters (Lachesis), Asia pit vipers (Trimeresurus), Malaya pit vipers(Calloselasma
rhodo stoma). These snakes are the most important once, which are responsible for the
highest incidents of the snake bite globally.
Elapidae: front-fanged snakes which includes cobras (Naja, Ohiophagus Hannah),
kraits (Bungarus), mambas (Dendroaspis), coralsnakes (Leptomecrurus, Micruroides,
Micrurus, Calliophis, Sinomicrurus), and sea snakes (Emydocephalus annulutus, Pelamis
platurus). These are the second most important family, which causes snake bites around the
world.
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Atractaspididae: side fanged-vipers from Africa and the Middle East such as
Capecentipede-eater (Aparallactus capensis), spotted harlequin snake (Homorosel apslacteus), southern stiletto snake (Atractaspis bibroni).
Colubridae: back-fanged species snakes Aesculapian snakes (Elaphe longissima),
American green snakes (Opheodrys), American garter snakes (Thamnophis): only few of
them are dangerous.
After, Malcolm Smith’s contribution regarding Indian Serpentes as the third volume
to the Fauna of British India in 1943, no major work on the subject has come out. Deoras
in 1965 has listed about 216 species of snakes out of which 52 are poisonous. Recently
"Romulus Whitaker and Ashok Captain (2004) have now provided a comprehensive list of
275 snakes recorded in the various parts of Indian subcontinent. A list of venomous snakes
identified in India is given in Table 1.01. Among venomous snakes only four pose threat to
human beings as they are found in the vicinity of human settlement. These four venomous
snakes are called Big Four- the Spectacled Cobra, Common krait, Russell’s viper and Sawscaled viper. According to the WHO guidelines, Indian venomous snakes along with these
big four snakes King cobra is also included. Apart from these snakes the other venomous
snakes that strikes human beings are Banded krait (Bungarus fasciatus) and the Indian
(Monocled) Cobra (Naja naja kaouthia). Branded Krait is commonly found in Assam,
Bengal, Bihar, Orissa and also in parts of Madhya Pradesh, Andhra Pradesh and Uttar
Pradesh. Monocled Cobras are a sub-species most commonly found in northwest India,
parts of Uttar Pradesh, Bihar, Orissa and the Andamans, all of Bengal and Assam.
Apart from these Big Five snakes, Indian subcontinent has many endemic snakes
whose habitat is limited to a particular region and some of them are venomous. The list of
Indian endemic snakes, which are venomous, is given in Table 1.02. These venomous
endemic snakes are not lethal but cause severe local tissue damage. Few endemic snakes
such as Trimeresurus gramineus and Bungarus andamanensis are found to be lethal.
Studies on the venom toxicities of endemic snakes allover the world including Indian subcontinent endemic snakes are only few.
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In general two types of toxins are identified in snake venoms, neurotoxins and
hemotoxins. Neurotoxin venom attacks the victim's central nervous system and usually
results in heart failure and/or breathing difficulties. Cobras, mambas, sea snakes, kraits and
coral snakes are examples of snakes that contain mainly neurotoxic venom. Hemotoxic
venom attacks the circulatory system and muscle tissue causing excessive scarring,
gangrene, permanent disuse of motor skills, and sometimes leads to amputation of the
affected area. The viperidae snakes family such as rattlesnakes, copperheads, and
cottonmouths are good examples of snakes that employ mostly hemotoxic strategy. Some
snake venoms contain combinations of both neurotoxins and hemotoxins.
Snake venom
Snake venoms are the most amazing and unique adaptations of animal evolution
and have developed one of the most effective and efficient weapon systems of the animal
kingdom. The principal, offensive as well as defensive armament of a venomous snake is
its venom. Venom is designed to immobilize, kill and digest the prey (Dufton 1993) and
also used secondarily as a defense system. Snake venom is an important natural product,
which is evolved as a specialized secretary product of exocrine gland. It is synthesized in
the venom gland located along the upper jaw and it is injected their secretion via a pair of
fangs. Snake venoms are generally a thick liquid composed of both organic and inorganic
constituents of enzymatic and non-enzymatic protein and peptide toxins.
The inorganic constituents of the venom include metal ions like Ca2+, Cu2+, Fe2+,
Mg2+, Na+, Zn2+ (Markland, 1998) not all of which are found in every snake venom. While
some are required for catalysis by venom enzymes others are thought to be essential for
stabilizing certain proteins. The organic constituents of venom can be broadly divided into
proteinaceous and non-proteinaceous components. The majority of the crude venom is
composed
of proteinous
components. The
non-proteinous
components
include
carbohydrates, lipids, bioactive amines like serotonin and acetylcholine, which are
predominant in viperid venom, nucleotides and amino acids (Freitas et al., 1992; Markland,
1998). Citrate was identified as the major constituents found in many venoms. It is found in
greater than 5 % of dry weight of venom of Crotalous atrox and Bothrops asper (Freitas et
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al., 1992). Snake venoms have several enzymes that depend on metalions for activity. For
example, Phospholipase A2 (PLA2) requires Ca2+ and metalloproteases and hemorrhagins
requires Zn 2+. These are kept in an inactive form by the chelating effect of citrate. Other
than this, citrate act as a buffer component and also as a negative counter ion for basic
proteins and polyamines.
The venom components are fairly common and are similar to one another within
each family of snakes. The proteins give venom the ability to attack wide variety of targets
inflicting different kind of toxicities. However, snake venoms exhibit marked variation in
their potency and extent of inducing toxic properties. The variability of venom composition
has been considered at several levels: Inter family, inter genus, interspecies, and inter
subspecies and intra species. While intra species variability may be due to geographical
distribution, seasonal and age dependent change, diet and variation due to sexual
dimorphism (Jayanthi and Gowda, 1988; Chipaux et al., 1991; Daltry et al., 1996 a, b;
Shishidhar murthy et al., 2002).
The most toxic component of the venom is the proteinaceous component. Peptides
and proteins together contribute to more than 90 % of the dry weight of the venom. The
proteins are both enzymatic and non-enzymatic. However the initial contribution of several
researchers (Weiland and Konz, 1936; Ghosh et al., 1941), it becomes evident that there are
several non-enzymatic proteins in snake venoms (Table 1.03), which posse’s important
biological activities and cannot be ignored. They are known to induce neurotoxicity,
cardiotoxicity, platelet aggregation, nerve growth factors and bradykinin potentiating
peptides are reported from snake venoms (Kini et al., 1988, Markland Jr, 1997; Cornitskaia
et al., 2003).
Exhaustive work has been carried out to understand the mode of action of these
toxins. Though venom research initially started with the intention of producing effective
antivenins, it is now realized that venom are a source of several biologically active
components that have found uses elsewhere in medicine and research. Snake venom
proteins have also been used as models to understand the intriguing structure function
relationship of proteins. Now it is well established that elapid venoms contain many low
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molecular weight toxins and are poor in enzymes. In contrast, viperid venoms contain high
molecular weight toxins and are rich in enzymes (Mebs, 1969).
Snake venom enzymes
Snake venoms contain several different enzymes. As many as 26 enzymes have
been identified in snake venoms (Iwanaga and Suzuki, 1979). Most of these have been
isolated and characterized in detail. A list of enzymes found in snake venom and their
general properties are given in Table 1.04 and Table 1.05 respectively. The distribution of
the enzymes varies from one snake species to another. Some enzymes like L-amino acid
oxidase, phospholipases, phosphodiesterase are found in almost all snake venoms
(Rosenberg, 1979). The remaining enzymes are usually confined to certain taxonomic
groups of snakes (Russell, 1980; Iwanaga and Suzuki, 1979). For example, Viperid venom
contains proteolytic enzymes like endopeptidases, arginine ester hydrolases, thrombin like
enzymes, kininogenases and pro-coagulant enzymes, which are not commonly found in
Elapid venoms (Zeller, 1948). Proteolytic and peptidase activities have been identified in
some of the Elapid venoms like Naja nigricollis (Evans, 1984) and Naja atra (Boumrah,
1993).
The enzymes of snake venoms generally act in the following ways: PLA2 cause
neuromuscular blockages resulting in neurotoxicity. Proteinases, arginine ester hydrolases,
hyaluronidases and some PLA2 cause tissue necrosis and local capillary damage (Gutierrez
and Ownby, 2003; Girish et al., 2004; Petan et al., 2005); Proteinases and phospholipases
are procoagulant or anticoagulant (Jadadeesha et al., 2002; White, 2005; Lu et al., 2005);
Kininogenases release bioactive peptides, which cause acute hypotension (Markland,
1998). Apart from these, there are other enzymes like 5`nucleotidase, phosphodiesterase,
choline esterase and L-amino oxidase, which may have weak pharmacological activities.
The enzymes mainly involved in various pharmacological activities are PLA2,
hyaluronidases and proteases.
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Protease
Proteases [E.C. 3.4.21.40] are present in most of the venoms except for hydrophidae
venoms. All viperid venoms are reported to be rich in proteolytic enzymes. The majority of
toxic effects of viperid (pit vipers, including Rattlesnakes, Water moccasins, Puff adder)
envenomation are due to “proteases”. These are actually hyrdolases that primarily act to
breakdown proteins and thus are also serve a digestive role. Further they are responsible
for most of the local tissue damage following envenomation. The important proteolytic
enzymes are endopeptidases, peptidases, arginine ester hydrolases, kininogenases,
procoagulants and anticoagulants. Some of them are known to induce various
pharmacological effects. For example: Proteinase and arginine ester hydrolases induce
local capillary damage and tissue necrosis (Kini and Evans, 1992; Gutierrez et al., 2005).
Proteases in addition, also have coagulant and hemorrhagic effects (Markland, 1998; Lu et
al., 2005). Kinin releasing enzymes (kininogenase) are responsible for the induction of pain
and acute hypotension due to the release of vasoactive peptides (Matsui et al., 2000;
Felicori et al., 2003; White, 2005).
Endopeptidases are mainly found in viperid venoms. A common feature of venom
endopeptidase is that they are metalloproteases, capable of hydrolyzing peptide bonds with
amino groups contributed by leucine and phenylalanine residues. Endopeptidases can easily
be inactivated by EDTA and reducing agent such as cysteine (Iwanaga and Suzuki, 1979).
Venom endopeptidase catalyzes the hydrolysis of peptide bonds of a variety of natural and
synthetic substrates, including casein, hemoglobin, gelatin, elastin, collagen, fibrinogen,
insulin, glucagons and bradykinin (Liu and Huang, 1997; Gutierrez et al., 2005).
Endopeptidases, which exhibit hemorrhagic activity, have been isolated from several
venoms such as Trimeresurus gramineus (Ouyang and Shiau, 1970), Agkistrodon acutus
(Xu et al., 1981), Crotalus horridus (Civello et al., 1983), Bothrops neuwiedi (Mandelbaum
et al., 1984) and Crotalus atrox (Hagihara et al., 1985). The hemorrhagic effect is attributed
to enzymatic disruption of the basement membrane with loss of integrity of the vessel wall
(Hati et al., 1999; Gutierrez and Rucavado, 2000). However, still it has to be established
whether the hemorrhagic activity is due to direct action of basement membrane or
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indirectly by the release of a tissue factors which can be responsible for the disruption. The
details of venom hemorrhagic toxins will be discussed later under hemorrhage.
The venoms of snakes belonging to Elapidae and Hydrophidae do not show any
arginine ester hydrolase activities. The venom of Ophiophagus hannah and Naja
melanoleuca are exceptions, which showed weak activity towards arginine esters such as
N-α-benzoyl-L-arginine ethyl ester (BAEE), p-tosyl-L-arginine-methyl ester (TAME), Lα-acetyl tyrosine ethyl ester (ATEE) and N- α-benzoyl-L-arginine p-nitroanilide (BAPNA).
The substrate specificities of arginine ester hydrolases are strictly directed towards the
hydrolysis of ester or peptide linkage to which an arginine residue contributes to the
carbonyl group (Iwanaga and Suzuki, 1979). Sato et al., (1965) classified these enzymes
from the venom of A. halys blomhoffi into three categories according to their biological
functions: (i) clotting (ii) bradykinin releasing and (iii) increasing permeability.
Snake venom proteases are a heterogeneous group of proteins with a wide range of
molecular masses between 15 – 380 kDa (Kini and Evans, 1992). They are single chain
proteins (Evans, 1984) and several other enzymes are multi subunits proteins (Zaganelli et
al., 1996; Fry, 1999). Proteases so far isolated are generally classified by the structure into
(1) serine proteases and (2) metalloproteases. There is only a weak or indirect evidence for
the presence of thiol proteases and aspartic proteases in the venoms. Some of them are seen
to degrade mammalian tissue proteins at the site of bites in a non-specific manner to
immobilize the victims. A number of them, however, cleave some of plasma proteins of the
victims in a relatively specific manner to give potent effects, as either the activators or the
inhibitors, on their hemostasis and thrombosis, such as blood coagulation, fibrionolysis and
platelet aggregation (Matusi et al., 2000; Andrews et al., 2004; Marsh and Williams, 2005).
According to the recent inventory of snake venom proteases, more than 150
different proteases have been so far purified, either completely or partially. The complete
amino acid sequences of about 40 of those proteases have been determined by protein
sequencing or deduced from the nucleotide sequence of the cDNA. Recently, the threedimensional (3D) structures of five venom proteases, four metalloproteinases (Gomis-Ruth
et al., 1993; Kumasaka et al., 1996; Gong et al., 1998) and one serine protease (Parry et al.,
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1998), have been determined by X-ray crystallographic analysis and this has made it
possible to understand their structure-function relationship in more detail.
Local manifestations
Local changes are the earliest manifestations of snakebite (Reid, 1979). Features
are noted within 6-8 minutes but may have onset upto 30 min (Reddy, 1980; Reid and
Theakston, 1983). Local pain with radiation and tenderness and the development of small
reddish wheal are the first to occur. This is followed by edema (Paul, 1993) and swelling
which can progress quite rapidly and extensively even involving the trunk (Saini et al.,
1984). Tingling and numbness over the tongue, mouth, scalp and paraesthesias around the
wound occur mostly in viper bites (Reddy, 1980). Local bleeding including ptechial and/or
purpuric rash is also seen most commonly with this family. Crotalid and Viperid venoms
are known to cause local effects, which frequently include pain, swelling, echymoses and
local hemorrhage are usually apparent within minutes of the bite.
Such signs are
sometimes followed by liquefaction of the area surrounding the bite. The local area of bite
may become devascularized with features of necrosis predisposing to onset of gangrenous
changes. Secondary infection including tetanus and gas gangrene may also result (Tu,
1991; Philip, 1994).
Hemorrhagins
Hemorrhage or bleeding is a common phenomenon in the victims of Viperidae
envenomation (Warrell, 1996). “Hemorrhagins” the term was introduced by Grotto et al.,
(1967). The main factors responsible for hemorrhage are hemorrhagins, which comprise a
major group of active principles in viperid venom. These toxins act directly on the
endothelial cells and the under lying basement membrane to induce local and systemic
hemorrhage depending on the severity of envenomation. In mild envenomation, their action
is limited to the site of the bite. However, in severe envenomation, hemorrhage can be wide
spread involving the whole extremity concerned and even organs distant from the site of
the bite, such a s heart, lungs, kidney, intestine and brain.
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Hemorrhagic activity has been associated with enzyme proteolytic activity.
Chelation of the zinc atom abolishes both proteolytic and hemorrhagic effects (Bjarnason
and Fox, 1988; 1994). Of the 65 hemorrhagic toxins, 12 have been analyzed for their metal
content, all of them have been found to contain zinc and many more are inhibited by metal
chealtors. Ten of the twelve toxins contained approximately 1 mole of zinc per mole of
toxin (Bjarnason and Fox, 1994). Therefore, that venom induced hemorrhage is primarily
caused by metal dependent, proteolytic activities of the hemorrhagic toxins, probably
acting on connective tissue and basement membrane components.
Most of the hemorrhagins are found to be absent in the venom of juvenile snakes
and appear only in adult snakes (Mackessay et al., 1996). A search for the signal that
initiates the appearance of these toxins in the venom of snakes at a particularly age in such
species may be considerable academic as well practical interest. Although hemorrhagins
are the main causative agents of hemorrhage, several other components residing in the
crude venom can act also as secondary factors to augment the process. Components that
cause fibrinogenolysis render blood almost completely incoagulable. Anticoagulant factors
directly block the clotting phenomenon. There are platelet aggregation inhibitors and
enzymes that release kinin from kininogen. In the absence of blood coagulation and platelet
aggregation, the two principle phenomena that occur following damage to blood vessels,
hemorrhage initiated by hemorrhagins can go on unchecked with massive extravasation of
RBCs into surrounding tissues, giving rise to swelling, blistering and edema (Bjarnason
and Fox, 1994).
In addition some hemorrhagins also possess other biological activities. For
example, myonecrosis (Bilitoxin and baH1), fibrinogenolytic (Atrolysin f, Jararhagin),
inhibition of platelet aggregation (Atrolysin a) etc. (Ownby et al., 1990; Kamiguti et al.,
1991; Gutierrez et al., 1995; Jia et al., 1997). Many hemorrhagic toxins have been purified
and characterized biochemically from the venoms of Bothrops asper (Franceschi et al.,
2000), Bothrops jararacussu (de Roodt et al., 2004) and Bothrops lanceolatus (Neto and
Marques, 2005).
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Structure and classification
The pathogenesis of venom induced hemorrhage involves the direct damage to
microvessels, performed by hemorrhagic toxins, combined with a wide variety of effects
that viperid venom exert on hemostasis (Bjarnason and Fox, 1994; Markland, 1998). Thus
microvessel disruption and hemostatic disturbances act synertically to provoke profuse
bleeding in viperid snakebites, although hemorrhagic toxins by themselves are able to
induce bleeding in the absence of hemostatic alterations (Kamiguti et al., 1996; Escalante et
al., 2000). Snake venom hemorrhagic toxins are zinc dependent metalloproteinases which
belong on the family of ‘metzincins’, together with astacins, serralysins, matrix
metalloproteinases
(MMPs)
and
ADAMs
(enzymes
with
a
disintegrin
and
metalloproteinases domains).
Snake venom metalloproteinases have been classified into four groups according to
the domain constitution: (i) P -I class SVMPs has only a metalloproteinase domain apart
from the pre and pro sequences. Their molecular masses vary from 20 -30 kDa. These
exhibit low hemorrhagic activity but with strong direct acting fibrinogenolytic activity.
These are mostly weakly acidic proteins; (ii) P -II class SVMPs includes enzymes
presenting the metalloproteinase domain followed by a disintegrin-like domain. The
molecular mass is 30 to 60 kDa and their hemorrhagic potency is low; (iii) P -III class
SVMPs contains a cysteine-rich domain in addition to metalloproteinase and disintegrin
like domain. The molecular mass is 60 - 90 kDa and with strong hemorrhagic potency; (iv)
P -IV class SVMPs is comprised by enzymes with two subunits, one constituted by the
three domains characteristic of P -III enzymes and another being a C-type lectin protein,
linked through disulfide bridges to the first one. The molecular mass is 90 -120 kDa and
hemorrhagic potency is very low. (Bjarnason and Fox, 1994; Hite et al.,1994). Figure 1.01
show the schematic structures of snake venom metallo -proteinases.
SVMPs are synthesized as zymogens, with a cysteine switch mechanism that
inhibits catalytic activity (Bjarnason and Fox, 1994). Cleavage of the pro-sequence results
in enzyme activation. Hemorrhagic effect is dependent on proteolytic activity in these
enzymes, since zinc chelation by EDTA salts, o-phenanthoroline or synthetic
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peptidomimetic hydrxamates completely abrogates this effect. The role of others domains
in the toxicity of high molecular weight enzymes is not clear, although it has shown that
large hemorrhage metalloproteinases having disintegrin like and high cysteine domains, are
more active in inducing hemorrhage than enzymes comprising only the metalloproteinase
domain (Escalante et al., 2000). Not all SVMPs induce hemorrhage in experimental
animals. In general terms, P-III class SVMPs is more potent hemorrhagic toxins than
SVMPs of other groups, particularly of class P-I, where a number on non-hemorrhagic
enzymes have been characterized.
Interestingly, there are metalloproteinases in snake venoms, which are devoid of
hemorrhagic activity (Willis and Tu, 1988, Markland, 1998). The structural basis of this
observation is not clear, although some comparative studies have identified residues, which
may be required to exert this activity (Hite et al., 1999; Ramos and Selistre-de-Araujo,
2004). Table 1.06 presents a summary of some of the biological activities that have been
associated with some of the SVMPs. This table is by no means exhaustive, but does
illustrate the diverse activities that have been attributed to the SVMPs. Most of the
functional activities associated with the SVMPs are associated with the disruption of
hemostasis; essentially pro or anti coagulatory.
Hemorrhagic metalloproteinases play another fundamental role in snake venom
induced muscle pathology, since they drastically affect skeletal muscle regeneration. After
a variety of injuries leading to necrosis, skeletal muscle tissue can regenerate due to
activation of satellite cells, which are myogenic cells located beneath of basal lamina of
muscle fibers. Besides inducing hemorrhage, myonecrosis and skin pathology, venom
metalloproteinases play a relevant role in the complex and multifactorial inflammatory
response characteristic of snakebite envenomation. In addition, metalloproteases degrade
extracellular matrix components and impair the regeneration of affected skeletal muscle.
Some of them also affect platelet function, through their disintegrin-like domain, and
degrade blood-clotting factors, precluding a normal hemostatic response after microvessel
damage. Figure 1.02 summarizes the multiple roles of snake venom metalloproteinases in
the pathogenesis of local tissue damages.
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Due to the protagonic role of metalloproteinases in the pathogenesis of venom
induced local effects, their inhibition by antivenoms and natural and synthetic inhibitors is
a key aspect in the treatment of these envenomations. Due to the rapid onset of these local
effects, and to the frequent delay in antivenom administration, neutralization of these
effects by antivenoms is only partial (Gutierrez et al., 1990; 1995), even when using
antibody fragments (Leon et al., 2000). The development of potent synthetic matrix
metalloproteinase inhibitors, some of which are being tested in clinical trials of other
pathologies opens the possibility of using them in snakebite envenomations (Gutierrez et
al., 1996). It has been recently shown that batimastat, a synthetic metalloproteinase
inhibitor, is effective at counteracting the local tissue damage induced my Bothrops asper
metalloproteinase BaPI, provided the inhibitor is administered at the site of venom
injection rapidly after toxin injection (Escalante et al., 2000). The search of new
alternatives to reduce local effects medicated my metalloproteinases in snakebites is a
highly relevant task.
Mechanism of hemorrhage
As soon as it was established that the hemorrhagins are metalloproteases, the
enzymatic action was given primary emphasis in elucidating the factor(s) responsible for
the leakage of blood from the vessels. Subsequently, it became evident that the enzymatic
disruption of the basement membrane (BM) underlying the endothelial cells of the
capillaries (which have been found to be prime target of the hemorrhagins) is main factor
responsible for hemorrhage. However, in-depth studies reveal certain other factors that may
or actually do facilitate thisprocess.
A. Enzymatic disruption of basement membrane
Basement membranes are extracellular sheets consisting of certain proteins such as
type IV collagen, laminin, nidogen (entactin), fibronectin and heparan sulfate
proteioglycans (Inoue, 1989; Yurchenco et al., 1990). BMs, also known as basal lamina,
are placed beneath the epithelia (under capillary endothelium also). The chief constituent
is type IV collagen, the structure of which is more flexible when compared with the
fibrillar form. The specialized orientation pattern of these molecules results in the
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formation of a basic frame like meshwork to which the other constituents bind by means of
specific associations. Laminin is a flexible complex of three long polypeptide chains and
short arms of laminin can also bind to collagen (Martin, 1987). The molecules of nidogen
are of special interest regarding the assembly and degradation of BM. Thus, it is thought to
act as a bridge between the collagen type IV and laminin networks. Secondly, idogen has
been found to be highly influenced by Zn 2+ and also highly susceptible to proteolytic
degradation, which can allow rapid disruption of the BM structure.
Both in vitro biochemical studies as well as in vivo microscopic observation have
confirmed that hemorrhagins cause local hemorrhage by proteolytic digestion of the BM
proteins. Thus, the most effective way to degrade BM is to attack type IV collagen, “the
scaffolding structure”, or nidogen, “the bridging molecule”. In fact, hemorrhagins can
effectively degrade both, as confirmed by in vitro studies. In addition, they can hydrolyze
laminin and fibronectin but not the proteoglycans. These capabilities have made them very
effective toxins, mediating disruption of BMs resulting in the hemorrhage.
B. Enzymatic disruption of Capillary endothelial cells
Capillaries, with a single cell thick wall, are the main targets of the hemorrhagic
toxins. Exposure to these toxins induces a disturbance in the endothelial cells (ECs), the
degree of which varies from a simple fall-off from the substratum (BM) to complete lysis.
Once this was established, investigations turned to explore whether the extravasation is by
a per rhexis (through the cell by disrupting the plasma membrane and the integrity of the
cell) or a per diapedesis (through the gaps between the cells, keeping them viable and
intact) mechanism. Interestingly, hemorrhagins have adopted both of them, some through
the lysis of the cells (per rhexis), and the others through the formation of gaps (per
diapedesis) between the cells (Table 1.07).
Hemorrhage per rhexis
The pathogenesis of local hemorrhage has been investigated with a number of
purified hemorrhage metalloproteases at the ultra-structural level. In the majority of the
cases, a per rhexis mechanism has been described in which endothelial cells of capillary
blood vessels become affected rapidly after metalloproteinases injection, with the
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development of gaps or lesions within these cells thorough which erythrocytes, and other
blood components, escape to the interstitial space (Ownby et al., 1990; Moreira et al.,
1994). No alterations were observed at the intercellular junctions of endothelial cells in
these studies (Moreira et al., 1994). The sequence in which endothelial cell damage and
BM degradation occurs or whether both of them occur concomitantly has not yet been
determined concussively. Apart from direct mechanism of cell damage, some indirect ones
have also been suggested. BaH1 and BaP1 (Bothrops asper) have been studied in detail,
and Rucavado et al. (1995) have suggested that EC degeneration in vivo is only a secondary
event resulting from disturbance in the interaction between these cells and the surrounding
BM.
Hemorrhage per diapedesis
In contrast to the mechanism described earlier, erythrocytes escape through
widened intercellular junctions instead of gaps in endothelial cell cytoplasm (Ohsaka.,
1979). This apparent discrepancy in the process of extravasation might be due to actual
differences in the mechanism of action of hemorrhagic toxins, although it is more likely a
consequence of variations in the methodologies and the types of microvessels examined.
Conflicting results have been also reported concerning the cytotoxic activity of
hemorrhagic metalloproteinases on endothelial cells. Despites observations of endothelial
cell pathology in vivo after injection of Bothrops asper venom metalloproteinases BaH1
and BaP1 (Moreira et al., 1994; Lomonte et al., 1994a), these toxins are devoid of
cytotoxicity on endothelial cells in culture, as judged by the lack of release of intracellular
enzymes (Lomonte et al., 1994a). The only effect observed in vitro was a dose dependent
detachment of these cells from their substratum, probably due to proteolytic degradation of
extracellular matrix components. Such effects were abolished when metalloproteinases
were incubated with chelating agents that inhibit enzymatic activity (Borkow et al., 1995).
Summarizing, it is suggested that hemorrhage metalloproteinases induce bleeding
mainly by a per rhexis mechanism in capillary blood vessels, in which endothelial cells
undergo degeneration and rupture, with the appearance of gaps through which erythrocytes
and other blood components escape. However, extravasation in venules might also occur as
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a consequence of pharmacologically induced endothelial cell contraction and widening of
intercellular junctions, with the consequent passive escape capillaries and venules affected
by metalloproteinases are needed to address this hypothesis.
Myotoxicity
Myotoxicity is a common and often a serious consequence of snake venom
poisoning. Local hemorrhage and necrosis affecting the skin and muscle layers are the
chief manifestations of myotoxicity. Myotoxicity due to direct action of myotoxins
(enzymatic / non-enzymatic) on muscle cells, cause extensive muscle damage resulting in
weakness of muscle and pain full restriction of movements with muscle tenderness. The
magnitude of nefarious systemic effects directly rely on the concentration and also diffuse
into systemic circulation from the site of injections and intern to their sites of action.
However, this precedes local effects, with accomplished local tissue damage due to
degradation of extracellualr matrix connective tissue surrounding blood vessels and
capillaries by enzyme such as hyaluronidase and hemorrhagic metalloproteinases.
Myotoxicity may be due to the vascular degeneration and ischemia caused by
venom metalloproteinases, or it may result from a direct action of myotoxins upon the
plasma membrane of muscle cells, which is evident from the rapid release of cytoplasmic
markers, creatine kinase (CK) and lactate dehydrogenase (LDH) accompanied by the
prominent increase in total muscle calcium ion (Rucavado and Lomonte, 1996; Gutierrez
and Lomonte, 1989; Gopalkrishnakone et al., 1997; Souza et al., 2000). The increased
influx of calcium ion leads to the cell death (Mebs and Samejima, 1980). Intramuscular
injection of many hemorrhagic metalloproteinases results in acute muscle cell damage, i.e.,
myonecrosis (Gutierrez et al., 1995; Franceschi et al., 2000). The mechanism by which
venom metalloproteinases induce muscle damage has not been fully elucidated. However,
Gutierrez et al., (1995), investigating the action of hemorrhagic metalloproteinases BaG1
from Bothrops asper venom, suggests that muscle damage was secondary to the ischemia
that ensues in skeletal muscle as a consequence of bleeding. Several observations supported
this hypothesis: Myonecrosis was observed only in vivo, and no cell damage occurred when
isolated gastrocnemius muscle was incubated with BaH1 in vitro, in conditions of adequate
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oxygen supply. There was an increment in the muscle contents of lactic acid, a biochemical
indicator of ischemia.Histopathological evidence of myonecrosis was observed at relatively
late time intervals after BaH1 injection, i.e., after 6 hr, whereas hemorrhage develops
within minutes. This observation suggests that muscle damage occurs secondarily to
hemorrhage.
Myotoxicity is associated with many presynaptically acting neurotoxins
(Gopalakrishnakone et al., 1980; Ziolkowske and Bieber, 1992). In addition, several
myonecrotic polypeptides and myotoxic PLA2 enzymes have been isolated and
characterized from various snake venoms (Fohlman and Eaker, 1977; Harris and Maltin,
1982; Mebs, 1986; Mebs and Samejima, 1986; Kasturi and Gowda, 1989; Weinstein et al.,
1992; Geh et al., 1992; Lomonte et al., 1994a,b; Thwin et al., 1995; Ownby et al., 1997;
Radis-Baptista et al., 1999; Nunez et al., 2001).
Edema inducing activity
Swelling and edema are early clinical features observed in snake venom poisoning
at the affected part of the victim. The edema is a result of increased vascular permeability
resulting in the accumulation of fluids in the interstitial space. The action on the vessels is
brought about by either the direct action of venom toxins affecting the microvasculature
(Chaves et al., 1995) or more commonly by the formation of autocoids and other
vasoactive compounds by the PLA2 action of the toxins. The edema induced by Bothrops
jararaca venom is mediated by cyclooxygenase and lipoxygenase eicosanoid products, and
by the action of L1 and L2 adrenergic receptors (Trebien and Calixto, 1989). Pretreatment
with indomethacin, a well-known inhibitor of the cyclooxygenase pathway reduced the
edema induced by Bothrops asper and Bothrops jararaca venoms. It is suggested that
PLA2 induces by two different mechanisms (a) by releasing arachidonic acid as a
membrane phospholipids, leading to the biosynthesis of eicosanoids and (b) by directly
affecting the microvasculature, there by causing plasma exudation (Chaves et al., 1995).
PLA2s are cytotoxic to mast cells and cause their degranulation Degranulation
releases physiological mediators like histamine, serotonin, leukotriens, which increases
vascular permeability (Bhat et al., 1991; Kasturi and Gowda, 1992; Camargo et al., 2005).
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The venoms of Trimeresurus flavoviridis (Vishwanath et al., 1987; Yamaguchi et al.,
2001), Trimeresurus mucrosquamatus (Teng et al., 1989; Chiu et al., 1989), Vipera russelii
(Kasturi and Gowda, 1989; Prasad et al., 1996), Naja naja (Bhat and Gowda, 1989;
Basavarajappa and Gowda, 1992), Echis carinatus (Kemparaju et al., 1994), Bothrops
asper (Lomonte et al., 1993; Chaves et al., 1995) and Bothrops lanceolatus (de Faria et al.,
2001) are reported to induce edema.
Systemic manifestations
The systemic manifestations depend upon the pathophysiological changes induced
by the venom of that particular species. Elapid venoms produce symptoms as early as in 5
min (Paul, 1993) or as late as 10 hr (Reid, 1979) after bite, vipers take slightly longer the
mean duration of onset being 20 min (Paul, 1993). However, symptoms may be delayed
for several hrs. Sea snake bites almost always produce myotoxic features within 2 hr hence
they are reliably excluded if no symptoms are evident within this period (Paul, 1993). The
magnitude of systemic toxicity induced by toxins is directly dependent on the
concentration, efficiency and rate of diffusion of target specific toxins. Based on the
predominant constituents of venoms of a particular species, snakes were loosely classified
as neurotoxic (notably cobras and kraits), hemorrhagic (vipers) and myotoxic (sea snakes).
However it is now well recognized that such a strict categorization is not valid as each
species can result in any kind of manifestations (Estevao-Costa et al., 2000; Moura-da-silva
et al., 2003).
Neurotoxicity
The neurotoxins in snake venoms interfere in synaptic transmission. They can either
inhibit the release of neurotransmitter from exocytosis of synaptic vesicle at the presynaptic
site or bind to the neurotransmitter receptor at postsynaptic site. Neurotoxins are divided
into two types depending on the mode of action at the neuromuscular junction. Those that
act at the presynaptic site are called the β- neurotoxins or presynaptic neurotoxins and
those, which act at the postsynaptic junction, are called the α- neurotoxins or post synaptic
neurotoxins (Rossetto et al., 2004).
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Presynaptic action is generally a two-step process. The first step is the binding of
the toxin to receptors in the presynaptic site of the neuromuscular junction. The second step
is the action at the presynaptic membrane leading to the blocking of the neurotransmitter
release (Schivavo et al., 2000; Montecucco and Rossetto, 2000; Rossetto et al., 2004).
Presynaptic neurotoxins have been intensively studied over the last few decades. All known
venom presynaptic neurotoxins are PLA2s as an integral part of their structure (Hawgood
and Bon, 1990; Westerlund et al., 1992; Chen et al., 2004). Both monomeric and
multimeric β- neurotoxins are found in venoms. Notexin from Notechis scutatus (Kiss et
al., 2004), Caudoxin from Bitis caudalis (Viljoen et al., 1982) and Ammodytoxin from
Vipera ammodytes ammodytes (Ivanovski et al., 2004) are the very well studied single
chain toxins. Taipoxin (Poulsen et al., 2005), Textilotoxin from pseudonaja textiles
(Wilson et al., 1995), Mojave toxin (French et al., 2004), Crotoxin (Beghini et al., 2005)
and β- bungarotoxin (Samson et al., 2005) are extensively studied multimeric neurotoxins.
Postsynaptic neurotoxins bind specifically to the nicotinic acetylcholine receptor (nAchR)
at the motor end plate and produce a non-depolarizing block of neuromuscular transmission
(Hodgson and Wickramaratan, 2002). Over 100 highly homologous postsynaptic
neurotoxins have been sequenced. Cobrotoxin from Naja naja atra, α-bungarotoxin from
Bungarus multicinctus, crotoxin from Crotalus durissus terrificus, cerulotoxin from
Bungarus fasciatus, mojave toxin from Crotalus scutellatus scutellatus and erabutoxin-b
from Laticauda semifasciata venom are known to exhibit postsynaptic neurotoxicity.
(Gopalakrishnakone et al., 1980; Bon and Saliou, 1983; Chang, 1985; Tzeng et al., 1986;
Pergolizzi et al., 2005).
Cytotoxicity
Snake venom PLA2 are known to exhibit cytotoxic activity (Dufton and Hider,
1983; Fletcher and Jiang, 1993). Cytotoxic PLA2s have been isolated from Naja nigricollis
(Chwetzoff et al., 1989a; Gowda and Middlebrook, 1993), Naja naja (Basavarajappa and
Gowda, 1992) and Taipoxin from Oxyuranus scutellatus scutellatus (Poulsen et al., 2005).
The cytotoxic property of nigexine from Naja nigricollis venom was reported to be
independent of enzymatic activity (Rawan et al., 1991). Further it is reported that in vivo
toxicity of nigexine depends on simultaneous expression of esterase activity and non-
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enzymatic property, which alone is able to provoke the lysis of certain eukaryotic cells
(Chwetzoff, 1990).
Cardiotoxicity
Snake venom cardiotoxins are small molecular mass (5.5 – 7 kDa), highly basic
proteins and cross-linked by four disulfide bridges (Jang et al., 1997). Cardiotoxins isolated
from elapid snake venoms are basic proteins. They cause depolarization of the cardiac,
skeletal and smooth muscles resulting in muscle contraction and loss of excitability. They
are also involved in membrane fusion, hemolysis, cytotoxicity, selective killing of certain
type of tumour cells and inhibition of protein kinase C activity (Mirtschin, 1991; Kumar et
al., 1996; Cher et al., 2005).
Cardiotoxicity exhibited by PLA2 enzyme appears to be independent of its
enzymatic activity. A basic PLA2 from Naja nigricollis appears to excert its cardiotoxic
action by increasing intracellular calcium ion concentration (Lee et al., 1977). In addition,
several cardiotoxic polypeptides and cardiotoxic PLA2 enzymes have been isolated and
characterized from snake venoms (Huang et al., 1993; Bhaskaran et al., 1994; Jang et al.,
1997; Wang et al., 2001; Cher et al., 2005).
Hemolytic activities
Hemolysis is the disruption of red blood cells by the action of venom factors which
act either directly or thorough a complex multistep process. Snake venoms have been
classified into two major groups according to the mode of their hemolytic action.
The direct lytic venoms that are capable of hemolyzing washed red blood cells through
hydrolysis of phospholipids at different domains of erythrocytes. PLA2s causing direct
hemolysis have been reported from Agkistrodon halys blomhoffi (Hanahan et al., 1980),
Trimeresurus flavoviridis (Vishwanath and Gowda, 1987), Naja naja (Bhat and Gowda,
1989; Basavarajappa and Gowda, 1992). These enzymes probably act by the mechanism,
which perturb the membrane and affect both the outer and inner leaflets of the phospholipid
bilayer. The indirect hemolysis has been shown to be mediated by lysophospholipids and
hemolytic agents (Welzein, 1979). All PLA2s shows indirect hemolytic activity. This
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property has been frequently used to assay PLA2 activity by semi quantitatively (Bowman
and Kalletta, 1957).
Hypotensive activity
Most snake venoms employ a variety of means to induce rapid and profound
hypotension, leading to circulatory shock, prey immobilization and death (Bjarnason et al.,
1988, Andriao-Escarso et al., 2002; Lumsden et al., 2004; Joseph et al., 2004). The sudden
drop in blood pressure is due to the release of pharmacologically active autocoids like
histamine, 5-hydroxy tryptamine, leukotrienes (Andriao-Escarso et al., 2002). Many
crotaline venoms posses hypotensive peptides of 5-13 amino acids that are N-terminally
blocked with pyroglutamic acid. These peptides are generally known as bradykininpotentiating peptides (BPPs) because of their capacity to enhance the hypotensive effects of
bradykinin (Ferreira et al., 1992).
Acidic PLA2s from Bothrops jararacussu (BthA-I-PLA2) and Vipera russelii have been
shown to cause hypotension (Huang and Lee. 1984; Andriao-Escarso et al., 2002). BthA-IPLA2 acts by an enzyme activity dependent mechanism since chemical modification of
active site histidine abolished the hypotensive activity. Similarly, Indomethacin, a know
PLA2 inhibitor, significantly reduces the hypotensive action of various snake venom
phospholipases (Lobo and Hoult, 1994). This suggests that synthesis and subsequent
release of prostaglandins appears to be very critical in the PLA2 induced hypotension.
Convulsion activities
Death following cobra envenomation is often proceeded by convulsion due to
asphyxia arising from respiratory paralysis and other pre-agonial effects. The snake venom
components known to cause depletion of stored acetylcholine due to high influx of
potassium ions. The nerve does not release the neurotransmitter (Karlsson, 1979) there by
these acts as presynaptic neurotoxins. The convulsant activity has been reported from the
venoms of Naja naja (Lysz and Rosenberg, 1974; Bhat and Gowda, 1991), Vipera russelii
(Jayanthi and Gowda, 1990; Kasturi and Gowda, 1989) and Echis carinatus (Kemparaju et
al., 1994).
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Snake venom action on hemostasis
Snake venoms, particularly from the Viperidae and Elapidae families,
contain a number of components that interact with proteins of the coagulation cascade and
fibrinolytic pathway. Snake venom toxins act as either procoagulants or anticoagulants
(Kini, 2005). Figure 1.03 shows the coagulation cascade and major sites of action by
snake venom components. Many types of venom contain more than a single procoagulant
or anticoagulant agents. Venom proteins affecting coagulation factors may be classified as
Coagulant factors include Factor V activators, Factor X activators, Prothrombin activators
and Thrombin like enzymes (TLEs).
Anticoagulant factors includes Factor IX / X binding proteins, Protein C activators,
Thrombin inhibitors and Phospholipase A2.
Fibrinolysis includes fibrinolytic enzymes and plasminogen activator.
The anticoagulant and procoagulant activities of venom components exert their
action differently. They all interfere at different steps in the coagulation pathways. Based
on their specific action in the coagulation cascade the venom components are studied as
activation of inhibition molecules of coagulation cascade. Snake venoms useful as
therapeutics or diagnostic agents are indicated.
Factor V activator
Factor V activator is a multifunctional 330 kDa glycoprotein, with an important role
in both procoagulation and anticoagulation activities. Thrombin activates, factor V by
cleaving at 709, 1018 and 1545 to form factor Va, a heterodimer consisting of a 105 kDa
heavy chain and a 72 / 74 kDa light chain doublet. Factor Va acts as cofactor in factor Xa
catalyzed prothrombin activation and it enhances thrombin generation more than 1000
folds. Several factor V activators have been described from Bothrops atrox, vipera russelli,
vipera lebetina, vipera ursine, naja naja oxiana and naja nigricollis nigricollis venoms
(Rosing et al., 2001).
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Factor X activator
Factor X activators have been isolated from many viperidae venoms as well as from
elapid venoms. Factor X activators are either metalloproteinases or serine proteases (Tans
and Rosing, 2001). Russell’s viper venom contains potent activators of human blood
coagulation factor X (RVV-X) that has been well characterized (Kisiel et al., 1976; Furie
and Furie, 1976). Factor X activation has also been isolated from Bothrops atrox (Hofmann
and Bon, 1987) and several other snake species (Lee et al., 1995; Zhang et al., 1995).
Interestingly, the factor X activators from venom of the elapidae, king cobra (Ophiophagus
hannah) and banded krait (Bungarus faciatus), have been reported to be serine proteinase
unlike RVV-X, which, is noted, as a metalloproteinase.
Prothrombin activator
Prothrombin (also known as factor II) is a single chain glycoprotein with a
molecular weight of 72,000 Da (Rosing et al., 1988; Rosing and Tans, 1991, 1992). A large
number of snake venoms contain prothrombin activators, which convert prothrombin into
meizothrombin or thrombin (Rosing and Tans, 1992). Based on their structure, functional
characteristic and cofactor requirements, they are classified into four groups. Group A
prothrombin activators are metalloproteinases and activate prothrombin efficiently without
cofactors, such as phospholipids (PLs) or cofactor Va. Group B prothrombin activators are
Ca2+ dependent. They contain two subunits linked non-covalently: a metalloproteinase and
a C-type lectin like disulfide linked dimmer. Group C prothrombin activators are serine
proteases found in Australian Elapids requiring Ca2+, PLs or Factor Va for maximal
activity. Oscutarin from Oxyuranus scutellatus also activates factor VII. Group D
prothrombin activators are serine proteases and are strongly dependent on Ca2+, negatively
charged PL and factor Va.
Some venom prothrombin activators are real structural and functional
homologues of coagulation factors. Group D prothrombin activators, hopsarin D
(Hoplocephalus stephensi) (Rao et al., 2003) and trocarin D (Tropidechis carinatus)
(Venkatewarlu et al., 2002) are similar to coagulation factor Xa. Pseutarin C, a group C
prothrombin activator from Eastern Brown snake venom, Pseudonaja textills, is a multi-
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subunit protein complex containing catalytic and non-enzymatic subunits similar to factor
Xa and factor Va, respectively (Rao et al., 2004). Structural information on these classes of
prothrombin activators should contribute significantly toward understanding the
mechanism of factor Xa-mediated prothrombin activation.
Thrombin like enzymes
Thrombin has many activities, the ability of a group of snake venom enzymes to
clot fibrinogen has resulted in these enzymes being called thrombin like (Ouyang et al.,
1992; Hutton and Warrel, 1993; Marsh, 1994). Thrombin like enzymes can be classified
into three groups, venombin A, venombin B and venombin AB (Markland, 1998). They
also show some species specificity in efficiency of fibrinogen conversion. Thrombin like
enzymes are inhibited by serine protease inhibitors, but most are unaffected by thrombin
inhibitors like anti-thrombin III and hirudin. Consequently, the fibrin formed by thrombin
like enzymes is easily removed from the circulation allowing their clinical use as
defibrinogenating agents.
These enzymes are widely distributed, primarily in venoms of snakes from true
vipers (Bitis gabonica, Cerastes vipera) and pit vipers (Agkistrodon contortrix contortrix,
Crotalus adamanteus, Bothrops atrox). There are several groups of snake venom
fibrinogen clotting enzymes based on the rate of release of fibrinopeptides A and B from
fibrinogen. One group releases fibrinopeptide A preferentially (the venom A including
ancord from venom of the Malayan pit viper, Colloselasma rhodostoma); another group
releases both fibrinopeptides A and B (the venombin AB group including gabonase from
venom of the Gaboon viper, Bitis gabonica); and the third group releases fibrinopeptide B
preferentially (the venombin B group including venzyne from venom of the southern
copperhead, Agkistrodon contortrix contortrix) (Lu et al., 2005)
Factor IX / X inhibitors
Many anticoagulant C-type lectin-like proteins, interacting with factor IX and / or
FX, have been isolated from various snake species (Morita, 2004). Based on their ligand
recognition differences, these proteins can be classified as: blood coagulation factor IX / X-
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binding proteins, interacting with factor IX or factor X in a 1:1 molar ratio; factor IXbinding proteins, which do not interact with factor X; factor X binding proteins, binding
predominantly to factor X. An inhibitor of factor X was isolated from Deinagkistrodon
acutus, Trimeresurus flavoviridis, Bothrops jararaca, Echis carinatus leucogaster
(Markland, 1998).
Protein C activators
Protein C is a vitamin K-dependent, two chain zymogen activated by thrombin.
Activated protein C degrades factor Va and factor VIIIa and is therefore anticoagulant.
Most protein C activators were purified from Agkistrodon venoms. Others come from
Bothrops, Trimeresurus, or Cerastes venoms. Most venom protein C activators have
sequences highly similar to other venom serine proteases. Unlike thrombin-catalyzed
protein C activation, requires thrombomodin as a cofactor, venom activators directly
convert protein C into the active form. The fast-acting protein C activator ProtacR from
Agkistrodon contortrix contortrix venom is widely used to diagnose protein C pathway
disorders (Gempeler-Messina et al., 2001).
Thrombin inhibitors
A unique thrombin inhibitor was purified from Bothrops jararaca venom by Zingali
et al. (1993). This is the only report to date of snake venom inhibitor of this type. The
inhibitors, named bothrojarcin, is a 27 kDa C-type lectin like thrombin inhibitors composed
of the polypeptides chains of 13 and 15 kDa subunits linked by disulfide bridges.
Bothrojarcin is highly resistant to urea or DTT, requiring both agents to denature it fully.
Bothrojarcin has two independent mechanisms for anticoagulant action it binds strongly to
exosites I and II to form a non-covalent equimolar complex and inhibits thrombin induced
platelet aggregation and secretion, but does not interact with the by competitively inhibiting
the binding of thrombin to fibrinogen and it inhibits thrombin binding to thrombomodulin
and decreases the rate of protein C activation (Arocas et al., 1996). Secondly, it inhibits
prothrombin activation by interacting with proexosite I. In the absence of PLs, bothrojarcin
strongly inhibits the zymogen activation by factor Xa in the presence but not in the absence
of factor Va.
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Phospholipase A2
Snake venom PLA2 is extremely important and diverse group of protein affecting
hemostasis. The prothrombinase complex is composed of factors Va, Xa, phospholipid and
calcium ions. Snake venom phospholipases appear to inhibit formation of the
prothrombinase complex by degrading phospholipids involved in this complex. PLA2 have
been isolated from a number of snake venoms and have a number of pharmacological
action including effects on blood coagulation (Ouyang et al., 1992). It has been suggested
that the anticoagulant action results from the formation of a hydrolytic complex between
the phospholipase and phosphodidylserine on the platelet surface (Boffa and Boffa, 1976).
Based on the potency of their action, the phospholipases have been classified as strong,
weak or non-anticoagulant. The strong anticoagulant act to inhibit both the extrinsic factor
X and the prothrombin activation complexes. The weak anticoagulants, by comparison,
only inhibit the extrinsic factor X activation complex (Subburaju and Kini, 1997).
The anticoagulant activity of PLA2 enzymes has been shown in the venoms of
snakes, Vipera aspis, Vipera berus (Boffa et al., 1976), Trimeresurus mucrosquamatus
(Ouyang et al., 1978), Naja siamensis (Karlsson and Pongaswadi, 1980), Naja atra
(Rosenberg, 1986), Trimeresurus flavoviridis (Vishwanath et al., 1987), Pseudechis
papuanus (Laing et al., 1995) and Naja naja (Sathish et al., 2004).
Fibrinolytic proteinases
The substrates for the fibrinogenlytic enzymes, fibrinogen, appears as large
trinodular protein by electro microscopy. The protein contains two symmetric halfmolecules which are disulfide-linked. Each half contains three chains designated as Aα, Bβ
and γ with molecular weights of 63 500, 56 000 and 47 000 Da respectively. The
fibrinogen molecule has a molecular weight of 340 kDa (Bauer and Rosenberg, 1987).
Fibrinogen contains long stretches of amino acids, which are exposed to proteolytic
enzymes including the snake venom proteinases. Fibrin, however, has a cross-linked
structure and is much less susceptible to proteolysis.
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Fibrinogenlytic activity has been described in the venoms of members of the
Viperidae and Elapidae families (Markland Jr., 1991). These fibrinolytic enzymes are
divided into metalloproteinases and serine proteinases (Matusi et al., 2000). Most of the
first groups of enzymes were characterized as zinc metalloproteinases and degrade Aα
chain of fibrinogen preferentially. The second groups are serine proteases and most have
specificity toward the Bβ chain of fibrinogen. However, there are exceptions to these
generalizations and specificity for Aα or Bβ chains are not absolute, as there is substantial
degradation of alternate chain with time. Most of the metalloproteinases are fibrinolytic and
many of the serine proteinases are both fibrinogenolytic and fibrinolytic (Braud et al.,
2000). Fibrinogenolytic metalloproteinase enzymes cleaves amino-terminal to hydrophobic
amino acids, while serine fibrinogenolytic enzymes cleave carboxy-terminal to basic amino
acids.
Plasminogen activator
Snake venoms have been reported to stimulate the release of plasminogen activators
from endothelial cells. The activity was most pronounced in the venoms of the rattlesnakes
Crotalus atrox and Crotalus adamanteus (Kirshchbaum et al., 1999). Plasminogen
activators are also reported from lachesis muta muta and Agkistrodon halys, Trimeresurus
stegnegeri venoms (Zhang et al., 1998; Park et al., 1998; Sanchez et al., 1991).
Proteases interfering in hemostasis have tremendous pharmacological applications
and are therapeutically important in thrombosis disorders (Marsh, 1994; Baker Jr, 2003). A
number of snake venom proteases degrade fibrinogen and effect blood coagulation through
both pro and anticoagulant mechanisms (Matsui et al., 2000; Braud et al., 2000; Swenson
and Markland, 2005). The action of different snake venom proteases in hemostasis is
summarized in Table 1.08.
Importance of proteases affecting blood coagulation and fibrinolysis
Proteases interfering in hemostasis have invaluable importance in laboratory
diagnosis of hemostatic disorders and other clinical use. Pro-coagulant proteases are either
of thrombin like enzymes, prothrombin activators, factor X activators, factor V activators
or factor VII activators. Over 90 thrombin like enzymes from 35 snake species have been
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recorded (Pirkle, 1998) and the most widely studied thrombin like enzymes are batroxobin
from Bothrops atrox, ancord from Callosellasma rhodostoma and ACTE from Agkistrodon
c. contortrix. Snake venom thrombin-like enzymes (SVTLEs) are used for fibrinogen and
fibrinogen breakdown product assay and for the detection of fibrinogen dysfunction.
SVTLEs are not inhibited by heparin and can thus be used for assaying antithrombin III
and other hemostatic variables in heparin-containing samples. And also to remove
fibrinogen from samples containing heparin. Snake venoms are a rich source of
prothrombin activators. Ecarin from the saw-scale viper (Echis carinatus) venom (Kornalik
et al., 1969). Carinactivase, also from E. carinatus venom (Yamada et al., 1996). Textarin
from the Australian brown snake (Pseudonaja textilis) and the enzyme from the taipan
(Oxyuranus s. scutellatus) (Denson et al., 1971) are few among them. These are utilised in
prothrombin assays, for studying dysprothrombinaemias and for preparing meizothrombin
and non-enzymic forms of prothrombin. A serine protease from (RVV-V) Russell’s viper
(Daboia russelli) venom is factor V activator used to assay factor V (Kisiel and Canfield,
1981). Russell’s viper venom also contains a potent activator of factor X (RVV-X)
(MacFarlane and Barnett, 1934). RVV-X (Pentapharm) has been employed in a number of
clotting assays, notably for the measurement of factor X itself (Bachmann et al., 1958), for
distinguishing between factor VII and factor X and in lupus anticoagulant assay
(Thiagarajan et al., 1986).
Activated Protein C (APC) is an anticoagulant protease which inactivates factors Va
and VIIIa and plays a key role in controlling hemostasis. Protein C activators were isolated
from the venom of the Southern copperhead snake and Agkistrodon c. contortrix (Klein and
Walker, 1986; Stocker et al., 1987). The use of protein C activators from snake venoms for
diagnostic purposes has been reviewed (Gempeler-Messina et al., 2001). Protein C and
activated protein C resistance can be measured by means of RVV-V.
Snake venom proteases affecting hemostasis are also used in the therapeutic setting:
Ancrod, in particular, has been used as an anticoagulant to achieve ‘therapeutic
defibrination’. Other snake venom proteins show promise in the treatment of a range of
hemostatic disorders. Several snake venom proteins are in clinical trials at various stages
(Sherman et al., 2000; Sherman, 2002). Snake venom proteases with fibrinogenolytic, and
anti-clotting properties find potential application in drug development to treat thrombotic
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disorders, which result in fatal, heart attacks and strokes. Many R & D laboratories of many
pharmaceutical industries are looking for new such proteases with similar pharmacological
application.
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Table 1. 01: Venmous snakes of India
Common name
Scientific name
Family
Geographical distribution
Ornate flying snake
Chrysopelea ornate
Colubridae
Southwestern and Eastern part of India
Paradise flying snake
Chrysopelea paradise
Colubridae
Limited to Andaman Islands
Himalayan Kellback
Colubridae
Himalayas from Kashmir in West to Northeast
False Cobra
Rhabdophis
himalayanus
Pseudoxenodon macrops
Colubridae
Northeast from Darjeeling to Arunachal Pradesh
Large-spotted Cat snake
Boiga multomaculata
Colubridae
Northeast from Assam to Arunachal Pradesh
Tawny Cat snake
Boiga ochracea
Colubridae
Eastern part of India
Common Cat snake
Boiga trigonata
Colubridae
South Asia, Except in Andaman and Nicobar
Eastern Cat snake
Boiga gokool
Colubridae
Eastern part of India
Ceylon Cat snake
Boiga ceylonensis
Colubridae
Beddome`s Cat snake
Boiga beddomei
Colubridae
Andaman Cat snake
Boiga andamanensis
Colubridae
Western Ghats from Maharastra to Kerala and Tamil
Nadu
Western Ghats from Maharastra to Kerala and Tamil
Nadu
Restricted to Andaman islands
Green Cat snake
Boiga cyanea
Colubridae
Northeast from West Bengal to Arunachal pradesh
Many-banded Cat snake
Boiga multifasciata
Colubridae
Western and Eastern part of India
Eyed Cat snake
Boiga ocellata
Colubridae
Northeastern India from West Bengal through
Assam to Eastern Arunachal Pradesh
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Common name
Scientific name
Family
Geographical distribution
Forsten`s Cat snake
Boiga forsteni
Colubridae
Nicobar Cat snake
Boiga wallachi
Colubridae
Western Ghats from Gujarat to Kerala, Peninsular
India
Restricted to little and Great Nicobar Islands
Afro-Asian sand snake
Psammophis schokari
Colubridae
Rajasthan and Gujarat
Condanarus sand snake
Colubridae
Western and North Eastern part of India
Stout sand snake
Psammophis
condanarus
Psammophis longifrons
Colubridae
Maharastra and Gujarat
Leith`s sand snake
Psammophis leithii
Colubridae
Mock viper
Colubridae
Gunther`s vine snake
Psammodynastes
pulverulentus
Ahaetulla dispar
Jammu & Kashmir, Punjab, Rajasthan and Uttar
Pradesh
Northeastern part of India
Colubridae
Southern Western Ghats of India
Short-nosed vine snake
Ahaetulla prasina
Colubridae
Eastern part of India
Common vine snake
Ahaetulla nasuta
Colubridae
Brown vine snake
Ahaetulla pulverulenta
Colubridae
Throughout India (Except in Northwest and
Gangetic basin
Western Ghats from Gujarat to Kerala
Plumbeous smoothscaled water snake
Common smooth-scaled
water snake
Siebold`s smooth-scaled
water snake
Enhydris plumbea
Colubridae
Great Nicobar Island
Enhydris enhydris
Colubridae
North and South of Eastern part of India
Enhydris sieboldii
Colubridae
Northeastern part of India
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Common name
Scientific name
Family
Geographical distribution
Dog-faced water snake
Cerberus rynchops
Colubridae
Glossy marsh snake
Gerarda prevostiana
Colubridae
Along coastal and tidal rivers (Including Andaman
and Nicobar
Along coastal and tidal rivers
Crab-eating water snake
Fordonia leucobalia
Colubridae
West Bengal and Nicobar islands
Yellow-banded
Mangrove snake
Branded krait
Cantoria violacea
Colubridae
Middle and North Andaman
Bungarus fasiatus
Elapidae
Eastern part of India
Common krait
Bungarus caeruleus Delhi
Elapidae
Andaman krait
Bungarus andamanensis
Elapidae
Found all over India except Jammu and Kashmir
and Delhi
Restricted to Andaman Islands
Wall`s sind krait
Bungarus sindanus walli
Elapidae
Black krait
Bungarus niger
Elapidae
Slender coral snake
Calliophis melanurus
Elapidae
Striped coral snake
Calliophis nigrescens
Elapidae
Most of peninsular India (Except the extreme
Northwest)
Restricted to Coastal region of Western Ghats
Maccleland`s coral
snake
Spectacled Cobra
Sinomicrurus
macclellandi
Naja naja
Elapidae
Northeast part of India
Elapidae
Throughout mainland India (excluding the northeast)
Monocled Cobra
Naja kaouthia
Elapidae
Northeastern part of India
Andaman Cobra
Naja sagittifera
Elapidae
Restricted to Andaman Islands
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Found only in the gangetic plain, Central and
Western India
Eastern States of India
Common name
Scientific name
Family
Geographical distribution
Central Asian Cobra
Naja oxiana
Elapidae
Jammu and Kashmir, Himachal Pradesh
King Cobra
Ophiophagus hannah
Elapidae
Western Ghats of India and Northeastern states
Yellow-lipped sea Krait
Laticauda colubrine
Hydrophidae
Only in Andaman and Nicobar Islands
Jerdon`s sea snake
Kerilia jerdonii
Hydrophidae
West and East coast of India
Hook-nosed sea snake
Enhydrina schistose
Hydrophidae
Indian coast of India
Annulated sea snake
Hydrophis cynocinctus
Hydrophidae
Along coast line of India
Cochin banded sea
snake
Malacea sea snake
Hydrophis ornatus
Hydrophidae
Along coast line of India
Hydrophis caerulescens
Hydrophidae
Western and Eastern coastal lines
Short sea snake
Lapemis curtus
Hydrophidae
Along coast line of India
Large-headed sea snake
Astratia stokesii
Hydrophidae
Bay of Bengal
Black and yellow sea
snake
Russell’s viper
Pelamis platurus
Hydrophidae
Coastal waters and Andaman and Nicobar islands
Daboia russelii
Viperidae
Throughout India
Levantine viper
Macrovipera lebetina
Viperidae
Only in few localities in Jammu and Kashmir
Saw-scaled viper
Echis carinatus
Viperidae
Throughout Mainland of India
Gloydius himalayanus
Gloydius himalayanus
Viperidae
Western Himalayas
Levantine viper
Macrovipera lebetina
Viperidae
Only in few localities in Jammu and Kashmir
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Common name
Scientific name
Family
Geographical distribution
Hypnale hypnale
Hypnale hypnale
Viperidae
Western Ghats as far North as Belgaum
Saw-scaled viper
Echis carinatus
Viperidae
Throughout Mainland of India
Gloydius himalayanus
Gloydius himalayanus
Viperidae
Western Himalayas
Large-scaled pit viper
Viperidae
Restricted to few localities in South India
Mountain pit viper
Trimeresurus
macrolepis
Ovophis monticola
Viperidae
Eastern parts of India
Jerdon`s pit viper
Protobothrops jerdonii
Viperidae
Northeastern states of India
Malabar pit viper
Viperidae
Trimeresurus strigatus
Trimeresurus
malabaricus
Trimeresurus strigatus
Viperidae
Western Ghats from Maharastra South to
Kanyakumari
Found only in Southern parts of Western Ghats
Bamboo pit viper
Trimeresurus gramineus
Viperidae
Restricted to Western and Eastern Ghats of India
Medo pit viper
Trimeresurus medoensis
Viperidae
Only in Arunachal Pradesh
Pope`s pit viper
Viperidae
Found in Northeastern States
Cantor`s pit viper
Trimeresurus popeiorum
popeiorum
Trimeresurus cantori
Viperidae
Found only in the Central Nicobar group of Islands
Spot-tailed pit viper
Trimeresurus erythrurus
Viperidae
Northeastern states of India
White-lipped pit viper
Trimeresurus albolabris
Viperidae
Restricted to West Bengal and Assam
Nicobar pit viper
Trimeresurus labialis
Viperidae
Found only on the Nicobar Islands
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Table 1. 02: Endemic snakes of India
Common name
Mildly venomous
Scientific name
Geographical distribution
Family
Andaman Cat
snake
Boiga
andamanensis
Colubridae
Nicobar Cat snake
Boiga wallachi
Colubridae
Gunther`s vine
snake
Ahaetulla dispar
Colubridae
Andaman Islands
Little and Great Nicobar Islands
Southern Western Ghats- Tamil Nadu, Kerala.
Venomous
Restricted to Andaman Islands
Andaman krait
Bungarus
andamanensis
Elapidae
Wall`s Sind krait
Bungarus sindanus
walli
Elapidae
Found only in the gangetic plain, Central and
Western part of India
Striped coral snake
Calliophis
nigrescens
Elapidae
Restricted to coastal region of Western Ghats hills of
Kerala and Tamil Nadu
Andaman Cobra
Naja sagittifera
Elapidae
Large-scaled pit
viper
Trimeresurus
macrolepis
Viperidae
Malabar pit viper
Trimeresurus
malabaricus
Viperidae
Horseshoe pit
viper
Trimeresurus
strigatus
Viperidae
Bamboo pit viper
Trimeresurus
gramineus
Viperidae
Cantor`s pit viper
Trimeresurus
cantori
Viperidae
Andaman pit viper
Trimeresurus
andersoni
Viperidae
Nicobar pit viper
Trimeresurus
labialis labialis
Viperidae
Restricted to Andaman Islands
Restricted to few localities in South India; Tamil
Nadu and Kerala
Found only in Western Ghats from Maharastra
Found only in Southern parts of Western Ghats
Restricted to Western Ghats of India and also
occurs in the Eastern Ghats
Found only in the Central Nicobar group of Islands
Only found in Andaman Islands and Nicobar Islands
Found only on the Nicobar Islands
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Table 1. 03: Non-enzymatic toxic proteins/ peptides found in snake venoms
Non enzymatic toxins
Molecular
weight
Snake species
References
Elapidae
Neurotoxin (toxin a)
6,787
Karlsson et al., (1966)
Naja nigricollis
Cobramine A and Cobramine B
Dendrotoxin (DTX)
α-Neurotoxin, B.F.III
Cardiotoxin
Muscarinic toxin (MTxs)
Phospholipase Inhibitor (NN-I3)
6,400
7,077
6,500
7,000
7,500
6,500
Naja naja
Dendroaspis angusticeps
Bungarus fasciatus
Naja nigricollis
Dendroaspis angusticeps
Naja naja naja
Larsen and Wolff, (1968)
Harvey and Karlsson, (1980)
Ji et al., (1983)
Kini et al., (1987, 1988)
Adem et al., (1988)
Rudrammaji, (1994)
4,900
4,400
4,932
5,035
5,132
8,900
2,504
Crotalus durissus terrificus
Crotalus viridis viridis
Crotalus viridis helleri
Crotalus viridis concolor
Crotalus adamanteus
Trimeresurus wagleri
Trimeresurus wagleri
Laure, (1975)
Ownby et al., (1976)
Maeda et al., (1978)
Engle et al., (1983)
Samejima et al., (1988)
Tan and Tan, (1989)
Weinstein et al., (1991)
11,600
6,900
14,000
Vipera palaestinae
Vipera russelii
Vipera ammodytes
Moraz et al., (1967).
Jayanthi and Gowda, (1990).
Krizaj et al., (1991).
6,760
6,780
6,520
Laticauda semifasciata
Laticauda semifasciata
Laticauda laticaudata
Laticauda colubrina
Tamiya et al., (1967)
Tamiya et al., (1967)
Sato et al., (1969)
Viperidae:
Crotalinae
Crotamine
Myotoxin a
Peptide C
Myotoxin I
CAM-toxin
Wagleri toxin
Lethal peptide I
Viperinae
Neurotoxin
Trypsin Inhibitor (TI)
Ammodytin L (AMDL)
Hydrophidae
Erubutoxin a
Erubutoxin b
Neurotoxic peptide
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Table 1.04: Enzymes found in snake venoms
Enzymes found in all venoms
Phospholipase A2
Deoxyribonuclease
Phosphodiesterase
Adenosine triphosphatase
Phosphomonoesterase
NAD nucleosidase
L-amino acid oxidase
Ribonuclease
5` Nucleotidase
Hyaluronidase
Enzymes found mainly in Viperid venoms
Endopeptidase
Kininogenase
Arginine ester hydrolase
Thrombin like enzyme
Factor X activator
Prothrmbin activator
Enzymes found mainly in Elapid venoms
Acetylcholinesterase
Phospholipase B
Glycerophosphatase
Enzymes found in some venoms
Glutamate-pyruvate transaminase
Catalase
Amylase
Lactate dehydrogenase
Heparin like enzyme
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Table 1. 05: Properties of enzymes found in snake venoms
Trivial name
Typical substrate
Phospholipase A2
Phosphadylcholine
L- amino acid oxidase
L-amino acid
Phosphomonoesterase
Oligonucleotides
Molecular
Characteristics
weight
11000- 15000 Simple protein, histidine active site
10000-
Glycoprotein, 2 moles FAD per mole
130000
enzyme, heat unstable
115000
Heat labile, EDTA sensitive, acid
unstable, optimum at pH 9.0
Phosphodiesterase
5`-Mononucletides
100000
5` Nucleotidase
Adenosine
monophosphate
100000
Deoxyribonuclease and
Ribonuclease
DNA and RNA
RNA
Hyaluronidase
Hyaluronic acid
15900
Heat labile Zn 2+ sensitive, EDTA
sensitive, acid unstable, optimum at
pH 8.5
Heat labile Zn 2+ sensitive, EDTA
sensitive, acid unstable, optimum at
pH 8.5
Optimum at pH 5.0
Optimum at pH 7.0 – 9.0 specific
towards pyrimidine nucleotides
Heat labile, optimum at pH 4.6
resembles testicular enzyme
NAD-nucleosidase
NAD
100000
Heat labile, optimum at pH 7.5,
nicotinamide sensitive
Acryamidase
L-Leucine
naphthylamide
100000
Heat labile, SH-enzyme, PCMP
sensitive, optimum at pH 7.5
Endopeptidase
Casein,
hemoglobin
21400-95000
Thrombin like enzyme
Factor X activator
Fibrinogen, BAEE
Factor X
28000-33000
78000
Prothrombin activator
Prothrombin
56000
Glycoprotein, metal (Ca2+, Zn2+),
protease, EDTA sensitive, heat labile,
optimum at pH 8.0-9.0
Glycoprotein, heat stable, DEP
insensitive, EDTA sensitive,
activates also Factor IX
Glycoprotein, heat labile, DEF
insensitive, EDTA sensitive
Factor V activator
Factor V, BAEE
2000
DEF sensitive, heat stable
Acetylcholine esterase
Acetylcholine
12600
Heat labile, DEP sensitive, optimum
at pH 8.0 - 8.5
Phospholipase
Lysolecithin
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Heat stable, optimum at pH 10.0
Table 1. 06: Biological activities of selected SVMPs
SVMP
Activity
References
P-I Class
Atrolysin C
Hemorrhagic
Shannon et al., 1989; Zhang
et al., 1994
Acutolysin A
Hemorrhagic
Gong et al., 1998; Liu et al.,
1999
BapI
Hemorrhagic; myonecrotic; inflammatory
Gutierrez et al., 1995;
Rucavado et al., 1995
Fibrolase
Fibrinolytic
Markland, 1996
HT-2
Hemorrhagic
Mori et al., 1987; Takeya et
al., 1990
Atroxase
Fibrinolytic
Willis and Tu, 1988
LHF-II
Hemorrhage
Sanchez et al., 1991
H2 –Proteinase
Proteolytic; non-hemorrhagic
Takeya et al., 1993
HR2A
Hemorrhagic
Graminelysin I
Apoptotic
Takahashi and Osaka, 1970;
Yamada et al., 1999
Wu et al., 2001
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P-II Class
Atrolysin
Hemorrhagic
Hite et al., 1992; Shimokawa et al.,
1996
MT-d
Proteolytic
Jeon and Kim, 1999
Jerdonitin
Inhibition of platelet aggregation
Chen et al. 2003
Bilitoxin I
Hemorrhagic
Imai et al., 1989; Nikai et al., 2000
P-III Class
Atrolysin A
Hemorrhagic; inhibition of platelet aggregation
Fox and Bjarnason, 1995; Jia et
al., 1997
Catrocollastatin
Inhibition of platelet aggregation
Zhou et al., 1996
Jarahagin
Hemorrhagic; inhibition of platelet aggregation
Paine et al., 1992; Kamiguti et
al., 1996
HF3
HR1a
Hemorrhagic; activation of macrophage
phagocytosis
Hemorrhagic
Assakura et al., 1986; Silva et
al., 2004
Kishimoto and Takahashi, 2002
HR1b
Hemorrhagic
Kishimoto and Takahashi, 2002
Kaouthiagin
Cleavage vWF; inhibition of platelet aggregation
Hamako et al., 1998
VAP1
Apoptotic
Masuda et al., 2000
HV1
Apoptotic
Masuda et al., 2001
Acurhagin
Hemorrhagic; inhibition of platelet aggregation
Wang and Huang, 2002
Ecarin
Berythractivase
Activation of prothrombin
Activation of prothrombin
Nishida et al., 1995
Silva et al., 2003
P-IV
RVV-X
Activation of Factor X
Takeya et al., 1992; Gowda et al., 1994
VLFXA
Activation of Factor X
Siigur et al., 2004
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Table 1. 07: Mechanism of Extravasation by hemorrhagins
Toxin
ACI-1
Species
Mechanism
References
Per rhexis
Ownby et al., 1997
Per rhexis
Ownby et al., 1990
HT-1 and 2
Agkistrodon contortrix
laticinctus
Agkistrodon bilineatus
bilineatus
Critakys rubber rubber
Per rhexis
Obrig et al., 1993
Atrolysin a
Crotalus atrox
Per rhexis
Obrig et al., 1993
Proteinase IV
Crotalus horridus horridus
Per rhexis
Ownby and Green, 1978
Proteinase H
Crotalus adamenteus
Per rhexis
Anderson et al., 1977
HR-1, -2a, -2b
Trimeresurus flavoviridis
Per diapedesis
Ohasaka, 1976
BaH1
Bothrops asper
Per diapedesis
Borkow et al., 1995
Bulitoxin
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Table 1.08: Snake venom proteases affecting hemostasis
Snake venom
Protease (common
name)
Activity
Action
Type
Calloselasma
rhodostoma
Ancrod
Thrombin like
Procoagulant
Serine
Procoagulant
Serine
Procoagulant
Serine
Procoagulant
Serine
Procoagulant
Serine
Procoagulant
Serine
Procoagulant
Serine
Procoagulant
Metallo
enzyme
Bothrops atrox Batroxabin
moojeni
Thrombin like
Agkistrodon
bilineatus
Bilineobin
Thrombin like
Bothrops
jararaca
Bothrombin
Crotalus atrox
Calobin
enzyme
enzyme
Thrombin like
enzyme
Thrombin like
enzyme
Crotalus
adamanteus
Crotalase
Thrombin like
Trimeresurus
flavoviridis
Flavoxobin
Trimeresurus
malabaricus
Malabarin
Echis carinatus
Carinactivase-1
Prothrombin activator
Procoagulant
Metallo
Agkistrodon
halys blomhoffii
Halystase
Kallekrein like enzyme
Procoagulant
Serine
enzyme
Thrombin like
enzyme
Thrombin like
enzyme
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Agkistrodon
contortrix
ACC-C
Protein C activator
Anticoagulant
Serine
Vipera russelli
RVV-V
Factor V activator
Procoagulant
Serine
Vipera russelli
RVV-X
Factor X activator
Procoagulant
Metallo
Vipera labetina
Labetase
Fibrino(geno)lytic
Anticoagulant
Metallo
Crotalus atrox
Atrolysin F
Fibrino(geno)lytic
Anticoagulant
Metallo
Trimeresurus
flavoviridis
Trimerelysin I
Fibrino(geno)lytic
Anticoagulant
Metallo
Trimeresurus
flavoviridis
Trimerelysin II
Fibrino(geno)lytic
Anticoagulant
Metallo
Cerastes cerastes
Cerastase
Plasminogen activator
Anticoagulant
Metallo
Trimeresurus
stejnegeri
TSV-PA
Plasminogen activator
Anticoagulant
Serine
Crotalus atrox
Atroxase
Fibrinogenase,
Plasminogen activator
Anticoagulant
Metallo
Naja kouthia
Kouthiagin
VWF cleaving
Anticoagulant
Metallo
contortrix
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Figure 1. 01: Schematic structures of snake venom metalloproteinases
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Figure 1. 02: Summarizes the multiple roles of snake venom metalloproteinases in the
pathogenesis of local tissue damages
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Figure 1.03: Coagulation cascade and major sites of action by snake venom components
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AIM AND SCOPE
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Aim and scope of the Study
Venom of viperidae snakes are a rich source of novel compounds, which may have
applications in medicine and biochemistry. Envenomation by this snakes are frequently
associated with a complex pathophysiological conditions in which hemorrhage, tissue
necrosis and hemostatic alterations are frequently observed. Trimeresurus malabaricus
belonging to the family of viperidae snakes is endemic to Western Ghats of Indian
subcontinent. Even though exhaustive studies are made with the big-four venomus snakes,
not much work has been done with Indian endemic snakes. Some endemic snakes, which
pose serious threat, are T. malabaricus and H. hypnalae. T. malabaricus bite is not lethal
but it causes hemorrhage, tissue necrosis and hemostatic diorders in the experimental
animal. Based on the toxicity studies it has been found that the venom T. malabaricus is
rich source of proteolytic enzymes. These protease enzymes are responsible for many
pathological activities of the snakebite and it was inhibited by known protease inhibitors.
Nowadays many purified snake venom toxins are used for therapeutic purposes. Since the
venom is not lethal the purified toxins are of immense use therapeutically. In this thesis an
attempt has been made to purification and characterizes the component and may be used as
a biological tool to explore many facets of hemostasis.
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