CHAPTER I GENERAL INTRODUCTION -...

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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

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.

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

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.

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

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

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.

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,

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).

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

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

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.

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

(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

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).

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

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

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).

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).

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

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

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

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

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

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.

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

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

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

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

21400-95000

27000-30000

28000-33000

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

Characteristics Features

Catalyzes the Ca ^ dependent hydrolysis of

2-acyl ester bond in 3-Sn-phosphoglycerides

releasing lysophosphatide and free fatty acids.

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

Class I

A.acutus

Bitis arietans

Bothrops jararaca

Bothrops moojeni

C adamanteus

C.adamanteus

C.atrox

C.b. basiliscus

C. ruber ruber

C.ruber ruber

C.s.scutulatus

Lachesis muta muta

T.flavoviridis

(Okinawa)

T.flavoviridis

(Okinawa)

T.gramineus

T. mucrosquamatus

AaH-II

AaH-III

Protease I

Protease II

LHF-II

References

Ouyang and Huang, 1976, 1977.

Mori et al., 1984; Nikai et al., 1977

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

Bjamason and Tu, 1978; Bjamason et al., 1983

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

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

V.labetina

Cerastes cetastes

Class II

A.acutus

A.bilineatus

B.jararaca

B.jararacussu

B.neuwiedi

Calloselasma

rhodostoma

C.atrox

C.h.horridus

C.r.ruber

V.a.ammodytes

V.berus berus

V.palaestinae

T.flavoviridis

Atractaspis

engaddesis

Class III

A.halys blomhoffti

B.asper

Bitis arietans

B.jararaca

C.adamanteus

C.atrox

Lebetase

Cerastase F-4

Bilitoxin

Bothropsin

Bjussu MP-I

Jarahagin

Proteinase H

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

CivelloeM/., 1983a, b

Mon etal., 1987

Samel and Siigur, 1990

Ovadia, 1978a

Yonahdi etal., 1991

Ovadia, 1987

Oshimae/a/., 1972

Franceschi et al., 2000

Omori-Satoh e/a/., 1995

Omori etal., 1964; Oshima etal, 1972

Assakura et al., 1986

Paine e/a/., 1992

Kurecki and Kress, 1985

Bjamason and Tu, 1978; Bjamason et al. 1988

V.aspis aspis

T.flavoviridis

T.gramineus

Ophiophagus

Hannah

V.a.ammodytes

Class IV

A.h.blomhoffii

L.muta muta

T. mucrosquamatus

Trimeresurus

purpureomaculatus

Hannahtoxin

Mucrotoxin

Hemorrhagin

Omori-Satoh and Sadahiro, 1979

Omori-Satoh and Sadahiro, 1979; Takeya et al.,

Huang e? a/., 1984

Tan and Saifliddin, 1990

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)

Chapter I

Introduction.

iDii^ntigfih

MBMlopratBlVHW

EXiNioitn pnrareor

tigtiiTMiHiiirmais

0-t¥P8l«*ln

j - B S - ' moHlofirDMrMae wHhi

Figure 1.02 Schematic structures of snake venom metallo-proteinases

Bulitoxin

HT-1 and 2

Atrolysin a

Proteinase IV

Proteinase H

HR-l,-2a,-2b

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 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)

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)

6,400 7,077 6,500 7,000 7,500

4,400 4,932 5,035 5,132 8,900 2,504

11,600 6,900 14,000

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 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

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

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

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.

CHAPTER I GENERAL INTRODUCTION -...

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