CHAPTER I
GENERAL INTRODUCTION
Chapter I Introduction...
Introduction
Snakes and the venom of snakes have fascinated the mankind, since time
immemorial. Throughout the course of the history of civilization, venomous snakes have
held a imique and particular fascination for humans. Snakes have provoked deep interest,
curiosity and analogies in many paths of human life: inspiring symbols in religion (the
evil and original sin); science (pharmacy and medicine symbols) and others since ancient
times (Ramos and Araujo, 2005).
Venomous 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 all over 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 three families under the order: Serpentes
(Wuster 1996, 1997, 1998).
1. Viperidae, which includes Russell's viper. Saw scaled viper. Puff adder. Water
moccasins, Copper head. Pit vipers of Asia, European vipers, Gaboon vipers.
Horned viper of Sahara.
The family Viperidae is further classified into two-sub families, Viperinae and
Crotalinae. The subfamily of Viperinae includes Puff adder, Gaboon vipers, Russell's
viper. Homed viper of Sahara, Saw scaled viper, European vipers. The subfamily of
Crotalinae includes Rattlesnakes, Copper head. Water moccasins and Pit vipers of Asia.
2. Elapidae, which includes Cobras, Coral snakes, Mambas and Kraits.
3. Colubroidae, which includes all sea snakes.
In 1965, Deoras has listed about 216 species of snakes that are found in India out
of which 52 are poisonous. However, "Romulus Whitker and Ashok Captain (2004) have
provided a comprehensive list of 275 snakes recorded in the various parts of Indian
subcontinent. Among venomous snakes only four pose threat to human beings as they are
Chapter I Introduction...
found in the vicinity of human settlement, especially in rural areas, which are agricultural
and have rats in abundance. The four venomous snakes are called 'Big Four'-the
Spectacled Cobra, Common Krait, Russell's viper and Saw scaled viper. The distribution
of these big four snakes along with King Cobra in the Indian subcontinent is given in
Table 1.01.
Globally venomous snakebite is estimated to affect greater than 2.5 million
humans annually, of which more than 100,000 will die (Chippaux, 1998). The burden of
morbidity and mortality is greatest in the rural tropics (Lalloo et al., 1995; Laing et al.,
1995; Warrell et al., 1999) but snake bite is not confined to poorer rural tropical areas.
There is evidence that some of the most dangerous venomous snakes are invading urban
areas, putting new groups of humans at significant risk (Melgarejo and Aguiar, 1995;
Revault, 1995).
Snake envenomation
Snake envenomation is basically a subcutaneous or intramuscular injection of
venom into the prey/human victims. It employs three well integrated strategies. Two of
these are prey immobilization strategies and may denominated 'hypotensive' and
'paralytic' strategies. Both serve to limit prey flight, in the case of snake taxa which
strike release and then track their prey (most viperids), or to overcome pre resistance, in
the case of snakes that seize and bulldog their prey (many el^ids and all colubrids). The
third strategy is digestive and commences degradation of prey tissues internally, even
before the prey has been engulfed. Normally, all three strategies operate simultaneously
and individual venom constituents fi-equently participate in more than one of them. Each
of these strategies contains interchangeable mechanisms, elements or sub strategies.
Different venomous snake taxa employ different combinations of mechanisms and no
single species employs them all (Aird, 2002).
Snake venom
Snake venoms contain a multitude of biologically active toxins that work together
for the capture of prey. Their effects include blood coagulation (pro/anti), neurotoxicity
(pre/post synaptic), myotoxicity, nephrotoxicity, cardiotoxicity and necrotoxicity (local
tissue damage), hemorrhagic, edema inducing and possible direct action on vital organs.
Chapter I Introduction...
The proportionate mixing of these biological activities varies considerable from venom to
venom. The variability in venom composition is considered at several levels. The
intergenus, interspecies, intersubspecies, intraspecies, geographical distribution, seasonal
and age dependent change and variation due to sexual dimorphism (Chippaux et al.,
1991). In addition to protein/peptide toxins, snake venom is also composed of organic
and inorganic components such as metal ions like Ca " , Cu^ , Fe " , Mg " , Na " , Zn "
(Markland, 1998) and citrate, non-proteinaceous components include carbohydrates,
lipids, bioactive amines like serotonin, nucleotides and amino acids (Freitas et al., 1992).
Bioactive amines are predominantly found in viperid venoms (Hider et al., 1991; Freitas
et al., 1992). Citrate was identified as the major constituent found in many types of
venom. It is foimd in greater than 5% of dry weight of venom of Crotalous atrox and
Bothrops asper (Freitas et al., 1992). Snake venoms have several enzymes that depend on
metal ions for activity. For example, PLA2 requires Ca and metallo-proteases and
hemorrhagins requires Zn . These enzymes are kept in an inactive form by the chelating
effect of citrate. Other than this, citrate can also act as a buffering agent and also serve as
a negative counter ion for basic proteins and polyamines (Odell et al., 1999).
The biologically active protein and peptide toxins in snake venoms can be either
enzymatic or non-enzymatic in property. Earlier investigators tried to explain all the
biological activities of snake venoms based on the presence of enzymes or combination
of enzymes. However, the initial contributions of several researchers (Weiland and Konz,
1936; Slotta and Frankel-Conrat, 1938; Ghosh et ah, 1941), becomes evident that there
are several non-enzymatic proteins in snake venoms, which possess important biological
activities and cannot be ignored. They are known to induce neurotoxicity (Larsen and
wolf, 1968; Sato et al, 1969), myotoxicity (Ownby et al, 1976; Chang, 1979; Lomonte
and Gutierrez, 1989), cardiotoxicity and platelet aggregation (Kini et al, 1988). Nerve
growth factors (Oda et al, 1989; Kostiza and Meier, 1996) and bradykinin potentiating
peptides are also reported from snake venoms (Ferreira et al, 1970; Ondetti et al, 1971;
Aird, 2002).
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
Chapter I Introduction...
isolated and characterized in detail. A list of enzymes found in snake venom and their
general properties are given in Table 1.02 and 1.03 respectively. The distribution of the
enzymes varies from one snake species to another. Some enzymes like L-amino acid
oxidase, phospholipases, phosphodiesterases are foimd in almost all snake venoms
(Rosenberg, 1979). The remaining enzymes are usually confined to certain taxonomic
groups of snake (Russell, 1980; Iwanaga and Suzuki, 1979). For example viperid venom
contains proteolytic enzymes like endo-peptidases, arginine ester hydrolases, thrombin
like enzymes, kininogenases and procoagulant enzymes, which are not commonly found
in el^id 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.
a) 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)
b) Proteinases and phospholipases are procoagulant or anticoagulant (Jagadeesha et
al., 2002; White, 2005; Lu etal., 2005)
c) Kininogenase release bioactive peptides, which cause acute hypotension
(Markland, 1998).
Most of the snake venoms contain an interguing variety of enzymes. All snake
venoms contain L-amino acid oxidase (Zeller and Maritz, 1944), phosphodiesterase
(Pareira et al., 1971), PLA2 (Rosenberg, 1979). Certain enzymes are characteristic of
only a few species (Iwanaga and Suzuki, 1979; Russell, 1980). Acetylcholine esterase is
characteristic of elapid venoms, which is never found in viperid and crotalid venoms.
Arginine ester hydrolases, endopeptidase, thrombin like enzymes, kininogenase and
procoagulant enzymes are distributed in viperid and crotalid venoms but have not
detected in elapid venoms (Deutsch and Diniz, 1955). The enzymes mainly involved in
various pharmacological activities are L-amino acid oxidase, PLA2, hyaluronidases and
proteases (metallo/serine) listed in the Table 1.04.
Chapter I Introduction...
PhosphoUpase A2
PLA2 (E.C.3.1.1.4) enzymes specifically catalyzes the hydrolysis of fatty acid
ester bond at position 2 of 1, 2, diacyl-Sn-3 phosphoglycerides liberating free fatty acids
and lysophospholipids (Van deewen and Dehaas, 1963). These enzymes are ubiquitous in
nature and are found extra-cellularly and intra-cellularly. Extra-cellular PLA2S are found
in mammalian pancreatic tissue, as well as in snake, bee and scorpion venoms (Van eijk
et al., 1983). High levels of extra-cellular PLA2 enzymes have also been found at
inflamed sites as exudates in some experimental animals and himians with diseases (Hara
et al., 1991). PLA2 enzymes are usually small and stable as compared to most other
proteins. They withstand harsh conditions of extreme temperature and pH. The stability is
probably due to the presence of large number of (6-7) intramolecular disulfide bridges.
The venom PLA2 display molecular masses from 12-16 kDa and consists of 119-143
amino acid residues (Scott and Sigler, 1994). PLA2 have become a super family of
distinct enzymes that have pivotal role in various biological activities.
Among the enzymatic toxins, PLA2 constitutes the major part of the venom toxin
pool and is being attributed to be involved in abnost all the pharmacological effects of
snake venom. They are known to induce various pharmacological effects like
neurotoxicity, myotoxicity, cardiotoxicity, cytotoxicity edema inducing activity, anti
coagulant activity etc. (Kini, 1997). To date, ~280 PLA2S (enzymes/analogs) primary
amino acid sequences have been determined (Danse et al., 1997; Tan et al., 2003). They
share any where between 40-99 % homology in their primary amino acid sequence.
Three-dimensional structures of more than 20 PLAas from snake venoms have been
determined by X-ray crystallographic studies. They share similar three-dimensional
folding but exhibit wide differences in their pharmacological properties (Scott, 1997).
PLA2S have been sub-classified based on a number of parameters. A commonly
used and recently updated classification has been made by Six and Dennis (2000) which
divides PLA2 into ten main type or group based on molecular weight, amino acid
sequence, calcium dependent and cellular origin. It is however convenient to divide PLA2
into two large classes based on size, Ca " requirement and catalytic mechanism. The first
class consists of secretory enzymes that are small extensively disulfide cross-linked
secreted molecules which require millimolar concentration of Ca " for activity. The
Chapter I Introduction...
second class consists of mechanistically distinct large molecular weight enzymes, which
are Ca"*" independent or require micromolar concentration of Ca " for activity.
The PLA2S, so far isolated from snake venoms, all come imder the type I and II.
More than 300 PLA2S have been sequenced. Type I and IIPLA2S are highly homologous
and have similar kinetic behaviour. Both Type I and Type II PLA2's are 13-15 kDa
proteins with similar tertiary structure (Renetseder et al., 1985).
In addition, venom PLA2 enzymes also exhibit species specificity (Harris, 2003).
The neuromuscular blockages induced by crotoxin from Crotalus durissus terrificus,
taipoxin from Oxyuranus scutellatus scutellatus and p-bungarotoxin from Bungarus
multicinctus venom have similar characteristics but their potencies are species dependent.
Taipoxin is three times more potent than P-bungarotoxin and five times more potent than
crotoxin in blocking mouse phrenic nerve diaphragm transmission (Chang et al., 1977;
Howard and Gundersen, 1980). VRV-PL-VI from Daboia russelii (north) causes
hemorrhage in the intraperitonial cavity, pituitary and thyroid glands as well as liver and
kidney (Vishwanath et al, 1988a), on the other hand VRV-PL-VIII (south) causes
hemorrhage in the lungs (Uma and Gowda 2000). The most toxic VRV-PL-V from the
same venom is a presynaptic neurotoxin (Kasturi and Gowda, 1989b).
Hyaluronidase
Hyaluronidase, (E.C.3.2.1.35) an endoglycosidase, has been considered an
invariant factor in the venom of snakes and is frequently referred to as "Spreading
factor". Spreading activity has been evident from its ability to promote the local
hemorrhagic effect of a toxin from Trimeresums flavoviridis venom (Tu and Hendon,
1983). Distortion in the integrity of ECM of local tissue(s) due to degradation of
hyalxironan; dissemination of target specific toxins is presumed to be the critical event in
the enzyme mediated spreading process. Despite the key role played in local effect(s) of
envenomation, the enzyme has been explored exhaustively. It is evident from the limited
number of studies carried out on this enzyme (Xu et al, 1982; Pukrittayakamee et al,
1983; Kudo and Tu, 2001). Recently, Girish et al, (2004) demonsfrated the degradation
of extra-cellular matrix in humans and other tissue samples. This property of the
hyaluronidase is attributed for the fast diffusion of the lethal toxins. The action of
Chapter I Introduction...
hyaluronidase in the degradation of extra-cellular matrix is considered an important step
for assisting toxins inducing local tissue damage. Its toxic effect is due to the synergetic
action associated with the other venom toxins (Girish et al., 2004). Regulation of this
enzyme is highly beneficial in mitigating the venom toxicities. In addition, information
on the existence of isoforms of hyaluronidase in snake venoms is limited. Although,
there are reports on the isolation and characterization of this enzyme from the venom of
snakes such as Agkistrodon acutus (Xu et al, 1982), Daboia russelii (Pukrittayakamee et
al., 1983) Agkistrodon contortrix contortrix (Kudo and Tu, 2001) and the presence of
isoenzymes of hyaluronidase in Naja naja venom (Girish et al., 2004).
Proteolytic enzymes
Proteases (E.C.3.4.21.40) form the major toxin pool in the venom of viperid
family snakes. These proteases are esterases that hydrolase primarily proteins and thus
also serve as a digestive role. Further, they are responsible for most of the local tissue
damage following envenomation. The important proteolytic enzymes reported fi-om the
venom of snakes are: endo-peptidases, peptidases, arginine ester hydrolases,
kininogenases and some of them also possess pro/anticoagulant activities. Proteases with
variety of activities such as coagulant effects (Meume, 1966), hemorrhagic effect (Kini
and Evans, 1992; Matsue et al., 2000) and local tissue necrosis (Markland, 1998; Lu et
al., 2005) have been reported fi'om snake venoms.
Snake venom proteases are heterogeneous group of proteins with a wide range of
molecular masses between 15-380 kDa (Kini and Evans, 1992). Some of them are single
chain proteins (Evans, 1984) and several others are multi-subunits proteins (Zaganelli et
al, 1996; Fry, 1999). More than 150 different proteases have been isolated and about one
third of them structurally characterized. The complete amino acid sequence of about 40
of them have been determined either by protein sequencing or deduced from the
nucleotide sequence of the cDNA.
Chapter I Introduction...
Arginine ester hydrolases
Deutsch and Diniz (1955) first reported the arginine ester hydrolase in snake
venom. Later, several investigators reported the presence of arginine ester hydrolase
activity fi-om both crotalid (White and Gate, 1982; Schwartz et al., 1984 & Silva et al,
1985) and viperid (Samel et al, 1987) venoms. The venoms of Elapidae and
Hydrophidae generally do not show any arginine ester hydrolase activities. The venom of
Ophiophagus hannah and Noja melanoleuca are exceptions, which showed weak activity
towards arginine esters such as N-a-benzoyl-L-arginine ethyl ester (BAEE), /?-tosyl-L-
arginine-methyl ester (TAME), L- a-acetyl tyrosine ethyl ester (ATEE) and N- a-
benzoyl-L-arginine /7-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).
Kininogenases
All crotalid and viperid venoms contain kininogenase (bradykinin releasing
enzyme) activity. This enzyme specifically acts on plasma kininogen and liberates a
physiologically active (vasoactive) nanopeptide, bradykinin. The bradykinin lowers the
blood pressure (Iwanaga and Suzuki, 1979). It also hydrolyzes the synthetic arginine ester
substrates (BAEE, SAME and TAME). Kininogenase has been isolated and purified
fi-om the venom of Bothrops jararaca, Crotalus atrox, Agkistrodon halys blomhoffii,
Echis coloratus, Bitis gabonica (Iwanaga and Suzuki, 1979). Vipera labetina (Siigur et
al, 1982) and Cratalus atrox (Bjamason et al, 1983).
Coagulant activity (Pro/anticoagulant)
Blood coagulation is the result of a series of zymogen activation. Over 30
different substances that affect blood coagulation have been foxmd in the blood and
tissues. Some promote coagulation are called procoagulants and others that inhibit
coagulation are called anticoagulants; whether or not the blood will coagulate depends on
the degree of balance between these two groups of substances. Normally, the
anticoagulants predominate and thus the blood does not coagulate, but at the site of
trauma the activity of procoagulants become much greater than that of anticoagulants,
Chapter I Introduction...
and then clot does develop. Snake venoms contain many biologically active proteins,
which intervene at different steps in blood coagulation (Teng et al, 1985). Snake venom
proteins such as PLA2S, proteases, thrombin-like enzymes, pro-thrombin activators,
factor-V activators, factor-X activators, fibrin(ogen)olytic and factor-X binding proteins
and some non-enzymatic polypeptides are known to interfere in the blood coagulation
processes (Evans et al., 1980; Ouyang et al, 1992; Markland, 1998; Huang et al, 1999;
Matsui et al., 2000; Xu et al, 2001; Koh et al, 2001; Samel et al, 2002).
There are six sites in the blood coagulation cascade where the venom pro-
coagulants can interact.
1. Indirect factor-X activator
2. Activation of factor-X
3. Indirect pro-thrombin activator
4. Activator of factor-V
5. Conversion of pro-thrombin to thrombin
6. Direct conversion of fibrinogen to fibrin (thrombin like activity)
Factor-X activators are either metallo-proteinases or serine proteinases
(Tans and Rosing, 2001). The presence of factor-X activating enzymes have been
reported from both crotalid and viperid venoms (Williams and Esnouf, 1962, Denson et
al, 1972; Amphlett et al, 1982; Hofinann et al, 1983; Teng et al, 1984a; Hemker et al,
1984; Jayanthi, 1987). The mechanism of activation of factor-X by the purified pro-
coagulant protein fi"om Daboia russelii has been shown to be calcium dependent
(Fujikawa et al, 1972; Lindquist et al, 1978; Hofinann et al, 1983), Factor-X activators
fi-om Ophiophagus Hannah and Bungarus fasciatus have been reported to be serine
proteinases.
Factor-V activating enzyme has been identified in the venoms of Vipera aspis
(Boffa and Boffa, 1974). The physiological activator of pro-thrombin is thought to be a
complex of factor-Xa, factor-V, phospholipids and calcium. In addition it can also be
activated by certain snake venoms of the family elapid and viperid (Walker et al, 1980;
Chester and Crawford, 1982). Several factor-V activators have been described fi"om
Bothrops atrox, Vipera russelii, Vipera labetina, Vipera ursine, Naja naja oxiana and
Naja nigricollis venom (Rosing et al, 2001).
Chapter I Introduction...
Prothombin (also known as factor II) is a single chain glycoprotein with a
molecular weight of 72 kDa (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 metallo-proteinases and activate prothrombin efficiently
without cofactors, such as phospholipids (PLs) or cofactor Va. Group B prothrombin
activators are Ca " dependent. They contain two subunits linked non-covalently: a
metallo-proteinase and a C-type lectin like disulfide linked dimmer. Group C
prothrombin activators are serine proteases foimd in Australian Elapids requiring Ca^ ,
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 Ca , negatively charged PL and factor-Va,
Thrombin is a proteinase that activates or inactivates many different factors. It can
also initiates aggregation of blood platelets. Thrombin-like enzymes are widely
distributed in the venoms of the snakes belonging to Crotalidae and Viperidae. These are
glycoproteins with a molecular weight ranging from 29-35 kDa. They are similar to
thrombin in their physical and chemical properties but are not sensitive to thrombin
inhibitors such as heparin, anti-thrombin-III (Aronson, 1976) and soyabean trypsin
inhibitor. Unlike thrombin, thrombin-like enzymes do not activate factor-XIII (Stocker
and Barlow, 1976; Markland and Pirkle, 1977), which stabilizes the fibrin clot dimng
coagulation. The mechanism of fibrinogen clot formation by the venom thrombin-like
enzymes are different from that provoked by thrombin. The venom enzymes
preferentially release only fibrinopeptide A (or B) (Markland and Pirkle, 1977).
Endo-peptidases
Endo-peptidases are mainly found in viperid venoms. A common feature of
venom endo-peptidase is that they are metallo-proteases, capable of hydrolyzing peptide
bonds with amino groups contributed by leucine and phenylalanine residues. Endo-
peptidases can easily be inactivated by EDTA and reducing agent such as cysteine
(Iwanaga and Suzuki, 1979). Venom endo-peptidase catalyzes the hydrolysis of peptide
10
Chapter I Introduction...
bonds of a variety of natural and synthetic substrates, including casein, hemoglobin,
gelatin, elastin, collagen, fibrinogen, insulin, glucagon and bradykinin (Liu and Huang,
1997; Gutierrez et al., 2005).
Endo-peptidases, which exhibit hemorrhagic activity, have been isolated fi-om
several snake 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), Crotalus atrox (Hagihara et al., 1985), Crotalus
ruber ruber (Mori et al., 1987), Bothrops jararacussu (Mazzi et at., 2004), Bothrops
lanceolatus (Stroka et al., 2005) and Trimeresurus malabaricus (Raghavendra et al.,
2006). 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 on basement membrane or indirectly by the release of a tissue
factors which can be responsible for the disruption.
The venom proteases are generally classified by their structure into: (1) Serine
proteases and (2) Metallo-proteinases. There is only a weak or indirect evidence for the
presence of thiol 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, fibrinolysis and platelet
aggregation (Ouyang et al., 1990; Pirkle and Theodor, 1990; Pirkle and Stocker, 1991;
Markland Jr., 1991; Tu, 1996; Pirkle, 1998; Markland Jr., 1998).
Serine proteases
Some of the serine proteases have both fibrinogenolytic and fibrinolytic activities.
But a number of them have only fibrinogenolytic activity and are also called 'thrombin-
like' proteases if they show 'fibrinogen clotting activity' (Pirkle and Theodor, 1990;
Pirkle, 1990; Pirkle, 1991; Markland Jr., 1991, 1998). However, their actions toward
fibrinogen as well as the other substrates of thrombin are not exactly identical to those of
11
Chapter I Introduction...
thrombin. Instead of fibrin(ogen)olytic activity for releasing bradykinin from kininogen is
like mammalian kallikrein (or kininogenase) [Iwanaga, et al., 1976; Bjamason et al.,
1983] and are also called 'kallikrein-like' proteases (Bjamason et al., 1983). In addition,
there have been some reports on the snake venom serine proteases (S VSPs) with a unique
activity, such as the activation of factor-V (Tokunaga et al., 1988), protein C (Kisiel et
al, 1987), plasminogen (Zhang et al, 1995,1997) or platelets (Serrano et al., 1995).
Most SVSPs are glycoprotein showing a variable number of N- or O-
glycosylation sites in sequence. The type and site of glycosylation vary in SVSPs. They
contain twelve cysteine residues, ten of which form five disulfide bonds, based on the
homology with trypsin (Itoh et ah, 1987), the remaining two cysteines form a unique and
conserved bridge among SVSPs, involving Cys245 (chymotrypsinogen numbering),
found in the C-terminal extension (Parry et al., 1998).
Metallo-proteinases
The snake venoms contain a variety of metallo-proteases that are highly toxic
resulting in severe bleeding by interfering with the blood coagulation and hemostatic plug
formation or by degrading the basement membrane or extra-cellular matrix components
of the victims (Iwanaga and Takeya, 1993; Bjamason and Fox, 1994; Marsh, 1994).
More than 100 metallo-proteinases, including the isozymes from the same species, have
been isolated and the amino acid sequences of about 20 enzymes have been determined.
They are all Zn " metallo-proteinases with a Zn^* binding motif of HEXXHXXGXXH
and belong to the metzincin family as well as matrixins such as mammalian matrix
metallo-proteinases (Stocker et al., 1995). Snake venom metallo-proteinases (SVMPs)
are involved in the pathogenesis of local tissue damage, myonecrosis, edema and other
reactions associated with inflammation (Gutierrez et al, 1995b; Rucavado et al, 1998,
2002; Clissa et al, 2001; Costa et al, 2002; Laing et al, 2003). The multiple roles of
SVMPs in the pathogenesis of local tissue damage are summarized in the Fig. 1.01.
Snake Venom Hemorrhagins
Hemorrhage is a serious manifestation of snake bite, causing prolonged and
sometimes permanent disability. Hemorrhage is principally caused by metallo-
proteinases, enzymes that are responsible for degrading proteins of extra-cellular matrix.
12
Chapter I Introduction...
they also have cytotoxic effect on endothelial cells and act on components of the
hemostatic system (Kamiguti et al., 1996). Hemorrhage is a common phenomenon in the
victims of crotalidae and viperidae envenomation (Arnold, 1982). Hemorrhagins (the
term was introduced by Grotto, 1967) act directly on the capillary basement membrane
and the endothelial cells to cause internal hemorrhage. In mild envenomation, 'their
action is limited to the site of the bite' however, in severe cases hemorrhage can be
widespread involving the whole extremity concerned and even organs distant from the
site of the bite, such as heart, limg, kidney, intestine and brain.
Venom induced hemorrhage has been shown to be caused primarily by zmc
dependent proteolytic action of hemorrhagic toxins, capable of degrading extra-cellular
matrix proteins and blood clotting factors (Baramova er a/., 1989,1990; Markland, 1998;
Gutierrez and Rucavado, 2000). Hemorrhagic activity has been associated with
proteolytic activity. Chelation of the zinc atom abolishes both proteolytic and
hemorrhagic effects (Bjamason and Fox, 1988; 1994). Of the 65 hemorrhagic toxins, 12
have been analyzed for their metal content, all of them have been foimd to contain zinc
and many more are inhibited by metal chelators. Ten of the twelve toxins contained
approximately 1 mole of zinc per mole of toxin (Bjamason 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.
Although, hemorrhagins are the main causative agents of hemorrhage, several
other components residing in the crude venom can act 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 imchecked with massive extravasation of RBCs into surrounding tissues, giving rise to
swelling, blistering and edema (Bjamason and Fox, 1994). Some hemorrhagins also
induce pharmacological effects such as myonecrosis (bilitoxin and ba HI),
fibrinogenolysis (Atrolysin f, jarahagin), inhibiton of platelet aggregation (atrolysin a) etc
13
Chapter I Introduction...
(Nikai et al., 1984; Ownby et al, 1990; Kamiguti et al. 1994; Gutierrez et ai, 1995; Jia
e/a/., 1997).
Structure and classification of hemorrhagins
More than 65 hemorrhagins from 24 different species of snakes have been
purified and characterized. Most of these have been found to be metallo-proteinases
(Table 1.05). Snake venom hemorrhagic toxins are zinc dependent metallo-proteinases
which belong to the family of 'metzincins', together with astacins, serralysins, matrix
metallo-proteinases (MMPs) and ADAMs (enzymes with a disintegrin and metallo-
proteinases domains). With few exceptions, these proteinases contain similar zinc binding
motif on their catalytic domain, characterized by the sequence HEXXHXXGXXH,
followed by a Met-tum (Bode et al., 1993).
On the basis of the domain structure, SVMPs have been classified into four main
groups according to the domain constitution: (i) P-I class SVMPs has only a metallo-
proteinase domain apart from the pre- and pro-sequences. Their molecular masses vary
from 20-30 kDa. They exhibit low hemorrhagic activity but with strong direct acting
fibrinogenolytic activity. These are mostly weakly acidic proteins; (ii) P-II class SVMPs
include enzymes presenting the metallo-proteinase domain followed by a disintegrin-like
domain. The molecular mass is 30-60 kDa and their hemorrhagic potency is also low;
(iii) P-III class SVMPs contain a cysteine-rich domain in addition to metallo-proteinase
and disintegrin like domain. The molecular mass is 60-90 kDa, they possess strong
hemorrhagic potency; (iv) P-IV class SVMPs are 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 ranges from 90-120 kDa and they possess very low hemorrhagic
potency (Bjamason and Fox, 1994; Hite et al., 1994). Fig. 1.02 shows the schematic
structures of snake venom metallo-proteinases.
Mechanism of hemorrhage
In hemorrhagic action of metallo-proteinases the enzymatic fiinction is given
primary emphasis that helps in elucidating the factor(s) responsible for the leakage of
14
Chapter I Introduction...
blood from the vessels. Subsequently, it became evident that the enzymatic disruption of
the basement membrane (BM) xmderlying 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 they may facilitate the
process hemorrhage.
A. Enzymatic disruption of basement membrane
Both in vitro biochemical studies as well as in vivo microscopic observations have
confirmed that hemorrhagins cause local hemorrhage by proteolytic digestion of the BM
proteins. Basement membranes are extra-cellular sheets consisting of certain proteins
such as type-IV collagen, laminin, nidogen (entactin), fibronectin and heparan sulfate
proteoglycans (Inoue, 1989; Yurchenco et ai, 1990). BMs, also known as basal lamina,
are placed beneath the epithelia (under capillary endothelium also).
The chief constituent is type-IV collagen, the structxire of which is more flexible
when compared with the fibrillar form. The specialized orientation pattern of these
molecules result in the formation of a basic frame like mesh work 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, nidogen has been found to be highly influenced by
Zn and also highly susceptible to proteolytic degradation, which can allow rapid
disruption of the BM structure. In fact, hemorrhagins can effectively degrade both type-
IV collagen and nidogen. In addition, they can hydrolyze laminin and fibronectin but not
the proteoglycans. These capabilities have made them very effective toxins mediating
disruption of BMs to cause the hemorrhage. In hemorrhagins, such as sfrolysins the
relationship between the hemorrhagic and general proteolytic potency is not always
parallel and may even be inverse. All these observations have necessitated a search for
mechanisms of hemorrhage other than BM degradation. The hemorrhagic potency of the
toxin is related to the action on specific substrates such as BM proteins rather than
general non-specific substrates (Baramova etal., 1989).
15
Chapter I Introduction...
B. Enzymatic disruption of Capillary endothelial cells
Capillaries, with a single cell thick wall, are the main targets of the hemorrhagic
toxins. Exposure of capillaries 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 hyper rhexis (through the cell by disrupting the plasma membrane and
the integrity of the cell) or 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. Mechanism of extravasation by
some of the hemorrhagins are listed in the Table 1.06 (Hati et al, 1999)
Hemorrhage/;£r rhcMS
The pathogenesis of local hemorrhage has been investigated with a number of
purified hemorrhage causing metallo-proteases at the ultra-structural level. In the
majority of the cases,/?er rhexis mechanism has been described in which endothelial cells
of capillary blood vessels become affected rapidly after metallo-proteinases injection.
This is characterized by an initial swelling of cells, followed by formation of
blebs from the luminal plasma membrane that occurs within a short interval of time.
Transmission electron microscopic observations frequently depict swollen mitochondria,
but intercellular junctions remain unaltered. The cells get detached from the substratum
with subsequent to, prior rupture of the plasma membrane, allowing the blood to pass
through the damaged cells into the surrounding tissue space. Capillary basement
membrane, at the same time, gets disorganized and is often wholly or partially absent.
Many larger vessels in most cases have been foimd to be congested with
erythrocytes and platelets. Persistent hemorrhage occurs in capillaries in the form of
extravasated and hemolyzed erythrocytes. Capillaries are also congested with platelets.
With advancement of time they become very obscure due to extensive damage to the
cells. In some capillaries platelets appear outside the lumen, suggesting direct damage to
16
Chapter I Introduction...
the plasma membrane. A large amoimt of intravascular as well as extravascular fibrin is
also present.
The sequence in which endothelial cell damage and BM degradation occurs or
whether both of them occur concomitantly has not yet been determined conclusively.
Apart fi-om direct mechanism of cell damage, some indirect mechanisms have also been
suggested. Rucavado et al. (1995) after studying BaHl and BaPl (Bothrops asper) in
detail, have suggested that extracellular degeneration in vivo is only a secondary event
resulting fi-om disturbance in the interaction between these cells and the surrounding
basement membrane.
Hemorrhage p^r diapedesis
In contrast, the mechanism described earlier suggest that erythrocytes escape
through widened intercellular junctions instead of gaps in endothelial cell cytoplasm
(Ohsaka et al, 1975; 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 micro-vessels examined.
Conflicting results have been also reported concerning the cytotoxic activity of
hemorrhagic metallo-proteinases on endothelial cells. Despite, observations of
endothelial cell pathology in vivo after injection of Bothrops asper venom metallo-
proteinases BaHl and BaPl (Moreira et al, 1994; Lomonte et al, 1994a), these toxins
are shown to be devoid of cytotoxicity on endothelial cells in culture, as judged by the
lack of release of intra-cellular enzymes (Obrig et al, 1993; 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 extra-cellular matrix components.
Such effects were abolished when metallo-proteinases were incubated with chelating
agents that inhibit enzymatic activity (Borkow et al, 1995).
Simultaneously, hemorrhagic toxins devoid of proteolytic activities have also
been reported (Omori et al, 1964; Toom et al, 1969). Vishwanath et al, 1987b purified
and characterized a PLA2 (VRV-PL-VI) from Vipera russelii (North) venom, which
17
Chapter I Introduction...
induced necrosis, hemorrhage in the liver, kidney, pituitary and thyroid glands,
hemorrhage and bleeding in the peritoneal cavity and edema in the footpad of mice. TF-
PL-Ia and TF-PL-Ib from Trimeresurus flavoviridis venom was non-proteolytic but
induced hemorrhagic spot on the iimer surface of the ventrolateral side of the skin at the
site of injection (Vishwanath et al., 1987). VRV-PLV-III from Vipera russelii showed
lung hemorrhage (Kasturi and Gowda, 1989). While metallo-proteases which are devoid
of hemorrhagic activity also reported from Russell's viper {Vipera russelii) and Indian
cobra (Naja naja) venom (Jayanthi et al., 1990; Jagadeesha et al., 2002).
To complicate the situation, hemorrhagins have often been found to exert
additional toxic effects, such as edema, myotoxicity, platelet aggregation and fibrinogen
depletion, which considered as local and systemic manifestations.
Local manifestations
Changes at the site of envenomation are the earliest manifestations of snakebite
(Reid, 1979). Features are noted within 6-8 min but may have onset up to 30 min
(Reddy, 1980; Reid and Theakston, 1983). Localized pain with radiation, tenderness at
the site of bite and the development of small reddish wheal, occur at first. 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 piupuric rash is also seen, most
conmionly at the site of bite with this family of snakes. 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 infections including tetanus and gas gangrene may also
result (Tu, 1991; Philip, 1994).
18
Chapter I Introduction...
Edema inducing activity
Swelling and edema are often the chief early symptoms of snake venom poisoning
at the affected part of the victim. Snakebite leads to increase in the capillary permeability,
which may cause loss of blood and plasma volume into the extra-cellular space.
Accumulation of fluid in the interstitial space is responsible for edema. The edema
induced by Bothrops jararaca venom is mediated by cyclooxygenase and lipoxygenase
eicosanoid products, and by the action of Li 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. This suggested the role of eicosanoids formed in the edema induction
phenomena (Trebian and Calixto, 1989; Campbell, 1990). The venoms of Trimeresurus
flavoviridis (Vishwanath et al., 1987; Yamaguchi et al., 2001), Trimeresurus mucro
squamatus (Teng et ah, 1989; Chiu et al., 1989), Naja 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 on the pathophysiological changes induced
by the venom of that particular species. Elapid venoms produce symptoms as early as 5
minutes (Paul, 1993) or as late as 10 h (Reid, 1979) after bite, whereas vipers take
slightly longer time, the mean duration of onset being 20 min. (Paul, 1993). However,
symptoms may be delayed for several hours. Sea snake bites invariably produce
myotoxic features within 2 h so that they are reliably excluded if no symptoms are
evident within this period (Paul, 1993). The magnitude of systemic toxicity induced by
toxins are directly relay 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 induce any kind of manifestations
(Estevao-Costa et al., 2000; Moura-da-silva et al., 2003).
19
Chapter I Introduction...
Myotoxicity
Myotoxicity is one of the common and a serious consequence of snake venom
poisoning. Local hemorrhage and necrosis affecting the skin and muscle tissues are the
chief manifestations of myotoxicity. Myonecrosis may be due to the vascular
degeneration and ischemia caused by venom metallo-proteinases, or it may result from
the direct action of myotoxins upon the plasma membrane of muscle cells, which is
evident from the rapid release of cytoplasmic markers creatine kinase (CK), creatine
phosphokinase (CPK) and lactate dehydrogenase (LDH) accompanied by the prominent
increase in total muscle calcium ion (Rucavado and Lomonte, 1996; Gutierrez and
Lomonte, 1997; Gopalakrishnakone et al., 1997; Salvini et al., 2001; Souza et al., 2002).
The increased influx of calcium ion leads to the cell death (Mebs and Samejima, 1980).
Myotoxicity is associated with many pre-synaptically acting neurotoxins
(Gopalakrishnakone et al., 1980; Ziolkowske and Bieber, 1992). In addition, several
myonecrotic polypeptides and myotoxic PLAa 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 tt al.,
1997; Radis-Baptista etal, 1999; Nunez et al., 2001).
Intramuscular injection of many hemorrhagic metallo-proteinases results in acute
muscle cell damage, i.e., myonecrosis (Gutierrez et al., 1995; Franceschi et al., 2000).
The mechanism by which venom metallo-proteinases induce muscle damage has not been
fully elucidated. However, Gutierrez et al., (1995), investigating the action of
hemorrhagic metallo-proteinases BaGl from Bothrops asper venom, suggested that
muscle damage was secondary to the ischemia that ensue in skeletal muscle as a
consequence of bleeding.
Platelet aggregation
Platelet aggregation means platelets sticking to each other rather to a different
surface. In the circulating blood, discoidal platelets are considered to be in the resting
state and in this state; they do not readily adhere to any surface. They become sticky
when there is any damage to the vascular endotheliimi. They aggregate into a mass at the
20
Chapter I Introduction...
site of vascular injury and form hemostatic plug, which seals off the break in the blood
vessel.
Platelets become sticky upon stimulation by diverse agonists which includes small
molecular weight compounds such as AD?, arachidonate, serotonin and epinephrine;
enzymes such as thrombin and trypsin; particulate materials, such as collagen and
antigen-antibody complexes; lipids, such as platelet activating factor (PAF-acether) and
ionophores, such as A23187 (Zucker, 1989). Stimulation by these diverse agonists
initiates a series of cellular responses such as adhesion, change in platelets shape from
disc to sphere and release of various substances (Kini and Evans, 1990). Enzymatic and
non-enzymatic platelet aggregating factors have been isolated from so many different
snake venoms (Kini and Evans, 1990 and references therein; Ouyang et al., 1992). Snake
venom components such as PLA2S, hemorrhagic metallo-proteinases, RGD containing
disintegrins, GPI6-binding proteins, lectin-like proteins and antiplatelet polypeptides
(Kamiguti et al., 1998; Kemparaju et al., 1999; Siigur and Siigur, 2000; Jagadeesha et al.,
2002; Wei et al., 2002) are known to interfere in platelet function.
Fibrinogenolytic proteinases
Fibrinogenolytic activity has been described in the venoms of members of the
Viperidae and Elapidae families (Markland Jr., 1991). The substrates for the
fibrinogenolytic enzymes is fibrinogen, appears as large trinodular protein by electro
microscopy. The protein contains two symmetric half-molecules which are disulfide-
linked. Each half contains three chains designated as A-a, B-P and y with molecular
weights of 63.5, 56 and 47 kDa 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.
Most of the enzymes characterized are zinc metallo-proteinases and degrade A-a
chain of fibrinogen preferentially. Serine proteases have specificity toward the B-p chain
21
Chapter I Introduction...
of fibrinogen. However, there are exceptions to these generalizations and specificity for
Aa or BP chains, as there is substantial degradation of alternate chain with time. Most of
the metallo-proteinases are fibrinolytic and many of the serine proteinases are both
fibrinogenolytic and fibrinolytic (Brand et al., 2000). Fibrinogenolytic metallo-proteinase
cleave amino-terminal to hydrophobic amino acids, while fibrinogenolytic serine protease
cleave carboxy-terminal to basic amino acids.
Non-enzymatic venom components
Most of the deleterious pharmacological effects of snake venoms are attributed to
enzymes present in the venom. However, the polypeptides without enzyme activity
contribute significantly towards the lethal potency and also elaborate several
pharmacological properties of the venom. Several polypeptides, possessing important
pharmacological properties have been identified, purified and characterized fi-om
different snake venoms (Tablel.07). So far more than 100 non-enzymatic proteins have
been characterized, and these protein toxins are grouped into well recognized families as
follows: 1) three-finger toxins (includes neurotoxins and cardiotoxins), 2) serine protease
inhibitors, 3) lectins, 4) sarafatoxins, 5) nerve growth factors, 6) atrial natriuretic
peptides, 7) bradykinin-potentiating peptides, 8) disintegrins, and 9) helveprins/CRISP
(Mebs and Claus,1991; Kini, R. M. 2002 ; McLane, et al, 1998).
In addition to independently acting toxic peptides, there are reports of non-
enzymatic non-toxic peptides that interact synergistically with PLA2S or other venom
proteins and thereby enhance the toxicity of interacting protein (Jayanthi and Gowda,
1990). Other important non-enzymatic components are inhibitors of acetylcholine
esterase, phospholipase and proteases.
Enzyme inhibitors from snake venoms
Inhibitors of enzymes such as acetylcholine esterase, phospholipase and proteases
have been reported fi-om several snake venoms.
Naturally occurring phospholipase-inhibitor complexes have been shown to be
present in the venom of both Elapidae and Crotalidae (Braganca et al., 1970; Vidal and
22
Chapter I Introduction...
Stoppani, 1971; Breithaupt, 1976; Simon and Bdolah, 1980). Rudrammaji, 1994 has
reported the isolation and characterization of PLA2 inhibitors from the venom of Indian
Cobra, Naja naja naja. These inhibitors are small peptides and appear to have ionic
interaction with phospholipases.
Potent inhibitors of serine proteases have been detected in venoms such as Vipera
russelii (Takahashi et al., 1974), Naja nivea, Haemachatus haemachatus (Hokoma et al.,
1976), Bungarus fasciatus (Liu et al, 1983). These inhibitors are low molecular weight
basic polypeptides and are homologous to bovine basic pancreatic trypsin inhibitor
(BPTT).
Inhibitors of carboxypeptidase (angiotension converting enzyme or bradykinin)
have been reported from Bothrops jararaca venom. These inhibitors inhibit the
bradykininase activity and potentiate the activity of the peptide hormone bradykinin.
Hence they are named as Bradykinin potentiating factors (BPF) [Greene, 1974].
The inhibitors of thrombin like enzyme factor-X activating enzyme and
prothrombin activator have been reported from the venoms of Agkistrodon halys
blomhoffii, Bothrops jararaca and Ophiophagus scutellatus scutellatus respectively
(Ohshima et al., 1969; Walker et al., 1980)
Inhibitors of hemorrhagins (antihemorrhagins) foimd in Crotalus atrox were
characterized as acidic glycoproteins with a molecular weight ranging from 65 to 80 kDa
(Weissenberg et al., 1991). Omori-Satoh et al., 1972 isolated an anti hemorrhagic factor
from the serum of the habu snake, Trimeresurus flavoviridis. The purified serum factor
inhibited two immonologically distinct hemorrhagic principles, HRi and HR2 in the
venom.
Application of snake venom components
The wide range of activities of snake venom proteins on human physiological
system has provoked researchers to look for potential use of venom components. Snake
venom proteins are used for the treatment of pathological conditions such as leprosy,
epilepsy, chronic pains of the nervous system, cancer, neuritis, migraine, neuralgia,
arthralgia, tuberculosis etc. Some components are used as therapeutic agents while others
23
Chapter I Introduction...
have served as research tools to understand some of the physiological functions. Snake
venoms are rich source of enzymes, many of which like phosphodiesterases, LAO,
thrombin like enzymes are purified and sold commercially. Post-synaptic neurotoxins act
as antagonists to acetyl choline. They have been used to quantitative determination of
acetyl choline receptors (ACHR) in neuromuscular junctions. Cobra venom factor (CVF)
has found application during organ transplantation due to its anti-compliment factors.
Nerve growth factor (NGF) stimulates the growth of nerve fibers in vitro.
Fibrinogenolytic enzymes of venoms are used to dissolve blood clots without causing
hemorrhage. PLA2 is also being used for preparation of lyso-phospholipids, as catalysts
to synthesize phospholipids and in studies on membrane asymmetry.
Treatment of snake bite
Snake venom poisoning is a serious medical, social and economic problem in
many tropical countries especially in Africa, South America and Southeast Asia including
India. Snake venom poisoning can simultaneously, sequentially and disjimctively exert
toxic and lethal effects on local tissues, blood, cardiovascular, respiratory and nervous
system (Ohsaka, 1979); hence it is a medical emergency. There are impressive number of
stories about the treatment of snakebite in mythology and folklore. All of them display
similar themes in different cultures over the span of centuries (Russell, 1980). Though,
these methods have historical value none of them are effective in curing snake venom
poisoning.
Botanical cure is the most popular and widely used of all folklore remedies for
snakebite. The qjplication of various plants, most often the root extract, either in the form
of poultice to the bitten region or orally is still in practice. A large number of plants
(Morton, 1981; Duke, 1985; Mors et al., 2000) and its components are claimed to
antagonize the action of snake venoms. In India, the plants that have been used for the
snakebite include Acalypha indica, Achyranthus aspera, Achyranthus superba. Capsicum
annuum. Datura fastuosa, Strychnos colubrine, Rauwolfia serpentina, Hemidesmus
indicus, Aristolochia radix. Mimosa pudica, Withania somnifera and Tamarindus indica
(Chopra e/a/., 1958; Nadkami, 1976; Sathyavathi etal, 1976; Gowda, 1997; Alam and
24
Chapter I Introduction...
Gomes, 1998; Mahanta and Mukherjee, 2001; Deepa and Gowda, 2002; Ushanandini et
al., 2006). Even now attempts are continuously being made in this direction for
neutralization of venom toxicity using active principles isolated from various plants.
The mortality due to snake venom poisoning is reduced markedly by the use of
antivenoms, which are the most useful and best remedy available until today for
snakebite treatment (Gutierrez et al., 1985; Lomonte et al., 1996; Leon et al., 1997;
Gutierrez et al., 1998; Leon et al., 1999; Leon et al., 2000; Rucavado et al., 2000). Since
Calmette prepared cobra antivenom in 1884 (See, Grasset, 1957), antisera against various
kinds of snake venoms have been prepared and their effectiveness in treatment of snake
venom poisoning has been widely accepted. Monovalent (prepared against single species
of snake venom) and polyvalent (prepared against mixture of selected species of snake
venoms) antivenoms are produced commercially by several laboratories all over the
world (Theakston and Warrell, 1991). Though the mortality due to snake venom
poisoning is reduced markedly by the use of anti-venoms, there are several inherent
drawbacks associated with it, some of them are; their limited availability, specificity,
storage, dosage, solubility and sensitivity of individuals towards antivenoms. Excess
infusion of anti-venom increases the potential risk of serum sickness, which can lead to
arthritis, vasculitis and nephritis.
25
Chapter I Introduction.
Common name
Indian Spectacled Cobra
King Cobra
Common icrait
Russell's viper
Saw-scaled viper
Scientific name
Naja naja naja
Ophiophagus hannah
Bungarus caeruleus
Vipera russelii
Echis carinatus
Family
Elapidae
Elapidae
Elapidae
Viperidae
Viperidae
Distribution
Throughout India, sea level up to 4000 m (in the Himalayas) Confined to the dense forests of the Western Ghats and the Northern hill forests. Himalayan foot hills (up to 2000 m). Forests of Assam, Orissa, Bihar, West Bengal and the Andamans.
Throughout India, sea level up to 1700 m.
Hills and plains throughout India Up to 3,000 m.
Throughout India, sea level up to 2000 m.
Table 1.01 Distribution of Big Four snakes in India
Enzymes found in all venoms Phospholipase A2 Phosphodiesterase Phosphomonoesterase L-amino acid oxidase 5' Nucleotidase
Enzymes found mainly in Viperid venoms Endopeptidase Arginine ester hydrolase Factor X activator
Enzymes found mainly in Elapid venoms Acetylcholinesterase Glycerophosphatase
Enzymes found in some venoms Glutamate-pyruvate transaminase Amylase Heparin like enzyme
Deoxyribonuclease Adenosine triphosphatase NAD nucleotidase Ribonuclease Hyaluronidase
Kininogenase Thrombin like enzyme Prothrombin activator
Phospholipase B
Catalase Lactate dehydrogenase
Table 1.02 Enzymatic proteins found in snake venoms
26
Chapter I Introduction.
Trivial name Phospholipase A2
L- amino acid oxidase Phosphodiesterase
5'-Nucleotidase
Phosphomonoesteras e
Deoxyribonuclease Ribonuclease Hyaluronidase
NAD-nucleosidase
Arylamidase
Endopeptidase
Arginine ester hydrolase Kininogenase
Thromobin-like enzyme Factor X activator
Prothrombin activator
Factor V activator Acetylcholine-esterase Phospholipase B
Trivial name Phosphatidylcholine
L-amino acid
Oligonucleotides
5'-Mononucleotides
p-Nitrophenyl-phosphate
DNA RNA Hyaluronan
NAD
L-Leucine napthylamide Casein, Hemoglobin
BAEE, TAME
Plasma kininogen, BAEE, Fibrinogen, BAEE Factor X
Prothrombin
Factor V, BAEE Acetylcholine
Lysolecithin
M.Wt (Daltons) 11000-15000
100000-130000
115000
100000
100000
15900
100000
100000
21400-95000
27000-30000
33500
28000-33000
78000
56000
20000 126000
Characteristics Simple protein, histidine at active site
Glycoprotein, 2 moles FAD per mole enzyme, heat labile Heat labile, EDTA sensitive, acid unstable, optimum at pH 9 Heat labile, EDTA sensitive, acid unstable, optimum at pH 8.5-9.0 Heat labile, EDTA sensitive, acid unstable, optimum at pH 8.5-9.0 Optimum at pH 7 - 9, specific towards pyrimidine nucleotides Heat labile. Optimum at pH 5-6, resembles testicular enzyme Heat labile, optimum at pH 7.5, nicotinamide sensitive Heat labile, SH-enzyme, PCMB sensitive, optimimi at pH 8.5 Glycoprotein, metal (Ca^*, Zn " protease, EDTA sensitive, heat labile, optimum at pH 8-9 Glycoprotein, heat stable, DFP sensitive, optimum at pH 8-9 Heat stable, DFP sensitive, specific towards kininogen
Glycoprotein, heat stable DFP sensitive Glycoprotein, heat labile, DFP insensitive, EDTA sensitive, activates Factor DC. Glycoprotein, heat labile, DFP insensitive, EDTA sensitive. DFP sensitive, heat stable Heat labile, DFP sensitive, optimum at pH 8.0-8.5 Heat stable, optimum at pH 10
Table 1.03 Some of the properties of enzymes found in snake venoms
27
Chapter I Introduction.
Characteristics Features
PLA2
Catalyzes the Ca ^ dependent hydrolysis of
2-acyl ester bond in 3-Sn-phosphoglycerides
releasing lysophosphatide and free fatty acids.
LAO
Glycoprotein, 2 moles FAD per mole
enzyme, heat unstable, releases hydrogen
peroxide
Serine protease
Most SVSPs are glycoproteins, the kind and
site of glycosylation differ from one SVSPs
to other. They contain twelve cysteine
residues
Metallo-proteinase
They belongs to Reprolysin subfamily, They
are zinc-dependent metallo-proteinases
Hyaluronidase
Endoglycosidase, hydrolytic and ubiquitous
enzyme. It catalyzes the cleavage of internal
glycosidic bonds of ceratin acid
mucopolysaccharides
Pharmacological activities
Neurotoxicity (post/pre), myotoxicity.
Hemorrhage, hypotensive, cardiotoxic, platelet
aggregation, edema inducing and convulsant.
Exact role is not known, but it possesses
antibacterial, apoptosis, anti-platelet activities.
They act on specific factors of the coagulation
cascade systems to cause imbalance of the
haemostatic system of the prey.
They are known to induce hemorrhage,
myonecrosis and inflammatory reactions
They are spreading factors, which promote
local tissue damage
Table 1.04 Major snake venom proteins and their function
28
Chapter I Introduction.
Class I
A.acutus
A.acutus
A.acutus
A.acutus
A.acutus
A.acutus
A.acutus
Bitis arietans
Bothrops jararaca
Bothrops moojeni
C adamanteus
C.adamanteus
C.atrox
C.atrox
C.atrox
C.atrox
C.b. basiliscus
C.b. basiliscus
C. ruber ruber
C.ruber ruber
C.s.scutulatus
Lachesis muta muta
T.flavoviridis
T.flavoviridis
T.flavoviridis
(Okinawa)
T.flavoviridis
(Okinawa)
T.gramineus
T. mucrosquamatus
T. mucrosquamatus
Toxin
EP
Ac-1
Ac-2
Ac-5
AaH-I
AaH-II
AaH-III
HT-1
HF-1
MP-A
Protease I
Protease II
HT-b
HT-c
HT-d
HT-e
B-1
B-2
HT-2
HT-3
P-13
LHF-II
HR-2a
HR-2b
HR-2a
HR-2b
HR-1
HR-a
HR-b
M.Wt
(kDa)
24
24.5
25
24
22
22
22
ND
ND
22.5
24.6
23.7
24
24
24
25.7
27
27.5
24
25
27
23.5
23
24
24
19
24
15
27
MHD
(fig)
3.81
0.22
0.43
0.37
0.4
1.5
10.0
-
0.1
ND
ND
ND
3
8
11
1
<10
<10
0.3
1.4
1.2
16.2
0.07
0.07
-
-
-
1.7
1.3
References
Ouyang and Huang, 1976, 1977.
Mori et al., 1984; Nikai et al., 1977
Mori etal., 1984
Mori etal., 1984
Xue/a/., 1981
XueM/., 1981
Xuetal., 1981
Mebs and Panholzer, 1980
Assakura er a/., 1986
Assakura et al., 1985
Kurecki and Kress, 1985
Kurecki and Kress, 1985
Bjamason and Tu, 1978; Bjamason et al., 1983
Bjamason and Tu, 1978; Shannon et al., 1989
Bjamason and Tu, 1978; Shannon et al., 1989
Bjamason and Tu, 1978; Kite et al., 1992a
Molina ef a/., 1990
Molina e/a/., 1990
Mori et al., 1987; Takeya et al., 1990b
Mori et al., 1987; Takeya et al., 1990b
Martinez et al., 1990
Sanchez e a/., 1991
Takahashi and Ohsaka, 1980; Nikai et al., 1987
Takahashi and Ohsaka, 1980; Nikai et al., 1987;
Yonaha etal., 1991
Yonahae/a/., 1991
Ouyang and Shiau, 1970
Nikai e/fl/., 1985b
mkai etal., 1985b
29
Chapter I Introduction.
V.labetina
Cerastes cetastes
Class II
A.acutus
A.acutus
A.bilineatus
B.jararaca
B.jararacussu
B.neuwiedi
B.neuwiedi
Calloselasma
rhodostoma
C.atrox
C.atrox
C.h.horridus
C.r.ruber
V.a.ammodytes
V.a.ammodytes
V.a.ammodytes
V.berus berus
V.palaestinae
V.palaestinae
V.palaestinae
T.flavoviridis
Atractaspis
engaddesis
Class III
A.halys blomhoffti
B.asper
Bitis arietans
B.jararaca
B.jararaca
B.jararaca
C.adamanteus
C.atrox
Lebetase
Cerastase F-4
Ac-3
Ac-4
Bilitoxin
Bothropsin
Bjussu MP-I
NHF-a
NFF-b
HP-I
HT-f
HT-g
HP-rv
HT-1
HT-1
HT-2
HT-3
HMP
HR-1
HR-2
HR-3
HR-1
HT-1
HR-1
BaH4
BHRa
BHRb
HF-2
HF-3
Jarahagin
Proteinase H
HT-a
23.7
22.5
57
33
48
48
60
46
58
38
64
60
57
60
60
60
60
56.3
60
60
60
46
50
85
69
68
75
50
62
52
85.7
68
-
200
0.95
0.31
-
-
4.0
_
_
_
0.5
1.4
4.0
0.2
4.0
0.2
0.2
0.4
0.2
0.03
2.0
0.10
0.02
0.02
0.015
0.02
0.04
0.11
Siigur and Siigur, 1991
Daoudefa/., 1986a,b
Moii etal., 1984
Mode/a/., 1984
Imai et al., 1989
Mandelbaum e/a/., 1982
Mazzi et al., 2004
Mandelbaum e/a/., 1984
Mandelbaum et al., 1984
Bandoe/a/., 1991
Nikai et al., 1984
Nikai et al., 1984
CivelloeM/., 1983a, b
Mon etal., 1987
Samel and Siigur, 1990
Ovadia, 1978a
Ovadia, 1978a
Ovadia, 1978a
Yonahdi etal., 1991
Ovadia, 1987
Oshimae/a/., 1972
Franceschi et al., 2000
Omori-Satoh e/a/., 1995
Omori-Satoh e/a/., 1995
Omori etal., 1964; Oshima etal, 1972
Assakura et al., 1986
Assakura et al., 1986
Paine e/a/., 1992
Kurecki and Kress, 1985
Bjamason and Tu, 1978; Bjamason et al. 1988
30
Chapter I Introduction...
V.aspis aspis
T.flavoviridis
T.flavoviridis
T.gramineus
Ophiophagus
Hannah
V.a.ammodytes
Class IV
A.h.blomhoffii
L.muta muta
T. mucrosquamatus
Trimeresurus
purpureomaculatus
HT-1
HR-IA
HR-IB
HR-2
Hannahtoxin
VaHl
VaH2
HR-II
LHF-I
Mucrotoxin
Hemorrhagin
67
60
60
82
66
70
70
95
94
72
0.02
0.016
0.010
-
0.7
>1
>1
0.1
0.6
2.3
-
Omori-Satoh and Sadahiro, 1979
Omori-Satoh and Sadahiro, 1979; Takeya et al.,
1990
Huang e? a/., 1984
Tan and Saifliddin, 1990
Leonard! era/., 2001
Leonard! era/., 2001
Omori et al., 1964; Satake et al., 1963; Iwanaga
etal., 1965; Oshima etal., 1968, 1972
Sanchez er a/., 1987
Sugihara er a/., 1983
Khow et al., 2002
Tablet.05 List and proposed Classification of known Hemorrhagic Toxins isolated from snake venom
SNAKE VENOM METALLOPROTEINASES
Oogndaiion of MoodclotUng
(•ctort
ExtractHular nurtifx
dtgndition
Figure 1.01 Summary of the multiple roles of snake venom metallo-proteinases in the pathogenesis of
local tissue damages (Gutierrez and Rucavado, 2000)
31
Chapter I
m^^!^
Introduction.
P4
iDii^ntigfih
MBMlopratBlVHW
?'\\
Nil
R6&
EXiNioitn pnrareor
tigtiiTMiHiiirmais
p-rv
0-t¥P8l«*ln
j - B S - ' moHlofirDMrMae wHhi
Figure 1.02 Schematic structures of snake venom metallo-proteinases
Toxin
ACI-l
Bulitoxin
HT-1 and 2
Atrolysin a
Proteinase IV
Proteinase H
HR-l,-2a,-2b
BaHl
Species
Agkistrodon contortrix laticincttts Agkistrodon bilineatus bilineatus
Crotalus ruber ruber
Crotalus atrox
Crotalus horridus horridus
Crotalus adamenteus
Trimeresurus flavoviridis
Bothrops asper
Mechanism
Per rhexis
Per rhexis
Per rhexis
Per rhexis
Per rhexis
Per rhexis
Per diapedesis
Per diapedesis
References
Ovmby et al., 1997
Ownbyero/., 1990
Obng etal., 1993
Obrigera/., 1993
Ownby and Geren, 1978 Anderson et al., 1977
Ohasaka, 1976
Borkowe/o/., 1995
Table 1.06 Mechanism of Extravasation by hemorrhagins (Hati etal., 1999)
32
Chapter I Introduction.
Non enzymatic toxins
Elapidae
Neurotoxin (toxin a)
Cobramine A and Cobramine B Dendrotoxin (DTX) a-Neurotoxin, B.F.III Cardiotoxin Muscarinic toxin (MTxs) Phospholipase Inhibitor (NN-I3) Nawaprin Viperidae:
Crotalinae Crotamine
Myotoxin a Peptide C Myotoxin CAM-toxin Wagleri toxin Lethal peptide I
Viperinae Neurotoxin Trypsin Inhibitor (Tj) Ammodytin L (AMDL) Hydropliidae
Erubutoxin a
Erubutoxin b
Neurotoxic peptide
M.Wt (IcDa)
6,787
6,400 7,077 6,500 7,000 7,500
6,500
4,900
4,400 4,932 5,035 5,132 8,900 2,504
11,600 6,900 14,000
6,760
6,780
6,520
Snalce species
Naja nigricollis
Naja naja Dendroaspls angusticeps Bungarus fasciatus Naja nigricollis Dendroaspis angusticeps
Naja naja naja
Naja nigricollis
Crotalus durissus terrificus Crotalus viridis viridis Crotalus viridis helleri Crotalus viridis concolor Crotalus adamanteus Trimeresurus wagleri Trimeresurus wagleri
Vipera palaestinae Vipera russelii Vipera ammodytes
Laticauda semifasciata
Laticauda semifasciata
Laticauda laticaudata Laticauda colubrine
References
Karlssone/a/., (1966)
Larsen and Wolff (1968) Harvey and Karlsson (1980) Jie/a/.,(1983) Kini era/., (1989) Ademefa/., (1988)
Rudrammaji and Gowda (1998)
Torres, e/a/., (2003)
Laure, (1975)
Ownbyefa/.,(1976) Maedae/a/.,(1978) Biebere/a/., (1983) Samejimae/a/., (1988) Tan and Tan, (1989) Weinsteine/a/., (1991)
Morazefa/., (1967). Jayanthi and Gowda (1990). Krizaje/a/.,(1991).
Tamiyae/a/.,(1967)
Tamiya et al., (1967)
Sato ef a/., (1969)
Table 1.07 Non-enzymatic toxic proteins/ peptides found in snake venoms
33
Chapter I Introduction...
AIM AND SCOPE OF THE INVESTIGATION
Snake venom is a complex mixture of protein and peptide toxins with diverse
pharmacological activities. Snake bite is a subcutaneous/intramuscular injection into the
prey/human victim. The pathophysiology of envenomation includes both local and
systemic effects, which include hemorrhage, edema, myotoxicity, neurotoxicity,
cardiotoxicity, coagulant (pro/anti), hemostatic (activating/inhibiting) effects (Kini, 1997,
Shashidharamurthy et al., 2002, Aird, 2002). With some exceptions, snakes of the
viperidae family induce envenomation characterized by hemorrhage, in severe cases the
systemic hemorrhage (Tan and Ponnudurai, 1990; Mebs and Langeluddeke, 1992).
Venom enzymes such as hemorrhagic metallo-proteases, myotoxins
(enzymatic/non-enzymatic) and hyaluronidases are found to be the key toxins which are
responsible for causing local tissue damage and systemic toxicity as well (Gutierrez and
Lomonte, 1989; 1997; Gopalakrishnakone et al., 1997; Escalante et al., 2000; Anai et al.,
2002; Girish et al., 2002). However, snake venom do vary greatly with respect to the
relative abundance of these agents viz., myotoxic PLA2 and cardiotoxins are predominant
in cobra venom, while hemorrhagic metallo-proteases are rich in viper venoms
(Yingprasertchai et al., 2003).
Venom hemorrhagic metallo-proteinases provoke rapid spreading of venom
components from the injected area into systemic circulation, as well as causing local
tissue damage. Although the precise mechanism remains unclear, it is likely that the
venom components are easily differed to the tissue and absorbed into vessels by the
degradation of extra cellular matrix and vascular basement membrane by venom
hemorrhagic metallo-proteinases. Most of the snake venom hemorrhagic metallo-
proteinases are Zn " dependent metallo-proteinases, which is a characteristic of viperid
snake venoms. These hemorrhagic metallo-proteinases may play key roles in the
pathogenesis of both local and systemic complications resulting from snake
envenomation and therefore, may become the target molecule for study (Franceschi et al.,
2000).
Among the viperidae family Russell's viper {Daboia russelii) is one of the most
conmion poisonous snakes of India responsible for several thousand deaths and also
34
Chapter I Introduction...
perhaps, more people suffer from myonecrosis and hemorrhage due to its bite. A striking
geographical variation is observed in the composition of Russell's viper venom as well as
in the clinical manifestation by its bite.
Variation in the composition of Vipera russelii venom samples obtained from
northern, eastern, western and southern parts of India has been reported (Prasad et al.,
1999a). Venom samples from eastern region is characterized by high lethal potency,
hemorrhagic and proteolytic activities but less PLA2 activity compare to other regions.
The bite by Russell's viper in this region induces spontaneous hemorrhage (Chakrabarty
et al., 2000; Madhu kumar and Gowda, 2006).
Hemorrhagic metallo-proteases are the main contributors towards hemorrhagic
activity of the Russell's viper venom. Protease hemorrhagins like R W X and AE-II
(Jayanthi, 1987) and VRH-I (Chakrabarty et al., 1993) were purified from southern and
eastern regional Vipera russelii venoms respectively. No reports are available except
Uma, 1999 (Ph.D Thesis) for the presence of hemorrhagic complex in the eastern Daboia
russelii venom. Even the presence of weakly acidic/neutral PLA2 complex and its iso-
forms are not reported in the same venom till now.
In view of the above facts, following study was undertaken
i) Isolation and characterization of hemorrhagic complex (HC) from the eastern region
D. russelii venom
ii) Neufralization of pharmacological properties of HC and D.russelii venom against
antibody/antiserum raised (HC-IgG).
iii) Purification and characterization of weakly acidic/neutral PLA2 complex
(VRV-PL-II) and its three iso-forms VRV-PL-IIa, VRV-PL-IIb, VRV-PL-IIc.
D. russelii (east) venom is not abundant in systemic toxins but contains high
concenfration of hydrolytic enzymes, peptides and PLA2 enzymes. These enzymes are
responsible for many pathological activities of the snake bite. Nowadays many purified
snake venom toxins are used for therapeutic purposes. Thus the contribution of
hemorrhagins and PLA2 enzymes to the lethal potency and pharmacological properties of
the whole venom poisoning is not well known. Hence in the present study an attempt is
35
Chapter I Introduction...
made to isolate and characterize the HC and PLA2 iso-forms from D.russelii (east) venom
with a view to understand 1) the contribution of HC to the venom toxicity 2) to study the
effect of polyclonal antibodies/antiserum prepared against HC and whole venom on the
biological properties 3) the contribution of PLA2 enzymes to the lethal potency and
pharmacological properties of the whole venom.
36