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Snakes

Snakes fascinate. They repel. Some pose a danger. Most are harmless. And whether

they are seen as slimy creatures or colorful curiosities, snakes play important

environmental roles in the fragile ecosystems of the nation's wildlife areas.

Snakes have found a place in religion and rituals as a symbol of worship. But, our first

reaction on seeing a snake varies from panic, shock, to intense fear and thought to

exterminate it. Most of the fear about snakes prevails due to the presence of venom. Snakes

and their venoms have fascinated mankind since time immemorial.

Snakes are elongated, limbless, flexible reptiles. Their body shape depends on the

habitat in which they live. Aquatic snakes usually have a flattened body; those living in

trees are long and slender with a prehensile tail while burrowing snakes tend to be compact.

Snakes diet includs termites, rodents, birds, frogs, small deer and other reptiles. To keep

prey from escaping, snakes have rear- facing teeth that hold their prey in their mouths.

Venomous snakes inject their prey with venom, while constrictors squeeze their prey.

Snakes are first thought to have evolved some 100–150 million years ago.

Biologically, these ‘‘limbless tetrapods’’ are highly spec ialised and remarkably diverse,

inhabiting all major ecosystems outside of the polar regions (they are not found in Arctic,

New Zealand and Ireland (Deoras, 1965)) and representing the most common predators of

other vertebrates (Green, 1997). Modern snakes are divided into three superfamilies, the

Scolecophidia, the Henophidia and the Caenophidia. The Scolecophidia are all burrowing

snakes with primitive characteristics such as the presence of pelvic vestiges (Jacob et al.,

1998). The Henophidia contains several families that show a transition from primitive

forms, represented by the family Aniliidae, which still possess pelvic vestiges, to the more

advanced Achrochordidae that are more similar to the Caenophidia. The most highly

evolved snakes are represented in the Caenophidia superfamily; these include the families

Colubridae, Elapidae and Viperidae from whose ranks all the venomous species arise

(Heise et al., 1995; Vidal and Hedges, 2002; Fry et al., 2006).

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Venomous snakes

Venomous snakes possess one of the most sophisticated integrated weapons systems

in natural world. They make upto >80% of the ~2900 species of snake currently described

(Green 1997, Vidal 2002). Snake venom gland evolved a single time, at the base of the

colubrid radiation, 60-80 million years ago, with extensive subsequent “evolutionary

tinkering” (Vidal and Hedges 2002; Fry and Wuster 2004).

Snakes are highly evolved reptiles belonging to the phylum: Chordata, order:

Squamata and sub order: Serpentes. Among 2900 species of snakes have been identified

allover the world, 400 species of snakes are known to be venomous (Russell and Brodie,

1974; Philip, 1994, Fry, 2005). Based on their morphological characteristics like

arrangement of scales, dentition, osteology and sensory organs, these venomous snakes are

classified into different families. As per the recent update of classification, venomous

snakes have been grouped into three families under the order: Serpents (Wuster and

Harvey, 1996; Wuster et al., 1997; Fry, 2005).

1.Elapidae; which includes Cobras, Coral snakes, Mambas and Kraits.

2.Hydrophidae; which includes all sea snakes.

3.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, Goboon vipers, Russell’s

viper, Horned vipers of Sahara, Saw-scaled viper and European vipers. The subfamily of

Crotalinae includes Rattlesnakes, Copper head, Water moccasins and Pit viper of Asia.

No information is available regarding Indian snakes and their ecological

distribution. After Malcolm Smith’s contribution regarding Serpentes as the third volume

to the Fauna of British India in 1943, no major work on the subject has come out. Deoras

in 1965 has listed about 216 species of snakes out of which 52 are poisonous. Recently

"Romulus Whitaker and Ashok Captain (2004) have now provided a comprehensive list of

275 snakes recorded in the various parts of Indian subcontinent. 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

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and Saw-scaled viper. Apart from these snakes the other venomous snakes that strike

human beings are Banded krait (Bungarus fasciatus) and the Indian (Monocled) Cobra

(Naja naja kaouthia). Recently, Hypnale hypnale (Hump-nose pit viper) from Indian is

known to cause life-threatening envenomination symptoms upon snakebite victims (Joseph

et al., 2007). Now it is being bebated that this snake be included along with the “Big four”

snakes of medical importance (Simpson and Norris, 2007).

It is very difficult to estimate the incident of snakebite and mortality due to

snakebites. The figures from the hospitals vary between 15,000 to 20,000 deaths each year.

It is also argued in rural areas that people go for traditional medicine and deaths outside the

hospitals are not recorded and this number could go even very high. A study from the

Liverpool School of Tropical Medicine found out that 10 to 15 % of venomous bites end in

death. The possibility of survival, even without treatment, is incredibly good. There are

many reasons for this. One is that the snake often causes a dry bite without injecting any

venom. Sometimes, it might inject only a tiny bit of venom. The snake can inject the

quantity of venom it wants and it is an entirely voluntary process. But the amount injected

is only guesswork and arbitrarily related by the progress of the symptoms.

Snake venom

In snakes, venom is an evolutionary adaptation to immobilize, killing and it may

also play a role in pre-digestion of the intended meal (prey). It may also serve as a

defensive armament in protecting the snake against the predators and aggressors. It is

synthesized in the special oral glands called the Jacobson glands and is an exocrine

secretion. To provide victims with a lethal hit, venom gland synthesizes, stores and secretes

mixture of predominently protein/peptide components with different structures and

functions, as either the active or inactive precursor form into the site of their bite. The

precursor forms of components are activated by a special mechanism after the secretion. In

addition to protein/peptide toxins, the inorganic constituents of the venom include metal

ions like Ca2+, Cu2+, Fe2+, Mg2+, Na+, Zn2+ (Markland, 1998) not all of which are found in

every snake venom. While some are required for catalysis by venom enzymes others are

thought to be essential for stabilizing certain proteins. The organic constituents of venom

can be broadly divided into proteinaceous and non-proteinaceous components. The majority

of the crude venom is composed of proteinous components. The non-proteinous

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components include carbohydrates, lipids, bioactive amines like serotonin and

acetylcholine, which are predominant in viperid venom, nucleotides and amino acids

(Freitas et al., 1992; Markland, 1998). Citrate was identified as the major constituents

found in many venoms. It is found in greater than 5 % of dry weight of venom of Crotalous

atrox and Bothrops asper (Freitas et al., 1992). Snake venoms have several enzymes that

depend on metal ions for activity. For example, Phospholipase A2 (PLA2) requires Ca2+ and

metalloproteases and hemorrhagins requires Zn2+. These are kept in an inactive form by the

chelating effect of citrate. Other than this, citrate act as a buffer component and also as a

negative counter ion for basic proteins and polyamines.

The venom components seem to be fairly common and similar to one another within

each family of snakes. The target of snake venom toxins vary, while the elapid and

hydrophid venoms have mainly neurotoxic effects, while hemorrhagic and myonecrotic

toxins are generally found in the venoms of viperid and crotalid snakes, but are basically

different depending on each snake species (Tu, 1991). However, snake venoms exhibit

marked variation in their potency and extent of induction of toxic properties. The variability

of venom composition has been considered at several levels: Interfamily, intergenus,

interspecies, intersubspecies and intraspecies. While intraspecies variability may be due to

geographical distribution, seasonal and age dependent change, diet and variation due to

sexual dimorphism (Chippaux et al., 1991; Daltry et al., 1996a,b; Sasa, 1999; Shashidhara

murthy et al., 2002).

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 Fracnkel-Conrat, 1938; Ghosh et al., 1941), it becomes evident that there

are several non-enzymatic proteins in snake venoms (Table-1.02), 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). Enzymes found in venoms and their properties are given in Table.

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1.01. Table 1.02. Shows properties of some of the non-enzymatic toxic proteins/peptides

found in snake venoms.

Table 1.01: Enzymes found in snake venoms

Enzymes found in all venoms

Phospholipase A2

Deoxyribonuclease

Phosphodiesterase Adenosine triphosphatase

Phosphomonoesterase NAD nucleosidase

L-amino acid oxidase Ribonuclease

5` Nucleotidase Hyaluronidase

ATPase

Enzymes found mainly in Viperid venoms

Endopeptidase Kininogenase

Arginine ester hydrolase Thrombin like enzyme

Factor X activator Prothrmbin activator

Enzymes found mainly in Elapid venoms

Acetylcholinesterase Phospholipase B

Glycerophosphatase

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Table 1.02. Some of the non-enzymatic toxic proteins/peptides found in snake venoms.

Non enzymatic toxins Molecular

weight

Snake species References

Elapidae

Neurotoxin (toxin a) 6,787 Naja nigricollis Karlsson et al., (1966)

Cobramine A and Cobramine B

6,400 Naja naja Larsen and Wolff, (1968)

Dendrotoxin (DTX) 7,077 Dendroaspis

angusticeps

Harvey and Karlsson,

(1980)

-Neurotoxin, B.F.III 6,500 Bungarus fasciatus Ji et al., (1983)

Cardiotoxin 7,000 Naja nigricollis Kini et al., (1987, 1988)

Muscarinic toxin (MTxs) 7,500 Dendroaspis angusticeps

Adem et al., (1988)

Phospholipase Inhibitor

(NN-I3)

6,500 Naja naja naja Rudrammaji, (1994)

Viperidae:

Crotalinae

Crotamine 4,900 Crotalus durissus

terrificus

Laure, (1975)

Myotoxin a 4,400 Crotalus viridis viridis Ownby et al., (1976)

Peptide C 4,932 Crotalus viridis helleri Maeda et al., (1978)

Myotoxin I 5,035 Crotalus viridis concolor

Engle et al., (1983)

CAM-toxin 5,132 Crotalus adamanteus Samejima et al., (1988)

Wagleri toxin 8,900 Trimeresurus wagleri Tan and Tan, (1989)

Lethal peptide I 2,504 Trimeresurus wagleri Weinstein et al., (1991)

Viperinae

Neurotoxin 11,600 Vipera palaestinae Moraz et al., (1967).

Trypsin Inhibitor (TI) 6,900 Vipera russelii Jayanthi and Gowda,

(1990).

Ammodytin L (AMDL) 14,000 Vipera ammodytes Krizaj et al., (1991).

Hydrophidae

Erubutoxin a 6,760 Laticauda semifasciata Tamiya et al., (1967)

Erubutoxin b 6,780 Laticauda semifasciata Tamiya et al., (1967)

Neurotoxic peptide 6,520 Laticauda laticaudata

Laticauda colubrina

Sato et al., (1969)

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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 as ‘hypotensive’ and

‘paralytic’ strategies (Aird, 2002). Both serve to limit prey mobility. Some snakes strike,

release and then track their prey (most Viperids). In few cases they have to overcome prey

resistance in that case the snakes have to seize and bulldogs their prey (many Elapids and

all Colubrids). The third strategy is digestive and it commences degradation of prey tissues

internally, even before the prey has been engulfed. Normally, all three the strategies operate

simultaneously and individual venom constituents frequently participate in more than one

of strategies. Each of these strategies contains interchangeable mechanisms, elements or

sub strategies. Different venomous snake employ different combinations of mecha nisms

and no single species employs them all (Aird, 2002). The pathophysiology of snake

envenomation is a complex series of events that depend on the combined action of toxic

and non-toxic components (Warell, 1996). Snake envenomation includes both local and

systemic manifestations.

Systemic manifestations

The systemic manifestations depend upon the pathophysiological changes induced

by the venom of that particular species. Elapid venoms produce symptoms as early as in 5

min (Paul, 1993) or as late as 10 hr (Reid, 1979) after bite, vipers take slightly longer, the

mean duration of onset being 20 min (Paul, 1993). However, symptoms may be delayed

for several hrs. Sea snake bites almost always produce myotoxic features within 2 hr so that

they are reliably excluded if no symptoms are evident within this period (Paul, 1993). The

magnitude of systemic toxicity induced by toxins is directly relay on the concentration,

efficiency and rate of diffusion of target specific toxins. The systemic manifestations

include neurotoxicity, cytotoxicity, cardiotoxicity, hemolytic activitity, hypotensive

activity, convulusant activity and action/interference on hemostasis.

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Neurotoxicity

The neurotoxins of snake venoms interfere in synaptic transmission. They can either

inhibit the release of neurotransmitter from exocytosis of synaptic vesicle at the presynaptic

site or bind to the neurotransmitter receptor at postsynaptic site. Neurotoxins are divided

into two types depending on the mode of action at the neuromuscular junction. Those that

act at the presynaptic site are called the β- neurotoxins or presynaptic neurotoxins and

those, which act at the postsynaptic junction, are called the α- neurotoxins or post synaptic

neurotoxins (Rossetto et al., 2004).

Cytotoxicity

Snake venom PLA2 are known to exhibit cytotoxic activity (Dufton and Hider,

1983; Fletcher and Jiang, 1993). Cytotoxic PLA2s have been isolated from Naja nigricollis

(Chwetzoff et al., 1989a; Gowda and Middlebrook, 1993), Naja naja (Basavarajappa and

Gowda, 1992) and Taipoxin from Oxyuranus scutellatus scutellatus (Poulsen et al., 2005).

The cytotoxic property of nigexine from Naja nigricollis venom was reported to be

independent of enzymatic activity (Rawan et al., 1991). Further it is reported that in vivo

toxicity of nigexine depends on simultaneous expression of esterase activity and non-

enzymatic property, which alone is able to provoke the lysis of certain eukaryotic cells

(Chwetzoff, 1990).

Cardiotoxicity

Snake venom cardiotoxins are small molecular mass (5.5 – 7 kDa), highly basic

proteins and cross- linked by four disulfide bridges (Jang et al., 1997). Cardiotoxins isolated

from elapid snake venoms are basic proteins. They cause depolarization of the cardiac,

skeletal and smooth muscles resulting in muscle contraction and loss of excitability. They

are also involved in membrane fusion, hemolysis, cytotoxicity, selective killing of certain

type of tumour cells and inhibition of protein kinase C activity (Kumar et al., 1996; Cher et

al., 2005).

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Hypotensive activity

Most snake venoms employ a variety of means to induce rapid and profound

hypotension, leading to circulatory shock, prey immobilization and death (Bjarnason et al.,

1988, Andriao-Escarso et al., 2002; Lumsden et al., 2004; Joseph et al., 2004). The sudden

drop in blood pressure is due to the release of pharmacologically active autocoids like

histamine, 5-hydroxy tryptamine, leukotrienes (Andriao-Escarso et al., 2002). Many

crotaline venoms posses hypotensive peptides of 5-13 amino acids that are N-terminally

blocked with pyroglutamic acid. These peptides are generally known as bradykinin-

potentiating peptides (BPPs) because of their capacity to enhance the hypotensive effects of

bradykinin.

Convulsant activities

Death following cobra envenomation is often proceeded by convulsion due to

asphyxia arising from respiratory paralysis and other pre-agonial effects. The snake venom

components known to cause depletion of stored acetylcholine due to high influx of

potassium ions. The nerve does not release the neurotransmitter (Karlsson, 1979) there by

these acts as presynaptic neurotoxins. The convulsant activity has been reported from the

venoms of Naja naja (Lysz and Rosenberg, 1974; Bhat and Gowda, 1991), Vipera russelii

(Jayanthi and Gowda, 1990; Kasturi and Gowda, 1989) and Echis carinatus (Kemparaju et

al., 1994).

Snake venom proteins action on hemostasis

Snake venoms, particularly from the Viperidae and Elapidae families, contain a

number of components that interact with proteins of the coagulation cascade and

fibrinolytic pathway. Fontana (1987) was the first to describe the interactions between

snake venom and blood coagulation system. Snake venom toxins act as either

procoagulants or anticoagulants (Kini, 2005). Figure 1.01 shows the coagulation cascade

and major sites of action by snake venom components. Many types of venom contain more

than a single procoagulant or anticoagulant agents. Venom proteins affecting coagulation

factors may be classified as

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(i) Coagulant factors include Factor V activators, Factor X activators,

Prothrombin activators and Thrombin like enzymes (TLEs).

(ii) Anticoagulant factors includes Phospholipase A2, Metalloproteases,

serine proteases, L-amino acid oxidases, C-type lectin related

proteins, Three-finger toxins. Factor IX / X binding proteins, Protein

C activators, and Thrombin inhibitors

(iii) Fibrinolysis includes fibrinolytic enzymes and plasminogen

activator.

The anticoagulant and procoagulant activities of venom components exert their

action differently. They all interfere at different steps in the coagulation pathways and bring

about coagulation or anticoagulation activities; based on their specific action in the

coagulation cascade the venom components are studied as activation of inhibition

molecules of coagulation cascade.

Factor V activator

Factor V activator is a multifunctional 330 kDa glycoprotein, with an important role

in both procoagulation and anticoagulation activities. Thrombin activates, factor V by

cleaving at 709, 1018 and 1545 to form factor Va, a heterodimer consisting of a 105 kDa

heavy chain and a 72 / 74 kDa light chain doublet. Factor Va acts as cofactor in factor Xa

catalyzed prothrombin activation and it enhances thrombin generation more than 1000

folds. Several factor V activators have been described from Bothrops atrox, vipera russelli,

vipera lebetina, vipera ursine, naja naja oxiana and naja nigricollis nigricollis venoms

(Rosing et al., 2001).

Factor X activator

Factor X activators have been isolated from many viperidae venoms as we ll as from

elapid venoms. Factor X activators are either metalloproteinases or serine proteases (Tans

and Rosing, 2001). Russell’s viper venom contains potent activators of human blood

coagulation factor X (RVV-X), which has been well characterized (Kisiel et al., 1976; Furie

and Furie, 1976). Factor X activation has also been isolated from Bothrops atrox (Hofmann

and Bon, 1987) and several other snake species (Lee et al., 1995; Zhang et al., 1995).

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Interestingly, the factor X activators from venom of the e lapidae, king cobra (Ophiophagus

hannah) and banded krait (Bungarus faciatus), have been reported to be serine proteinase

unlike RVV-X, which, as noted, as a metalloproteinase.

Prothombin activator

Prothombin (also known as factor II) is a single chain glycoprotein with a molecular

weight of 72,000 Da (Rosing and Tans, 1991, 1992). A large number of snake venoms

contain prothrombin activators, which convert prothrombin into meizothrombin or

thrombin (Rosing and Tans, 1992). Based on their structure, functional characteristic and

cofactor requirements, they are classified into four groups. Group A prothrombin activators

are metalloproteinases and activate prothrombin efficiently without cofactors, such as

phospholipids (PLs) or cofactor Va. Group B prothrombin activators are Ca2+ dependent.

They contain two subunits linked non-covalently: a metalloproteinase and a C-type lectin

like disulfide linked dimmer. Group C prothrombin activators are serine proteases found in

Australian Elapids requiring Ca2+, PLs or Factor Va for maximal activity. Oscutarin from

Oxyuranus scutellatus also activates factor VII. Group D prothrombin activators are serine

proteases and are strongly dependent on Ca2+, negatively charged PL and factor Va.

Some venom prothrombin activators are real structural and functional homologues

of coagulation factors. Group D prothrombin activators, hopsarin D (Hoplocephalus

stephensi) (Rao et al., 2003) and trocarin D (Tropidechis carinatus) (Venkatewarlu et al.,

2002) are similar to coagulation factor Xa. Pseutarin C, a group C prothrombin activator

from Eastern Brown snake venom, Pseudonaja textills, is a multi-subunit protein complex

containing catalytic and non-enzymatic subunits similar to factor Xa and factor Va,

respectively (Rao et al., 2004). Structural information on these classes of prothrombin

activators should contribute significantly toward understanding the mechanism of factor

Xa-mediated prothrombin activation.

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Thrombin like enzymes

Thrombin has many activities; the ability of a group of snake venom enzymes to

clot fibrinogen has resulted in these enzymes being called thrombin like (Ouyang et al.,

1992; Hutton and Warrel, 1993; Marsh, 1994). Thrombin like enzymes can be classified

into three groups, venombin A, venombin B and venombin AB (Markland, 1998). They

also show some species specificity in efficiency of fibrinogen conversion. Thrombin like

enzymes are inhibited by serine protease inhibitors, but most are unaffected by thrombin

inhibitors like anti-thrombin III and hirudin. Consequently, the fibrin formed by thrombin

like enzymes is easily removed from the circulation allowing their clinical use as

defibrinogenating agents.

These enzymes are widely distributed, primarily in venoms of snakes from true

vipers (Bitis gabonica, Cerastes vipera) and pit vipers (Agkistrodon contortrix contortrix ,

Crotalus adamanteus, Bothrops atrox). There are several groups of snake venom fibrinogen

clotting enzymes based on the rate of release of fibrinopeptides A and B from fibrinogen.

One group releases fibrinopeptide A preferentially (the venom A including ancord from

venom of the Malayan pit viper, Colloselasma rhodostoma); another group releases both

fibrinopeptides A and B (the venombin AB group including gabonase from venom of the

Gaboon viper, Bitis gabonica); and the third group releases fibrinopeptide B preferentially

(the venombin B group including venzyne from venom of the southern copperhead,

Agkistrodon contortrix contortrix) (Lu et al., 2005). Various snake venom components

acting on coagulation cascade is shown in Fig. 1.01.

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Figure 1.01: Coagulation cascade and major sites of action by snake venom

components

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Anticoagulant factors from snake venom

Snake venom toxins that prolong blood coagulation are proteins or glycoproteins

with molecular masses ranging from 6 kDa to 350 kDa. These factors inhibit blood

coagulation by different mechanisms. Some of these anticoagulant proteins exhibit

enzymatic activities, such as PLA2 (phospholipase A2) and proteinase, whereas others do

not exhibit any enzymatic activity. The mechanism of anticoagulant activity of only a few

of these proteins is well understood. These are classified as Enzamatic and non-enzamatic

anticoagulant proteins.

Anticoagulant proteins with enzymatic activity

Several proteins with enzymatic activity, such as PLA2 and proteinases, inhibit

blood coagulation. Some of them inhibit clot formation by the physical destruction of a

factor that contributes directly to the coagulation. In these cases, the mechanisms appear to

be simple and are directly dependent on the respective enzymatic activity. The study of

such factors, in general, may not significantly contribute to our understanding of blood

coagulation. However, at times, a careful examination of their mechanisms may be not only

important, but also essential. For example, conventional wisdom suggests that PLA2

enzymes exert their anticoagulant effects by the hydrolysis and physical destruction of the

membrane surface required for the formation of coagulation complexes. Interestingly, the

anticoagulant activity of certain PLA2 enzymes is due to their interaction with blood

coagulation proteins and not phospholipid hydrolysis (for details, see below). Thus non-

enzymatic mechanisms of these enzymatic proteins cannot be ignored.

PLA2 enzymes

PLA2 enzymes are esterolytic enzymes which hydrolyse glycerophospholipids at the

sn−2 position of the glycerol backbone releasing lysophospholipids and fatty acids. Snake

venoms are rich sources of PLA2 enzymes. Several hundred snake venom PLA2 enzymes

have been purified and characterized. Amino acid sequences of over 280 PLA2 enzymes

have been determined. (A database is available at http://sdmc.lit.org.sg/Templar/

DB/snaketoxin PLA2/index.html.) They are approx. 13 kDa proteins and contain 116–124

amino acid residues and six or seven disulphide bonds. They are rarely glycosylated. So far,

threedimensional structures of more than 30 PLA2 enzymes have been determined (for a

comprehensive list, see (Kini, 2005). The structural data indicate that snake venom PLA2

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enzymes share strong structural similarity to mammalian pancreatic as well as secretory

PLA2 enzymes. They have a core of three α-helices, a distinctive backbone loop that binds

catalytically important calcium ions, and a β-wing that consists of a single loop of

antiparallel β-sheet. The C-terminal segment forms a semicircular ‘banister’, particularly in

viperid and crotalid PLA2 enzymes, around the Ca2+-binding loop. In addition, they have a

similar catalytic function in hydrolysing phospholipids at the sn−2 position. However, in

contrast with mammalian PLA2 enzymes, many snake venom PLA2 enzymes are toxic and

induce a wide spectrum of pharmacological effects (Harris, 1985; Rosenberg, 1990; Kini,

1997). These include neurotoxic, cardiotoxic, myotoxic, haemolytic, convulsive,

anticoagulant, antiplatelet, oedemainducing and tissue-damaging effects. Thus PLA2

enzymes alsoform a family of snake venom toxins, which share a common structural fold

but exhibit multiple functions. These factors make the structure–function relationships and

the mechanisms of action intriguing, and pose exciting challenges to scientists. Some snake

venom PLA2 enzymes inhibit blood coagulation (Boffa and Boffa, 1976; Verheji et al.,

1980; Boffa et al., 1980; Evans et al., 1980). Boffa and colleagues studied the anticoagulant

properties of a number of PLA2 enzymes and classified them into strongly, weakly and non-

anticoagulant enzymes. Strongly anticoagulant PLA2 enzymes inhibit blood coagulation at

concentrations below 2 µg/ml. weakly anticoagulant PLA2 enzymes show effects between 3

and 10 µg/ml. A number of venom PLA2 enzymes do not prolong the clotting times

significantly even at 15 µg/ml. Thus the anticoagulant activity of different PLA2 enzymes

varies significantly. Evans et al. (1980) purified three anticoagulant proteins (CM-I, CM-II

and CM-IV) from Naja nigricollis (black-necked spitting cobra) venom and showed their

identity with PLA2 enzymes. CM-IV shows at least 100-fold more potent anticoagulant

activity than CM-I and CM-II (Kini and Evans, 1987). On the basis of their anticoagulant

properties, they were classified as strongly (CM-IV) and weakly (CM-I, CMII)

anticoagulant PLA2 enzymes respectively. Since phospholipids play a crucial role in the

formation of several coagulation complexes, intuitively one might anticipate that the

destruction of phospholipid surface would be the primary mechanism to account for

anticoagulant effects of PLA2 enzymes. However, strongly anticoagulant PLA2 enzymes

also affect blood coagulation by mechanisms that are independent of phospholipid

hydrolysis (see below). To explain the functional specificity and mechanism of induction of

various pharmacological effects, the target model was proposed (Kini, 1997; Kini and

Evans, 1989; Kini, 2003). Accordingly, the susceptibility of a tissue to a particular PLA2

enzyme is due to the presence of specific ‘target sites’ on the surface of target cells or

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tissues. These target sites are recognized by specific ‘pharmacological sites’ on the PLA2

molecule that are complementary to ‘target sites’ in terms of charges, hydrophobicity and

van der Waals contact surfaces. (Kini, 1997; Kini and Evans, 1989; Kini, 2003). Proteins

(or glycoproteins) could act as specific target sites for PLA2 enzymes. The affinity between

PLA2 and its target protein is in the low nanomolar range, whereas the binding between

PLA2 and phospholipids is in the high micromolar range. Such a four to six orders of

magnitude difference in affinity between the protein–protein interaction and the protein–

phospholipids interaction explains why the interaction of PLA2 and its target protein

governs the pharmacological specificity (Kini and Evans, 1989; Kini, 2003). The target

proteins such as membrane-bound receptors/acceptors are identified through studies using

radiolabelled PLA2 enzymes and specific binding studies, as well as photoaffinity labelling

techniques. Anticoagulant PLA2 enzymes, on the other hand, target one or more soluble

proteins or their complexes in the coagulation cascade. Furthermore, the enzymes may

interact with the active, but not the zymogen, form of the coagulation factor. Therefore

different strategies have beenused to identify the soluble target protein in o rder to

understand the mechanism of anticoagulant effects of PLA2 enzymes.

Metalloproteinases

Snake venom metalloproteinases are endoproteolytic enzymes. Their catalytic

activity is dependent on Zn2+ ions. On the basis of size and domain structure

characteristics, they are classified into P-I, P-II, P-III and P-IV classes (Bjarnason and Fox,

1995; Fox and Serrano, 2005). P-I proteinases contain only a metalloproteinase domain, P-

II proteinases contain metalloproteinase and disintegrin domains, P-III proteinases contain

metalloproteinase, disintegrin- like and cysteine-rich domains, and P-IV proteinases contain

the P-III domain structure plus lectin- like domains connected by disulphide bonds.

Schematic structures of snake venom metalloproteinases is as shown in Fig. 1.02.To date,

the sequences of over 40 metalloproteinases from snake venoms have been determined

(Fox and Serrano, 2005). Six crystal structures of snake venom metalloproteinases are

available, but all of them are from the P-I class. They are structurally similar to elastases

and matrix metalloproteinases. They have a central core of a five-stranded β-sheet mixed

with α-helices. There is a characteristic methionineturn structure between the αD and αE

helices. The structure is organized as an upper and lower domain with the substratebinding

cleft running between them. In addition to their role in the digestion of prey, they exhibit

several biological effects, including haemorrhagic, pro-coagulant, anticoagulant and

17

antiplatelet effects (Fox and Serrano, 2005). Some of the snake venom metalloproteinases

inhibit blood coagulation. Most metalloproteinases are fibrinogenases and they release

peptides from the C-terminal of fibrinogen. They are classified into α- and β-fibrinogenases

on the basis of their specificity for the Aα or Bβ chain of fibrinogen. α-Fibrinogenases

inhibit blood coagulation, because truncated fibrinogen does not form as strong a fibrin clot

as the native fibrinogen. Thus the subtle physical destruction leads to the anticoagulant

action of metalloproteinases. The structure–function relationships of these

metalloproteinases with respect to their anticoagulant effects have not been studied yet.

Figure 1. 02: Schematic structures of snake venom metalloproteinases

Serine proteinases

Snake venom serine proteinases, in addition to their contribution to the digestion of

prey, affect various physiological functions. They affect platelet aggregation, blood

coagulation, fibrinolysis, the complement system, blood pressure and the nervous system

(Markland, 1998; Meier and Stocker, 1991; Braud et al., 2000; Kini, 2004; kornalik, 1991;

Kini et al., 2002a, Kini, 2005). Among the serine proteinases, only protein C activators

exhibit direct anticoagulant effects. Physiologically, the zymogen of protein C circulating in

the blood is activated by thrombin. This activated protein C degrades FV/FVa and FVIII/

FVIIIa, and releases a tissue-type plasminogen activator. It also stimulates fibrinolysis

through its interaction with plasminogen activator inhibitor (Fay and Owen, 1989; Moniwa,

1996; Sakamoto et al., 2003). Venoms from snake species belonging to the genus

Agkistrodon [copperhead snakes: A. contortrix contortrix (southern copperhead), A.

contortrix mokasen (northern copperhead), A. contortrix pictigaster (Trans-Pecos

18

copperhead), A. piscivorus (cottonmouth), A. piscivorus leucostoma (western cottonmouth),

A. halys halys (Siberian moccasin), A. blomhoffi ussuriensis (Ussurian mamushi) and A.

bilineatus (cantil)] contain protein C activators. These are glycoproteins with a molecular

mass of approx. 36–40 kDa. They activate protein C at low salt concentrations in the

absence of Ca2+ ions. High salt concentrations and the presence of Ca2+ ions inhibit their

ability to activate protein C (Klein and Walker, 1986; Kisiel et al., 1987; Bakker et al.,

1993). So far, the amino acid sequence of only the protein C activator from A.c. contortrix

venom has been determined (Memullen et al., 1989). They prolong clotting times (Stocker

et al., 1986; Stocker et al., 1987) and thrombus formation in the arteriovenous shunt (Kogan

et al., 1993) in vivo. So far, no significant data are available on the structure–function

relationshipsof this class of proteinases.

Another group of serine proteinases, namely TLEs (thrombinlike enzymes), deplete

the fibrinogen and makes the plasma unclottable. They are widely distributed within several

pit viper genera (Agkistrodon, Bothrops, Crotalus, Lachesis and Trimeresurus), as well as

some true vipers (Bitis and Cerastes) and the colubrid, Dispholidus typus (for an inventory

and reviews, see (Pirkle and Theodor, 1998; Bell, 1997; Pirkle and Stocker, 1991). They are

single-chain proteins or glycoproteins with a molecular mass of 26–33 kDa. They share a

high degree of sequence similarity among themselves (≈67%). However, they show less

than 40% similarity to human thrombin. They preferentially release either fibrinopeptide A

or B, although rarely both with equal efficiency, unlike thrombin (Bell, 1997; Aronson,

1976).Classical low-molecular-mass serine proteinase inhibitors inhibit them, but most are

not inhibited by thrombin inhibitors like antithrombin III and hirudin (Hutton and Warrel,

1993; Bell, 1997; Aronson, 1976). They act on blood plasma and induce friable and

translucent clots, presumably due to lack of cross- linking of fibrin by FXIIIa. They often

also act on the coagulation factor FXIII, but appear to degrade rather than activate it

(Hutton and Warrel, 1993). Unlike thrombin, they do not activate other coagulation factors

(Aronson, 1976). Thus, although TLEs ‘resemble’ thrombin to an extent, they are

structurally and functionally dissimilar to the coagulation factor (Hutton and Warrel, 1993;;

Kini et al., 2002b; Joseph and Kini, 2004; Bell, 1997). Furthermore, flavoxobin, a TLE

from Trimeresurus flavoviridis (Habu snake) venom, activates complement C3 protein and

acts as a heterologous C3 convertase (Yamamoto et al., 2002). These unique properties

enable their clinical use as defibrinogenating agents; for example, ancrod [Arvin®; from

Calloselasma rhodostoma (the Malayan pit viper)] and batroxobin [Defibrase®; from

Bothrops moojeni (the Brazilian lancehead snake)] (reviewed in Stocker and Barlow, 1976;

19

Stocker, 1998). Since the fibrin formed is not cross- linked, it is readily degraded by the

fibrinolytic system.Two anticoagulant serine fibrinogenases from Vipera lebetina (blunt-

nosed viper) venom have been characterized (Siigur et al., 2003). One is a basic (pI>10) α-

fibrinogenase, whereas the other is an acidic (pI<3) β-fibrinogenase (Siigur et al., 1991;

Mahar et al., 1987; Samuel et al., 2002). Both enzymes are structurally similar to other

snake venom serine proteinases (Siigur et al., 2003). They have the catalytic triad, and, in

both enzymes, Asp189, which is located in the bottom of the primary specificity pocket, is

replaced by Gly189.

L-Amino acid oxidases

L-Amino acid oxidases catalyse the oxidative deamination of a number of L-amino

acids and generate hydrogen peroxide (H2O2). It is widely known that these enzymes affect

haemostasis by modulating platelet function (Nathan et al., 1982; Sakurai et al., 2001).

Recently, Sakurai et al., (2003) showed that L-amino acid oxidase purified from

Agkistrodon halys blomhoffii exhibits anticoagulant activity. This enzyme affects only the

intrinsic pathway, having little effect on the extrinsic pathway. Furthermore, they showed

that it selectively inhibits FIX activity. H2O2 production does not appear to be involved in

the inactivation. Interestingly, L-amino acid oxidase does not bind or interact directly with

FIX, as shown by surface plasmon resonance (Sakurai et al., 2003). Further studies are

needed to clarify the mechanism of inactivation.

Non-enzymatic anticoagulant proteins

Several snake venom proteins with no ‘detectable’ (known or tested) enzymatic

activity inhibit blood coagulation. A number of non-enzymatic anticoagulant proteins have

been purified and characterized. These proteins inhibit the coagulation pro cess through

their direct interaction with a specific coagulation factor. The mechanisms appear to be

simple, and these proteins interfere in either complex formation or inhibit the activity of

one of the proteinases. The study of such factors significantly contributes to our

understanding of blood coagulation. Furthermore, the structure–function relationships of

these proteins and identi- fication of the functional sites may be useful in the development

of new anticoagulant agents.

20

C-type lectin-related proteins

C-type lectins are homodimers and possess the ability to agglutinate red blood cells

through their interaction with carbohydrate moieties. C-type lectin-related proteins, on the

other hand, are heterodimers or oligomeric complexes of heterodimers and do not possess

lectin- like activity (Drickamer et al., 1999; Morita, 2004; Ogawa et al., 2005). At times,

they are also found in the snake venom as a complex with metalloproteinases. C-type

lectin-related proteins form the integral part of pro-coagulant proteins, such as FX activator

from Daboia russelli (Russell’s viper; formerly Vipera russelli) venom and prothrombin

activators from Echis carinatus (saw-scaled viper) and Echis multisquamatus (Central

Asian sand viper) venoms (Takeya et al., 1992; Gowda et al., 1994; Yamada et al., 1996).

In all these cases, C-type lectin-related subunits act as regulatory subunits and are involved

in determining the substrate specificity in the presence of Ca2+ ions (for details, see Morita,

1998).

FX and FIX-binding proteins

Anticoagulant C-type lectin-related proteins were among the first non-enzymatic

proteins to be isolated and characterized from snake venoms. They were first isolated and

purified from Deinagkistrodon acutus (hundred-pace pit viper; formerly Agkistrodon

acutus) and Trimeresurus stejneri (Stejneger’s bamboo viper); formerly, Trimeresurus

gramineus) venoms (Ouyang and Teng, 1972; Ouyang and Yang, 1975). They showed that

these anticoagulant proteins inhibit prothrombin activation by non-enzymatic mechanisms

(Ouyang and Teng, 1973; Teng and Seegers, 1981). However, these studies were not

followed by detailed studies on their structure and mechanism of action. Atoda and Morita

(1993) purified an anticoagulant protein from T. flavoviridis venom using a FXaffinity

column. This protein binds to FX/FXa as well as to FIX/ FIXa. This anticoagulant was

shown to be the first C-type lectinrelated protein on the basis of its amino acid sequence

and disulphide linkages (Atoda and Morita, 1993). Subsequently, they also purified and

characterized a specific FIX-binding protein and FX-binding protein from T. flavoviridis

and D. acutus venoms respectively (Atoda et al., 1995; Atoda et al., 1998). These are

heterodimeric proteins with α- and β-chains. Both chains share the common structural

scaffold of C-type lectin (Mizuno et al., 1999). The core structure is similar to the

recognition domain of mannose-binding protein, a C-type lectin. A distinctive structural

feature among C-type lectin related proteins (Mizuno et al., 1999; Batuwangala, 2004) is

that the central loop of the individual subunit extends away from the core structure and

forms a large open loop. This central loop forms the dimeric interface through domain

21

swapping; a domain from the α subunit replaces essentially an identical domain in the β

subunit. At the same time, this domain from the β subunit is swapped for the same domain

in the α subunit. This is the first demonstrated example of three-dimensional swapping in

the central region, whereas all other domain swapping occurs at the N- or C-terminus (Liu

and Eisenberg, 2002). Furthermore, domain swapping is found mostly in the formation of

homodimers or homo-oligomers, but not in the formation of heterodimers, as in the case of

C-type lectin-related proteins (Mizuno et al., 1999; Hirotsu et al., 2001; Batuwangala,

2004). This swapped dimeric interface, along with core structures of the α and β subunits,

forms the concave ligandbinding site (see below).The snake venom anticoagulant C-type

lectin-related proteins inhibit the activity of the coagulation factors FIX and FX (Atoda and

Morita, 1993; Atoda et al., 1997). They bind to these coagulation factors with nanomolar

and subnanomolar affinities. The Gla (γ -carboxyglutamic acid) domain peptides of FX

(comprising residues 1–44 and 1–41) bind to FX-binding protein in the presence of Ca2+

with apparent dissociation constants of 1.0 and 100 nM respectively (Mizuno et al.,

2001).Thus most of the interaction occurs through the Gla domain. Interestingly, although

FIX/FX-binding protein interacts with both FIX and FX, it has a low affinity for FX Gla

domain peptides but binds to the Gla peptide of FIX-(1–46). The threedimensional

structures of the complexes (Mizuno et al., 2001) show that the Gla domains bind to the

concave ligand-binding site between the two subunits. The FX Gla domain has eight bound

Ca2+ ions (Mizuno et al., 2001). One of the Ca2+ ions participates in the binding interface

between the Gla domain and the FX-binding protein. There are nine salt-bridges between

the negatively charged Gla domain and the positively charged FX-binding protein, and 21

water molecules form an extensive network of hydrogen-bonds between the α-chain and the

Gla domain. Phe4, Leu5 and Val8 in the N-terminal loop of the Gla domain interact with

Arg112, Met113 and Ile114 of the β-chain. Thus salt-bridges along with hydrophobic

interactions and hydrogen bonds stabilize the complex between the Gla domain and the FX-

binding protein (for details, see Mizuno et al., 2001). This binding interferes in the Ca2+-

dependent binding of FIX and FX to phospholipid membranes, and hence exhibits potent

anticoagulant effects.

22

Three-finger toxins

This is a family of non-enzymatic polypeptides containing 60– 74 amino acid

residues (Endo and Tamiya, 1991). This family of proteins is found commonly in the

venoms of elapids (cobras, kraits and mambas) and hydrophids (sea snakes). Recently, they

have been found in colubrid venoms (Fry et al., 2003a, Fry et al., 2003b; Lumsden et al.,

2004; Mackessy, 2002; Lumsden et al., 2005), but not those of vipers and crotalids

(rattlesnakes) (Fry et al., 2003). They contain four or five disulphide bridges, of which four

are conserved in all the members (Endo and Tamiya 1991). Consequently, all proteins of

this family show a similar pattern of protein folding: three β-stranded loops extending from

a central core containing the four conserved disulphide bridges (Menez, 1998; Tsetlin,

1999). Because of this appearance, this family of proteins is called the three-finger toxin

family. Despite the overall similarity in structure, at times they differ from each other in

their biological activities. Members of this family include α-neurotoxins (Tsetlin, 1999;

Chang, 1979), κ-bungarotoxins (Grant and Chiappinelli, 1985),muscarinic toxins

(Jerusalinsky and Harvey, 1994), fasciculins (Le du et al., 1991), calciseptine (Deweille et

al., 1991; Albrand et al., 1995), cardiotoxins (cytotoxins) (Bilwes et al., 1994),

dendroaspins (Mcdowell et al., 1992) and anticoagulant proteins (Kini et al., 1988; Kini et

al., 1987). They exhibit such varied activities through interaction with different target

protein receptors/acceptors, ion channels or phospholipids (for details, see Kini, 2002).

Interestingly, several other non-venom proteins and polypeptides also belong to this

superfamily of proteins. Structure–function relationships of a number of these polypeptides

have been well elucidated, and their functional sites are located on distinct surfaces (for

details, see Kini, 2002).

Anticoagulant three-finger toxins

The anticoagulant and antiplatelet effects of three- finger toxins were first identified

in cardiotoxins isolated from Naja nigricollis crawshawii (spitting cobra) venom (Kini et

al., 1987; Kini et al., 1988]. The mechanism of antiplatelet action (Kini and Evans, 1988)

and structure–function relationships of these cardiotoxins (Kini and Evans, 1989a; Kini and

Evans, 1989b) have been well elucidated.

23

Hemextin AB complex

Recently, a novel anticoagulant complex was characterized from Hemachatus

haemachatus venom. It has two three-finger toxins, hemextin A and hemextin B, as

subunits. Individually, hemextin A prolongs blood coagulation, but hemextin B does not

show any effect on blood clotting. However, hemextin B forms a 1:1 complex and

synergistically enhances the anticoagulant effects of hemextin A. The dissection approach

was used to identify the coagulation step that is (are) inhibited by hemextin AB complex.

Hemextin A and hemextin AB complex prolong the prothrombin time, but not the Stypven

or the thrombin time, and hence we proposed that they inhibit the extrinsic tenase complex.

Hemextin A inhibits the reconstituted extrinsic tenase (TF– FVIIa) complex. As expected,

hemextin B by itself does not inhibit the complex, but through complex formation enhances

the inhibitory effects of hemextin A. Hemextin AB complex noncompetitively inhibits the

TF–FVIIa complex with a Ki value of 50 nM. Of the 12 serine proteinases tested, hemextin

A and hemextin AB complex specifically inhibit FVIIa and its complexes. In addition, they

mildly inhibit plasma kallikrein activity. Thus hemextin AB complex is a highly specific

natural inhibitor of the initiation of blood coagulation. It is also the first anticoagulant

complex isolated from snake venom.

Protein C activators

Protein C is a vitamin K-dependent, two chain zymogen activated by thrombin.

Activated protein C degrades factor Va and factor VIIIa and is therefore anticoagulant.

Most protein C activators were purified from Agkistrodon venoms. Others come from

Bothrops, Trimeresurus, or Cerastes venoms. Most venom protein C activators have

sequences highly similar to other venom serine proteases. Unlike thrombin-catalyzed

protein C activation, requires thrombomodin as a cofactor, venom activators directly

convert protein C into the active form. The fast-acting protein C activator ProtacR from

Agkistrodon contortrix contortrix venom is widely used to diagnose protein C pathway

disorders (Gempeler-Messina et al., 2001).

24

Thrombin inhibitors

A unique thrombin inhibitor was purified from Bothrops jararaca venom by Zingali

et al. (1993). This is the only report to date of snake venom inhibitor of this type. The

inhibitors, named bothrojarcin, is a 27 kDa C-type lectin like thrombin inhibitors composed

of the polypeptides chains of 13 and 15 kDa subunits linked by disulfide bridges.

Bothrojarcin is highly resistant to urea or DTT, requiring both agents to denature it fully.

Bothrojarcin has two independent mechanisms for anticoagulant action it binds strongly to

exosites I and II to form a non-covalent equimolar complex and inhibits thrombin induced

platelet aggregation and secretion, but does not interact with the by competitively inhibiting

the binding of thrombin to fibrinogen and it inhibits thrombin binding to thrombomodulin

and decreases the rate of protein C activation (Arocas et al., 1996). Secondly, it inhibits

prothrombin activation by interacting with proexosite I. In the absence of PLs, bothrojarcin

strongly inhibits the zymogen activation by factor Xa in the presence but not in the absence

of factor Va. Table.1.03 shows anticoagulant proteins from snake venom.

25

Table.1.03. Anticoagulant proteins from snake venom

Fibrinolytic proteinases

The substrates for the fibrinogenlytic enzymes, 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 Aα, Bβ

and γ with molecular weights of 63 500, 56 000 and 47 000 Da respectively. The fibrinogen

molecule has a molecular weight of 340 kDa (Bauer and Rosenberg, 1987). Fibrinogen

contains long stretches of amino acids, which are exposed to proteolytic enzymes including

26

the snake venom proteinases. Fibrin, however, has a cross- linked structure and is much less

susceptible to proteolysis.

Fibrinogenlytic activity has been described in the venoms of members of the

Viperidae and Elapidae families. These fibrinolytic enzymes are divided into

metalloproteinases and serine proteinases (Matusi et al., 2000). Most of the first groups of

enzymes were characterized as zinc metalloproteinases and degrade Aα chain of fibrinogen

preferentially. The second groups are serine proteases and most have specificity toward the

Bβ chain of fibrinogen. However, there are exceptions to these generalizations and

specificity for Aα or Bβ chains are not absolute, as there is substantial degradation of

alternate chain with time. Most of the metalloproteinases are fibrinolytic and many of the

serine proteinases are both fibrinogenolytic and fibrinolytic (Braud et al., 2000).

Fibrinogenolytic metalloproteinase enzymes cleaves amino-terminal to hydrophobic amino

acids, while serine fibrinogenolytic enzymes cleave carboxy-terminal to basic amino acids.

Plasminogen activator

Snake venoms have been reported to stimulate the release of plasminogen activators

from endothelial cells. The activity was most pronounced in the venoms of the rattlesnakes

Crotalus atrox and Crotalus adamanteus (Kirshchbaum et al., 1999). Plasminogen

activators are also reported from Lachesis muta muta and Agkistrodon halys, venoms

(Zhang et al., 1998; Park et al., 1998).

Venom proteins acting on platelets

C-type lectins

Many snake venom C-type lectins affecting platelets by binding to VWF or

receptors such as GPIb, 21 and GPVI have been characterized (Clemetson et al., 2001;

Andrews et al., 2004). Botrocetin and bitiscetin form trimolecular complexes with VWF

and GPIb to activate platelets. Recent results suggest that they interact with both proteins,

not simply by inducing conformational changes inVWFA1 (Fukuda et al., 2002; Maita et

al., 2003). TheC-type lectins acting viaGPIb fall into twocategories, thoseinhibiting platelet

activation by blocking binding of VWF/ ristocetin and/or thrombin and those either

agglutinating platelets or activating and aggregating platelets.Most inhibitory GPIb C-type

lectins are heterodimers, while most multimeric GPIb-binding venom proteins agglutinate

27

or aggregate platelets (Lu et al., 2004). GPIb-binding proteins may behave differently in

vitro and in vivo. Echicetin specifically binds platelet GPIb and blocks platelet interactions

with VWF and thrombin. It is cross- linked byIgMj to formmultimers that agglutinate

platelets in vivo (Navdev et al., 2001). Convulxin, stejnulxin and ophioluxin activate

platelets via GPVI (Polgar et al., 1997; Lee et al., 2003; Du et al., 2002). They are all

multimeric proteins composed of heterodimers. However, it has been reported that, like

alboaggregin- A (Dorman et al., 2001) and alboluxin (Du et al., 2002), convulxin also binds

to GPIb (Du et al., 2002; Kanaji et al., 2003). EMS16 binds to the collagen receptor,

integrin a2b1 and specifically inhibits collagen binding. EMS16 may bind specifically to

the a-I domain in a metal ion- independent fashion (Hori et al., 2004). Rhodocetin also binds

toa2b1 and is unusual in that the subunits are not covalently linked (Eble et al., 2001).

Aggretin (Navdev et al., 2001) and bilinexin (Du et al., 2001) activate platelets via 21

and GPIb. However, aggretin may use other unidentified receptor(s). Thus, venom C-type

lectins often use more than one platelet receptor.

Disintegrins

Disintegrins inhibit integrins of the b1 and b3 subfamilies including the fibrinogen

receptor GPIIb/IIIa (II3), vitronectin receptor (avb3) and the fibronectin receptor

(51). More than 50 disintegrins have been purified from various venoms from the viper

or pit viper families. GPIIb/IIIa antagonists inhibit aggregation caused by agonists

including ADP, thrombin, collagen and arachidonic acid. Disintegrins are either single

chain molecules of 40–80 amino acid residues or multimeric.Disintegrins contain an RGD

or KGDsequence in the carboxyl-terminal half of the molecule, which is essential for

blocking integrin interactions with ligands. Disintegrin structures are characterized by

irregular turns and loops that form a rigid core and the RGsequence is stabilized by two

disulfide bonds. The RGD sequence is highly mobile, allowing rapid binding to the

integrin-binding site within GPIIIa residues 217–302 (Fugii et al., 2003). The width and

shape of the RGD loop may be an important structural feature to fit into the binding pockets

of integrins aIIbb3 and avb3. Additional structural features may be important for the

selectivity and affinity of disintegrins (Hantgan et al., 2004). The structure of a

homodimeric disintegrin indicates that the N termini anchored two chains of the dimmer

diverge at their C termini exposing the RGD motif in opposite directions to enhance

binding efficiency for integrins (Bilgrami et al., 2004).

28

Proteinases

One group of proteinases acting directly on platelets is the TLEs. Some of these

mimic thrombin by activating platelets through cleavage of PAR or binding to GPIb. The

action of cerastocytin resembles thrombin, as rabbit platelets desensitized by pretreatment

with thrombin do not aggregate to cerastocytin (Marrakchi et al., 1997). Furthermore, its

activity was inhibited by antibodies to thrombin binding sites on GPIb. The activity of

thrombocytin and PA-BJ was blocked by monoclonal antibodies against PAR1 and by

heparin, but not by hirudin or thrombomodulin (Santos et al., 2000). Both PA-BJ and

thrombocytin cleave PARs like thrombin. Other proteinases affecting platelet function are

metalloproteinases.

Some metalloproteinases bind to collagen or collagen receptors on platelets by their

disintegrins-like or cysteine-rich domains to inhibit platelet aggregation (Jia et al., 1997;

Shimokawa et al., 1997). For example, jararhagin binds to a2 (Kamiguti et al., 1996),

catrocollastatin (Zhou et al., 1996) and crovidisin (Liu et al., 1997) bind to collagen.

Kaouthiagin cleaves VWF (Hamako et al., 1998), Mocarhagin (Ward et al., 1996) and

triflamp (Tseng et al., 2004) cleave GPIb, while crotalin cleaves both GPIb and vWF. Some

metalloproteinases activate platelets by binding to platelet receptors. Alborhagin (Andrews

et al., 2001) from T. albolabris activates platelet through GPVI via a different binding site

than convulxin.

Phospholipase A2

Venom PLA2s affect platelet functions by at least three mechanisms (Mounier et al.,

2001; Kini and Evans, 1990). One group of PLA2s induce platelet aggregation by cleaving

platelet membrane PLs releasing arachidonic acid and forming arachidonic acid metabolites

such as thromboxane A2. Another group of venom PLA2s inhibit platelet aggregation via

the cleavage products. Some venom phospholipases have biphasic effects on washed

platelets (Teng et al., 1984). The first phase is reversible aggregation and the second phase

is an inhibitory effect on platelet aggregation induced by arachidonic acid, ADP or

collagen. The aggregating effect may be due to thromboxane formation and the inhibition

due to effects of cleaved products from arachidonic acid metabolites. Effects on platelets

may also be independent of enzymatic activity. A phospholipase A2 with potent platelet

inhibitory activity from Ophiophagus hannah venom is only partially dependent on the

phospholipase activity of the enzyme (Huang et al., 1997). The anti-platelet activity appears

29

to be partially mediated by a dramatic change in the cytoskeleton. A PLA2 from Papuan

black snake (Pseudechis papuanus) venom also induced a change in platelet morphology

including a disruption of the cytoskeleton (Laing et al., 1995). The anti-platelet site of the

O. hannah PLA2 is in its pancreatic loop.

5`-nucleotidases

5`-nucleotidase activity is widely distributed in many viper and pit viper venoms

(Tan and Ponnuduri, 1992). Trimeresurus gramineus venom contains a 74 kDa

thermostable, single chain 5` nucleotidase. Activity was inhibited by EDTA but supported

by Zn2+ or Co2+. In rabbit platelet-rich plasma, 5` nucleotidase completely inhibited platelet

aggregation induced by ADP, sodium arachadonate or collagen, most likely by ADP and

possibly by generation of adenosine (Ouang and Huang, 1983).

L-Amino acid oxidases

Venom L-amino acid oxidases (LAAOs) are homodimeric flavoenzymes, which

catalyse the oxidative deamination of an L-amino acid substrate to aa-keto acid along

withammonia and hydrogen peroxide. They are widely distributed in Viperidae, Crotalidae

and Elapidae (Du and Clemtson, 2002). Each subunit has three domains: an FAD-binding

domain, a substrate-binding domain and a helical domain (Pawelek et al., 2000). The

reported effects of LAAOs on platelet function are quite controversial. LAAO from Echis

colorata inhibits ADP-induced platelet aggregation (Nathan et al., 1982). Agkistrodon halys

blomhoffii and Naja naja kaouthia LAAOs, inhibits agonist- or shear stress- induced

platelet aggregation (Takatsuka et al., 2001; Sakurai et al., 2001). The authors suggested

that the interaction between activated platelet integrin GPIIb/IIIa and fibrinogen was

inhibited by the continuous generation of H2O2. LAAOs from other snakeshave been

reported to have totally the opposite effect on platelets. LAAOs from Eristocophis

macmahoni, O. hannah, B. alternatus and Trimeresurus jerdonii induce human platelet

aggregation through formation of H2O2 (Du and Clemtson, 2002; Stabeli et al., 2004; Lu et

al., 2002). It is still not clear how H2O2 functions in LAAOs-induced platelet aggregation. It

is also possible that LAAOs activate platelets in a receptor dependent way as LAAO from

A. halys showed various binding and cytotoxic effects on different cell lines (Zhang et al.,

2004). Action of various venom proteins on platelets is as shown in Fig. 1.03.

30

Fig.1.03. Action of venom proteins on platelets. Venom proteins are shaded.Double -

headed arrow, binding; single arrow, enzymatic cleavage; straight line, activation;

dashed line, inhibition.

Local effects/manifestations

Local changes are the earliest manifestations of snakebite (Reid, 1979). Features

are noted within 6-8 minutes but may have onset upto 30 min (Reddy, 1980; Reid and

Theakston, 1983). Local pain with radiation and tenderness and the development of small

reddish wheal are the first to occur. This is followed by edema (Paul, 1993) and swelling

which can progress quite rapidly and extensively even involving the trunk (Saini et al.,

1984). Tingling and numbness over the tongue, mouth, scalp and paraesthesias around the

wound occur mostly in viper bites (Reddy, 1980). Local bleeding including ptechial and/or

purpuric rash is also seen most commonly with this family. Crotalid and Viperid venoms

are known to cause local effects, which frequently include pain, swelling, echymoses and

local hemorrhage are usually apparent within minutes of the bite. Such signs are sometimes

followed by liquefaction of the area surrounding the bite. The local area of bite may

become devascularized with features of necrosis predisposing to onset of gangrenous

31

changes. Secondary infection including tetanus and gas gangrene may also result (Tu, 1991;

Philip, 1994).

Hemorrhage

Hemorrhage or bleeding is a common phenomenon in the victims of Viperidae

envenomation (Warrell, 1996). “Hemorrhagins” the term was introduced by Grotto et al.,

(1967). The main factors responsible for hemorrhage are hemorrhagins, which comprise a

major group of active principles in viperid venom. These toxins act directly on the

endothelial cells and the under lying basement membrane to induce local and systemic

hemorrhage depending on the severity of envenomation. In mild envenomation, their action

is limited to the site of the bite. However, in severe envenomation, hemorrhage can be wide

spread involving the whole extremity concerned and even organs distant from the site of the

bite, such a s heart, lungs, kidney, intestine and brain.

Hemorrhagic activity has been associated with enzyme proteolytic activity. Chelation of

the zinc atom abolishes both proteolytic and hemorrhagic effects (Bjarnason and Fox, 1988;

1994). Of the 65 hemorrhagic toxins, 12 have been analyzed for their metal content, all of

them have been found to contain zinc and many more are inhibited by metal chealtors. Ten

of the twelve toxins contained approximately 1 mole of zinc per mole of toxin (Bjarnason

and Fox, 1994). Therefore, that venom induced hemorrhage is primarily caused by metal

dependent, proteolytic activities of the hemorrhagic toxins, probably acting on connective

tissue and basement membrane components.

Most of the hemorrhagins are found to be absent in the venom of juvenile snakes and

appear only in adult snakes. A search for the signal that initiates the appearance of these

toxins in the venom of snakes at a particularly age in such species may be considerable

academic as well practical interest. Although hemorrhagins are the main causative agents of

hemorrhage, several other components residing in the crude venom can act also as

secondary factors to augment the process. Components that cause fibrinogenolysis render

blood almost completely incoagulable. Anticoagulant factors directly block the clotting

phenomenon. There are platelet aggregation inhibitors and enzymes that release kinin from

kininogen. In the absence of blood coagulation and platelet aggregation, the two principle

phenomena that occur following damage to blood vessels, hemorrhage initiated by

hemorrhagins can go on unchecked with massive extravasation of RBCs into surrounding

tissues, giving rise to swelling, blistering and edema (Bjarnason and Fox, 1994).

32

In addition some hemorrhagins also possess other biological activities. For example,

myonecrosis (Bilitoxin and baH1), fibrinogenolytic (Atrolysin f, Jararhagin), inhibition of

platelet aggregation (Atrolysin a) etc. (Ownby et al., 1990; Kamiguti et al., 1991; Gutierrez

et al., 1995; Jia et al., 1997). Many hemorrhagic toxins have been purified and

characterized biochemically from the venoms of Bothrops asper (Franceschi et al., 2000),

and Bothrops lanceolatus (Neto and Marques, 2005).

Protease

Proteases [E.C. 3.4.21.40] are present in most of the venoms except for hydrophidae

venoms. All viperid venoms are reported to be rich in proteolytic enzymes. The majority of

toxic effects of viperid (pit vipers, including Rattlesnakes, Water moccasins, Puff adder)

envenomation are due to “proteases”. These are actually hyrdolases that primarily act to

breakdown proteins and thus are also serve a digestive role. Further they are responsible

for most of the local tissue damage following envenomation. The important proteolytic

enzymes are endopeptidases, peptidases, arginine ester hydrolases, kininogenases,

procoagulants and anticoagulants. Some of them are known to induce various

pharmacological effects. For example: Proteinase and arginine ester hydrolases induce local

capillary damage and tissue necrosis (Kini and Evans, 1992, Matsue et al., 2000; Gutierrez

and Rucavado 2000; Gutierrez et al., 2005). Proteases in addition, also have coagulant and

hemorrhagic effects (Markland, 1998; Lu et al., 2005). Kinin releasing enzymes

(kininogenase) are responsible for the induction of pain and acute hypotension due to the

release of vasoactive peptides (Matsui et al., 2000; Felicori et al., 2003; White, 2005).

Endopeptidases are mainly found in viperid venoms. A common feature of venom

endopeptidase is that they are metalloproteases, capable of hydrolyzing peptide bonds with

amino groups contributed by leucine and phenylalanine residues. Endopeptidases can easily

be inactivated by EDTA and reducing agent such as cysteine (Iwanaga and Suzuki, 1979).

Venom endopeptidase catalyzes the hydrolysis of peptide bonds of a variety of natural and

synthetic substrates, including casein, hemoglobin, gelatin, elastin, co llagen, fibrinogen,

insulin, glucagons and bradykinin (Liu and Huang, 1997; Gutierrez et al., 2005).

Endopeptidases, which exhibit hemorrhagic activity, have been isolated from several

venoms such as Trimeresurus gramineus (Ouyang and Shiau, 1970), Agkistrodon acutus

(Xu et al., 1981), Crotalus horridus (Civello et al., 1983), Bothrops neuwiedi (Mandelbaum

33

et al., 1984) and Crotalus atrox (Hagihara et al., 1985). The hemorrhagic effect is attributed

to enzymatic disruption of the basement membrane with loss of integrity of the vessel wall

(Hati et al., 1999; Gutierrez and Rucavado, 2000). However, still it has to be established

whether the hemorrhagic activity is due to direct action of basement membrane or indirectly

by the release of a tissue factors which can be responsible for the disruption. The details of

venom hemorrhagic toxins will be discussed later under hemorrhage.

Snake venom proteases are a heterogeneous group of proteins with a wide range of

molecular masses between 15 – 380 kDa (Kini and Evans, 1992). They are single chain

proteins (Evans, 1984) and several other enzymes are multi subunits proteins (Zaganelli et

al., 1996; Fry, 1999). Proteases so far isolated are generally classified by the structure into

(1) serine proteases and (2) metalloproteases. There is only a weak or indirect evidence for

the presence of thiol proteases and aspartic proteases in the venoms. Some of them are seen

to degrade mammalian tissue proteins at the site of bites in a non-specific manner to

immobilize the victims. A number of them, however, cleave some of plasma proteins of the

victims in a relatively specific manner to give potent effects, as either the activators or the

inhibitors, on their hemostasis and thrombosis, such as blood coagulation, fibrionolysis and

platelet aggregation (Matusi et al., 2000; Andrews et al., 2004; Marsh and Williams, 2005).

According to the recent inventory of snake venom proteases, more than 150

different proteases have been so far purified, either completely or partially. The complete

amino acid sequences of about 40 of those proteases have been determined by protein

sequencing or deduced from the nucleotide sequence of the cDNA. Recently, the three-

dimensional (3D) structures of five venom proteases, four metalloproteinases (Gomis-Ruth

et al., 1993; Kumasaka et al., 1996; Gong et al., 1998) and one serine protease (Parry et al.,

1998), have been determined by X-ray crystallographic analysis and this has made it

possible to understand their structure-function relationship in more detail.

A number of venom proteases degrade fibrinogen and effect blood coagulation

through both pro and anticoagulant mechanisms (Markland, 1998; Andrews et al., 2004).

Proteases from the venoms of Trimeresurus flavovirides (Kosugi et al., 1986), Bitis

gabanica (Pirkle et al., 1986), Cerastes vipera (Farid et al., 1989), Trimeresurus stejnegeri

(Zhang et al., 1998) and Agkistrodon caliginosus (Cho et al., 2001) induce plasma

coagulation, while proteases from Naja nigricollis (Evans, 1981), Bothrops castelnaudi

(Kamiguti et al., 1985), Echis carinatus (Teng et al., 1985), Viper lebetina (Siigur and

Siigur, 1991) venoms prolongs the coagulation of plasma. However, snake venom proteases

34

with fibrinogenolytic, and anti-clotting properties find potential application in drug

development to treat thrombotic disorders, which result in fatal, heart attacks and strokes.

The pathogenesis of venom induced hemorrhage involves the direct damage to

microvessels, performed by hemorrhagic toxins, combined with a wide variety of effects

that viperid venom exert on hemostasis (Bjarnason and Fox, 1994; Markland, 1998). Thus

microvessel disruption and hemostatic disturbances act synertically to provoke profuse

bleeding in viperid snakebites, although hemorrhagic toxins by themselves are able to

induce bleeding in the absence of hemostatic alterations (Kamiguti et al., 1996; Escalante et

al., 2003). Snake venom hemorrhagic toxins are zinc dependent metalloproteinases which

belong on the family of ‘metzincins’, together with astacins, serralysins, matrix

metalloproteinases (MMPs) and ADAMs (enzymes with a disintegrin and

metalloproteinases domains). With few exceptions, these proteinases contain similar zinc

binding motif on their catalytic domain, characterized by the sequence HEXXHXXGXXH,

followed by a Met-turn (Bode et al., 1993).

Hemorrhagic metalloproteinases play another fundamental role in snake venom

induced muscle pathology, since they drastically affect skeletal muscle regeneration. After

a variety of injuries leading to necrosis, skeletal muscle tissue can regenerate due to

activation of satellite cells, which are myogenic cells located beneath of basal lamina of

muscle fibers. Besides inducing hemorrhage, myonecrosis and skin pathology, venom

metalloproteinases play a relevant role in the complex and multifactorial inflammatory

response characteristic of snakebite envenomation. In addition, metalloproteases degrade

extracellular matrix components and impair the regeneration of affected skeletal muscle.

Some of them also affect platelet function, through their disintegrin- like domain, and

degrade blood-clotting factors, precluding a normal hemostatic response after microvessel

damage.

Due to the protagonic role of metalloproteinases in the pathogenesis of venom

induced local effects, their inhibition by antivenoms and natural and synthetic inhibitors is a

key aspect in the treatment of these envenomations. Due to the rapid onset of these local

effects, and to the frequent delay in antivenom administration, neutralization of these

effects by antivenoms is only partial (Gutierrez et al., 1990; 1995), even when using

antibody fragments (Leon et al., 2000). The development of potent synthetic matrix

metalloproteinase inhibitors, some of which are being tested in clinical trials of other

pathologies opens the possibility of using them in snakebite envenomations (Gutierrez et

35

al., 1996). It has been recently shown that batimastat, a synthetic metalloproteinase

inhibitor, is effective at counteracting the local tissue damage induced my Bothrops asper

metalloproteinase BaPI, provided the inhibitor is administered at the site of venom injection

rapidly after toxin injection (Escalante et al., 2000). The search of new alternatives to

reduce local effects medicated my metalloproteinases in snakebites is a highly relevant task.

Myotoxicity

Myotoxicity is a common and often a serious consequence of snake venom

poisoning. Local hemorrhage and necrosis affecting the skin and muscle layers are the

chief manifestations of myotoxicity. Myotoxicity due to direct action of myotoxins

(enzymatic / non-enzymatic) on muscle cells, cause extensive muscle damage resulting in

weakness of muscle and pain full restriction of movements with muscle tenderness. The

magnitude of nefarious systemic effects directly rely on the concentration and also diffuse

into systemic circulation from the site of injections and intern to their sites of action.

However, this precedes local effects, with accomplished local tissue damage due to

degradation of extracellualr matrix connective tissue surrounding blood vessels and

capillaries by enzyme such as hyaluronidase and hemorrhagic metalloproteinases.

Myotoxicity may be due to the vascular degeneration and ischemia caused by

venom metalloproteinases, or it may result from a direct action of myotoxins upon the

plasma membrane of muscle cells, which is evident from the rapid release of cytoplasmic

markers, creatine kinase (CK) and lactate dehydrogenase (LDH) accompanied by the

prominent increase in total muscle calcium ion (Rucavado and Lomonte, 1996; Gutierrez

and Lomonte, 1989; Gopalkrishnakone et al., 1995; Souza et al., 2000). The increased

influx of calcium ion leads to the cell death (Mebs and Samejima, 1980). Intramuscular

injection of many hemorrhagic metalloproteinases results in acute muscle cell damage, i.e.,

myonecrosis (Gutierrez et al., 1995; Franceschi et al., 2000). The mechanism by which

venom metalloproteinases induce muscle damage has not been fully elucidated. However,

Gutierrez et al., (1995), investigating the action of hemorrhagic metalloproteinases BaG1

from Bothrops asper venom, suggests that muscle damage was secondary to the ischemia

that ensues in skeletal muscle as a consequence of bleeding. Several observations supported

this hypothesis:

36

a) Myonecrosis was observed only in vivo, and no cell

damage occurred when isolated gastrocnemius muscle was

incubated with BaH1 in vitro, in conditions of adequate

oxygen supply.

b) There was an increment in the muscle contents of lactic

acid, a biochemical indicator of ischemia.

c) Histopathological evidence of myonecrosis was observed

at relatively late time intervals after BaH1 injection, i.e.,

after 6 hr, whereas hemorrhage develops within minutes.

This observation suggests that muscle damage occurs

secondarily to hemorrhage.

Myotoxicity is associated with many presynaptically acting neurotoxins

(Gopalakrishnakone et al., 1980; Ziolkowske and Bieber, 1992). In addition, several

myonecrotic polypeptides and myotoxic PLA2 enzymes have been isolated and

characterized from various snake venoms (Fohlman and Eaker, 1977; Harris and Maltin,

1982; Mebs, 1986; Mebs and Samejima, 1986; Kasturi and Gowda, 1989; Weinstein et al.,

1992; Lomonte et al., 1994a,b; Thwin et al., 1995; Ownby et al., 1997; Radis-Baptista et

al., 1999; Nunez et al., 2001).

Edema inducing activity

Swelling and edema are early clinical features observed in snake venom poisoning

at the affected part of the victim. The edema is a result of increased vascular permeability

resulting in the accumulation of fluids in the interstitial space. The action on the vessels is

brought about by either the direct action of venom toxins affecting the microvasculature

(Chaves et al., 1995) or more commonly by the formation of autocoids and other vasoactive

compounds by the PLA2 action of the toxins.

The edema induced by Bothrops jararaca venom is mediated by cyclooxygenase

and lipoxygenase eicosanoid products, and by the action of L1 and L2 adrenergic receptors

(Trebien and Calixto, 1989).Pretreatment with indomethacin, a well-known inhibitor of the

cyclooxygenase pathway reduced the edema induced by Bothrops asper and Bothrops

jararaca venoms. It is suggested that PLA2 induce by two different mechanism (a) by

releasing arachidonic acid as a membrane phospholipids, leading to the biosynthesis of

37

eicosanoids and (b) by directly affecting the microvasculature, there by causing plasma

exudation (Chaves et al., 1995).

PLA2s are cytotoxic to mast cells and cause their degranulation. Degranulation

releases physiological mediators like histamine, serotonin, leukotriens, which increases

vascular permeability (Bhat et al., 1991; Kasturi and Gowda, 1992; Camargo et al., 2005).

The venoms of Trimeresurus flavoviridis (Vishwanath et al., 1987; Yamaguchi et al.,

2001), Trimeresurus mucrosquamatus (Teng et al., 1989), Vipera russelii (Kasturi and

Gowda, 1989), 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.

Hyaluronidase

Hyaluronidase [E.C. 3.2.2.35], an endoglycosidase has been considered as an

invariant factor in the venom of snakes and is frequently refereed as a “spreading factor”.

Kary Mayer introduced the term “ Hyaluronidase” in 1940 to denote the enzyme that

degrades hyaluronan. In general Hyaluronidase are groups of glycosidases that have

recently taken on greater significance due to the increasing attention being given to their

substrate hyaluronan (HA).

The spreading property has been evident from its ability to promote the local hemorrhagic

effect of a toxin from Trimeresurus flaoviridis venom (Tu and Hendon, 1983). Degradation

of hyaluronan in the extracellular matrix of local tissues is pressured to be the key event in

the enzyme mediated spreading process during snake envenomation. Although previous

studies report the in vitro degradation of hyaluronan (Xu et al., 1982; Pukrittayakamee et

al., 1983; Kudo and Tu, 2001). Recently, Girish et al., (2004) demonstrated the degradation

of extracellular matrix in humans and other tissue samples. This property has been

attributed for the fast diffusion of other lethal toxins. The spreading action of

hyaluronidases for venom hemorrhagic toxins has been studied in detail. Hyaluronidase,

because of the degradation of extracellular matrix is considered an important enzyme in

inducing local tissue damage. Its toxicity is due to the synergetic action associated with the

other venom toxins (Girish et al., 2004). Regulation of this enzyme is highly beneficial in

neutralizing the venom toxicities. In addition, information on the existence of isoforms of

hyaluronidase in snake venoms is highly restricted. Although few studies report the

38

isolation and characterization of this enzyme from the venom of snakes such as Agkistrodon

acutus (Xu et al., 1982), Vipera russelii (Pukrittayakamee et al., 1983) Agkistrodon

contortrix contortrix (Kudo and Tu, 2001) and Girish et al., (2004) reported the presence of

isoenzymes of hyaluronidase in Naja naja venom.

Endogenous Purine releasing enzymes in Snake venoms

Apart from these some of the ubiquitous enzymes present in venoms are known to

endogenously release purines a multi toxin, which is involved in snake envenomation

stratergies. The enzymes can be broadly classified into nucleases, phosphomonoesterases

and nucleotidases.

The lack of interest among toxinologists in these enzymes seems to be because of

the assumption that they were only involved in digestion and that they were non-toxic.

However, recently there is renewed interest among toxinologists in these enzymes, as they

are known to endogenously liberate purines, which acts as a multitoxin (Aird, 2002, 2005).

The identification of free purines as endogenous constituent of venoms has further

supported the role of purinergic signaling in envenomation (Lumnsden et al., 2004; Aird,

2005). Purines are known to potentiate venom-induced hypotension and paralysis (Aird,

2002) via purine receptors, which are ubiquitously distributed among various organisms

envenomed by snakes (Ralevic and Burnstock, 1998; Burnstock, 2006; Sawynok, 2007;

Aird, 2005). In addition, some of the reports also suggest toxic nature of these enzymes

either acting independently or synergistically with other toxins, contributing to the overall

lethal effects of venoms (Boffa and Boffa, 1974; Ouyang and Huang, 1983, 1986; Aird,

2002, 2005; Dhananjaya et al., 2006).

The distribution of these enzymes in snake venoms, their catalytic mechanisms and

assay systems to determine their activities have been described in detail in reviews

(Iwanaga and Suzuki, 1979; Mackessay, 1998; Rael, 1998). Only few reviews suggest the

possible pharmacological actions of these enzymes (Arid, 2002). The components that are

known to endogenously release purines can be classified as Nucleases (DNases, RNases,

phosphodiesterases), Nucleotidases (5`Nucleotidases, ADPases and ATPases) and

Phosphatases (Acid and alkaline phosphomonoesterases).

39

I. Nucleases

Nucleases are enzymes that act on nucleic acids (DNA/RNA) and their derivatives.

Snake venom nucleases are classified as endonucleases and exonucleases. Endonucleases

include DNases, which specifically hydrolyze DNA, and RNases, which specifically

hydrolyze RNA. Exonucleases include phosphodiesterases (PDE), which hydrolyze both

DNA and RNA. They are also known to exhibit endonuclease activity (Mori et al., 1987;

Stoynov et al., 1997). An endonuclease activity in snake venom was first reported by

Delezenne and Morel (1919). Differentiating between specific venom endonuclease activity

and PDE activity is difficult since endonuclease activity is an inherent property of venom

PDE (Mori et al., 1987; Stoynov et al., 1997). Hence, most of the reported endonuclease

activities may actually be due to PDE action (Sittenfeld et al., 1991; de Roodt et al., 2003).

In order to differentiate PDE from endonucleases, biochemical parameters have to be

considered in addition to substrate specificities.

Even though endonucleases and PDE hydrolyze both DNA and RNA they exhibit

distinct pH optima and metal ion requirement. An unique venom protein with an acidic pH

optimum that does not require divalent cations for the hydrolysis of DNA or RNA has been

considered as an endonuclease (Georgatsos and Laskowski, 1962; Mackessay, 1998)

whereas, all PDEs are active at basic pH and require divalent metal ion for activity

(Iwanaga and Suzuki, 1979; Mackessay, 1998). The DNase activity reported by Sittenfeld

et al., (1991) may be due to the action of phosphodiesterase, since the activity was

measured at pH 7.0 using calf thymus DNA. A More recent study by de Roodt et al., (2003)

showing DNase activity using plasmid and calf thymus DNA in a zymogram is likely to be

PDE rather than DNase since EDTA was shown to inhibit the act ivity. Specific

endonuclease activity with a pH optimum of 5.0 in addition to phosphodiesterase activity at

basic pH optimum of 8.9 in the same venoms has been reported (Georgatsos and

Laskowski, 1962; Vasilenko and Babkina, 1965; Vasilenko and Rait, 1975; Mahalakshmi

and Pandit, 1987; Mahalakshmi et al., 2000). These data clearly indicate that the PDEs are

distinctly different from endonucleases. However, PDE and exonuclease activity is also

difficult to differentiate since there are no reports describing exclusive exonuclease activity

in snake venoms. Thus, venom exonuclease activity is attributed to PDE.

40

DNases (E.C. 3.1.21.1)

Relatively few studies have been carried with regard to specific DNases; as a result

it is difficult to say how widely they are distributed among snake venoms. A DNase activity

with pH optimum of 5.0 was purified from Bothrops atrox venom (Georgatsos and

Laskowski, 1962). However, it was interesting to note that this preparation also showed

activity towards RNA and poly-AU, in addition to DNA. During the course of preparation

of PDE from C. adamentus venom, an endonuclease activity was separated from

exonuclease activity (Laskowski, 1980). Since the main aim of the author was to eliminate

contaminating nucleases activity, very little is known about this isolated enzyme. This

study is important as it indicates the presence of a DNase activity in venoms, distinct from

PDE. No biological activity has been assigned to venom DNases apart from its role in

digestion.

RNases (E.C. 3.1.21.-)

Like DNases, RNases are also not well characterized. A specific ribonuclease was

isolated from the venom of naja oxiana hydrolyzing double stranded RNA (now called as

RNase V1). The enzyme was shown to hydrolyze RNA without showing any base

preference and produced oligonucleotides of 2 - 4 bases, which terminated in a 5`-

phosphate (Vasilenko and Babkina, 1965; Vasilenko and Rait, 1975). More recently, an

RNase with specificity for polycytidine was purified from Naja naja venom (Mahalakshmi

and Pandit, 1987; Mahalakshmi et al., 2000). Both these enzymes had an apparent

molecular weight of ~14 to 16 kDa. Although the authors claim that the RNase preparation

from N. naja did not show phospholipase and phosphodiesterase activity, its N-terminal

sequence was identical to that of PLA2. None of the endonucleases are reported to exhibit

any pharmacological activities. The properties of endonucleases purified from various

snake venoms are given in Table 1.08.

Phosphodiesterase (EC. 3.1.4.1)

They are known to catalyze the hydrolysis of phosphodiester bonds in a progressive

fashion beginning at the 3’ end of polynucleotides, liberating 5` mononucleotides at basic

pH. Uzawa, (1932) was the first one to describe Phosphodiesterase (PDE) activity in snake

venoms. Since then, PDE activity has been surveyed among wide variety of taxa, and found

41

to be ubiquitously distributed in snake venoms (Iwanaga and Suzuki, 1979; Mackessay and

Tu, 1993; Mackessay, 1998, 2002; Aird, 2002 and references their in). Crotalid and viperid

venoms are known to contain higher PDE activity than elapid venoms (Mackessay, 1998;

Aird, 2005).

PDE act on several native substrates like DNA, rRNA, and tRNA without showing

any preference for purine or pyrimidine bases. It is shown that the native DNA is a better

substrate than denatured DNA (Iwanaga and Suzuki, 1979). They also hydrolyze

oligonucleotides including polyadenylic acid (Philipps, 1976) and cyclic nucleotides

(Iwanaga and Suzuki, 1979). In addition, PDE also hydrolyze adenosine 5`-tetraphosphate,

TDP-rhamnose, UDP-glucose, GDP-mannose, poly ADP-ribose, NAD+, NADP+, and other

nucleic acid derivatives (Iwanaga and Suzuki, 1979). They also hydrolyze ATP and ADP,

liberating adenosine (Perron et al., 1993; Mackessy, 1989).

Venom PDE have been isolated and characterized from numerous species of snakes.

The properties of several purified venom PDE`s are summarized in Table. 1.09. In general,

unlike RNase, PDE are high molecular mass (> 90 kDa), single polypeptide chain proteins.

However, some exist as homodimers (Perron et al., 1993; Mori et al., 1987; Mackessay,

1989). They may be present in multimolecular forms or in only one form (Phillipps, 1975;

Mori et al., 1987; Kini and Gowda, 1984). All PDE are metallo enzymes, as metal chelators

are generally known to inhibit PDE activity (Iwanaga and Suzuki, 1979; Francis et al.,

1992; Freitas et al., 1992; Mackessay, 1998 and references therein). Mori et al., (1987)

showed that Crotolus ruber ruber PDE contained 1.04 mol of zinc per mol of enzyme.

Further, zinc is also shown to be inhibitory at higher concentrations (Sugihara et al., 1986;

Mori et al., 1987; Valerio et al., 2003). It is suggested that zinc is necessary for catalysis

whereas calcium and magnesium are involved in substrate binding (Dolapchiev et al.,

1980). Isoforms of PDE are known to exist in Vipera palastinae and Trimeresures

flavoviridius venoms (Levy and Bdolah, 1976; Kini and Gowda, 1984). Although, PDEs

have been isolated from several venoms, there is no information on amino acid or full-

length cDNA sequence. However, The Expressed Sequence Tags (EST) generated from

cDNA library of Deinagkistrodon acutus, Lachesis muta and Sistrurus catenatus edwardsi

species were found to have representatives for PDE gene (Junqueirs-de-Azevedo et al.,

2006; Oinghua et al., 2006; Pahari et al., 2006). The accession number of

Phosphodiesterase EST`s from various snakes species are given in Table 1.10.

Although venom PDEs are widely distributed among several snake taxa, only few

studies have investigated the biological activity of this near ubiquitous venom component.

42

An earlier study by Russell et al., (1963) showed a reduction in mean arterial pressure

(MAP) and locomotor depression with partially purified PDE preparation from several

snake venoms. This rapid reduction in MAP and locomotor depression can be assumed to

be due to the reduction of cAMP levels. Although this preparation had contaminating

proteins, this study is significant because it indicates that even in the absence of cellular

disruption there is adequate substrate available for the enzyme PDE in the circulation to

cause profound hypotension. Though PDEs are known to hydrolyze wide variety of

biologically important nucleotides such as ATP, NAD+, NADP+ and GDP, this enzyme has

not been investigated for other potential pharmacological activities.

II. Phosphomonoesterases

These are enzymes that hydrolyze phosphate esters non-specifically. Uzawa (1932)

first described the existence of non-specific phosphomonoesterases in snake venoms. The

acid phosphomonoesterases (E.C.3.1.3.1) are active at pH 5.0 and alkaline

phosphomonoesterases (E.C. 3.1.3.2) are active at pH 9.5. It is apparent that the two

enzyme activities (acid/alkaline) pertain to different enzymes because some venom contains

both the activities while others contain only one (Tu and Chua, 1966; Iwanaga and Suzuki,

1979; Rael, 1998). Of the two phosphatases, alkaline phosphomonoesterase seems to be

widely distributed and abundant in snake venoms compared with acid

phosphomonoesterases (Uwatoko-Setoguchi, 1970; Iwanaga and Suzuki, 1979; Sifford et

al., 1996; Rael, 1998). ALP activity has been surveyed among wide variety of snake taxa

and found ubiquitously distributed in snake venoms (Iwanaga and Suzuki, 1979;

Mackessay and Tu, 1993; Rael, 1998; Mackessay, 2002; Aird, 2002 and references their

in). It has been found that crotalid and elapid venoms contain more ALP activity than

viperid venoms (Rael, 1998; Aird, 2005).

ALP is known to hydrolyze non-specifically ribo- and deoxy ribonucleotides with

different rates. It is known to hydrolyze 5`AMP, 5`-dAMP, 3`AMP, ribose 3-phosphate,

ATP, deoxy-dinucleotide phosphates, dGDP, FMN and 5`-phosphoribose 1- pyrophosphate

(Sulkowski et al., 1963).

Although, ALP is known to be ubiquitously present in snake venoms, only few

attempts have been made to purify it (Sulkowski et al., 1963; Suzuki and Iwanaga, 1958 a,

b). In general, snake venom ALP is known to be high molecular weight protein (> 90 kDa)

(Iwanaga and Suzuki, 1979; Acosta et al., 1994; Rael, 1998). They are metalloenzymes,

43

since their activities are inhibited by metal ion chelators (Iwanaga and Suzuki, 1979;

Hassan et al., 1981; Francis et al., 1992; Acosta et al., 1994; Rael, 1998).

In contrast, acid phosphomonoesterases have been only partially purified from sea

snakes (Uwatoko-Setoguchi, 1970). These also require metal ion for activity (Uwatoko-

Setoguchi, 1970; Sifford et al., 1996). The enzyme is known to be active on various

substrates and is known to differ from ALP in that glucose-1-phosphate, glucose-6-

phosphate and glycerophosphates are not hydrolyzed (Uwatoko-Setoguchi, 1970).

We have not come across any reports isolating and examining biological activity of acid or

alkaline phosphomonoesterases from snake venom. However, bee venom acid

phosphomonoesterases is considered as an allergen, and known to be potent releaser of

histamine from sensitized human basophils (Barboni et al., 1987; Grunwald et al., 2006).

III. Nucleotidases

Nucleotidases are enzymes that act upon nucleic acid derivatives/nucleic acid

related substrates like ATP, ADP and AMP. Since many enzymes in venoms are known to

act on similar substrates, specific differentiation of nucleotidases is again difficult. It has

been found that snake venoms contain both non-specific phosphomonoesterases as well as

5`Nucleotidases, which specifically liberate phosphate upon hydrolysis of nucleotides. It

has been observed that though 5`Nucleotidase selectively hydrolyzes 5`nucleotides to

nucleosides; these substrates are also acted upon by alkaline phosphomonoesterases (ALP)

present in venoms (Sulkowski et al., 1963). Further it has been shown that both are metal

ion dependent and are active at basic pH (Rael, 1998). However, these two enzymes are

differentiated based on their substrate specificity. 5`Nucleotidase is not active on 3`AMP,

ribose-5-phosphate, mononucleoside 3`, 5`-diphosphates or higher nucleotides, but these

are acted upon by ALP (Sulkowski et al., 1963; Rael, 1998). Other specific nucleotidases

found in venoms are ATPases and ADPases. There is uncertainty about the existence of

specific ATPase and ADPase, since venom PDE is also known to hydrolyze ATP and ADP

(Mackessy, 1989; Perron et al., 1993). Further, both ATPase and PDE are metal ion

dependent and active at basic pH (Kini and Gowda, 1982 a, b; Mackessay, 1998). Thus the

role of these enzymes is so controversial, that the inhibitory effect exhibited by purified

protein on platelet aggregation is attributed to PDE by some (Mackessay, 1998) and to

ADPases by others (Ouyang and Huang, 1986; Kini, 2004). Interestingly, a purified protein

44

had exhibited both PDE activity and ADPase activity along with a weak 5`Nucleotidase

activity (Ouyang and Huang, 1986). Also, differentiation of these nucleotidases has become

more complicated since T. gramineous 5`Nucleotidase is also known to exhibit ADPase

activity (Ouyang and Huang, 1983). Pereira Lima et al., (1971) claimed that ATPase is

distinct from PDE because they found disproportionate levels in different venoms;

however, others have found that these two enzymes are proportionately distributed

(Pfleiderer and Ortlanderl, 1963). From these studies it appears that in snake venoms either

a single protein could have different domains to perform different activities or truly specific

nucleotidases could exist in venoms. So far there are no reports characterizing a specific

5`Nucleotidases/ATPases/ADPases and demonstrating that they are distinct from each

other. Since they exhibit overlapping properties and substrate preferences, there is a

possibility that different laboratories could have reported the same enzyme differently.

Among nucleotidases, 5`Nucleotidase is better studied when compared with ATPases and

ADPases. Although it appears logical that immobilization of prey could be achieved by

depletion of ATP by the action of nucleotidases, this aspect has not been verified

experimentally.

ATPases (E.C. 3.6.1.-)

These enzymes hydrolyze ATP forming adenosine and pyrophosphate as reaction

products (Johnson et al., 1953; Iwanaga and Suzuki, 1979). Zeller, (1950), showed for the

first time that snake venom on incubation with ATP resulted in the liberation of

pyrophosphate. Since then ATPase activity has been reported from several snake venoms

(Zeller, 1950; Johnson et al., 1953; Schiripa and Schenberg, 1964; Setoguchi et al., 1968;

Pereira Lima et al., 1971; Wei et al., 1981; Kini and Gowda, 1982 a, b; Mukerjee et al.,

2000). Depending upon experimental conditions, the enzyme is known to hydrolyze ATP

either into AMP and pyrophosphate or ADP and phosphate (Zeller, 1950).

Though ATPase activity is widely distributed, only a few attempts have been made

to isolate and characterize it. Kini and Gowda (1982 a, b) partially purified toxic ATPases

from N. naja and Daboia russelii venoms. They observed that ATPases of D. russelii

(ATPase -I and -II) and N. naja venoms were Mg2+ ion dependent and basic in nature.

However, it was interesting to note that ATPase -I was a glycoprotein while, ATPase -II did

not contain any carbohydrate. However, characterization of the ATPases was not done in

detail by the authors since their aim was to study the interaction of plant isolates with toxic

venom proteins.

45

Since ATPase enzymes are not purified from snake venoms, specific biological

activity has not been assigned to them. Zeller (1950) termed ATPase to be “toxic” as

ATPase was thought to be involved in production of shock symptoms by depletion of ATP.

Although it appears logical that immobilization of prey/victim could be achieved by

depletion of ATP by the action of ATPases along with other nucleotidases, this aspect has

not been verified experimentally.

ADPases (E.C. 3.6.1.-)

They catalyze the hydrolysis of ADP to adenosine and orthophosphate as reaction

products (Johnson et al., 1953; Iwanaga and Suzuki, 1979). ADPase activity has been

observed in several snake venoms (Schiripa and Schenberg, 1964; Setoguchi et al., 1968;

Boffa and Boffa, 1974; Sekiya et al., 1975; Ouyang and Huang, 1986).

ADPase isolated from A. acutus venom, had a molecular weight of 94 kDa, it was

basic in nature and was known to inhibit platelet aggregation induced by ADP, collagen and

sodium arachidonate in platelet rich plasma. Although it strongly inhibited ADP induced

platelet aggregation, it did not inhibit thrombin- induced aggregation in PPP (Ouyang and

Huang, 1986). This protein was known to possess both phosphodiesterase and weak

5`Nucleotidase activities. The inhibition of platelet aggregation was assumed to be due to

the generation of adenosine, which is known to inhibit platelet aggregation. Vipera aspis

ADPase has been shown to be the most potent inhibitor of ADP induced platelet

aggregation among others (Boffa and Boffa 1974). It was found that inhibitory effect was

not disassociated from enzymatic activity. The inhibitory ac tion was explained by the

formation of inhibitory AMP or adenosine by the action of the enzyme. It is possible that a

synergistic interaction of ADPases with hemorrhagic proteases and fibrinogenases found in

the same venom occurs during envenomation, interfering with normal hemostatic

mechanisms, promoting blood loss and circulatory collapse in the prey/victim. Properties of

ADPase purified from various snake venoms are given in Table 1.11.

Snake venom 5`Nucleotidase (E.C. 3.1.3.5)

The enzyme 5`Nulcleotidase preferentially catalyses the hydrolysis of phosphate

esterified at carbon 5` of the ribose and deoxyribose of nucleotide molecules. Gulland and

Jackson (1938) were the first to show the presence of 5`nucelotidase activity in snake

venoms. Since then, 5`Nucleotidase activity has been surveyed among wide variety of taxa

46

and found ubiquitously distributed in snake venoms (Iwanaga and Suzuki, 1979;

Mackessay and Tu, 1993; Rael, 1998; Mackessay, 2002; Aird, 2002 and references their

in). It has been found that viperid and crotalid venoms contain more 5`Nucleotidase activity

than elapid venoms (Rael, 1998; Aird, 2005).

5`Nucleotidase is known to cleave a wide variety of ribose and deoxyribose mono

nucleotides, including 5`-AMP, 5`IMP, 5`UMP, 5`CMP, 5`GMP, 5`dAMP, 5`-dTMP, 5`-

dCMP, 5`dGMP, nicotinamide mononucleotide, and a number of hydroxylated, methylated

and halogenated substrates (Sulkowski et al., 1963; Rael, 1998). It has also been shown that

they hydrolyze ADP, thus exhibiting ADPase activity (Ouyang and Huang, 1983).

However, 5`Nucleotidase prefers 5`AMP as substrate, releasing adenosine as end product

(Rael, 1998; Aird, 2002, 2005; Dhananjaya et al., 2006). It does not cleave ribose 5`-

phosphate, 3`AMP, flavin mononucleotide or cAMP (Rael, 1998).

Only few studies have attempted to purify and characterize 5`Nucleotidase from

snake venom. The properties of 5`Nucleotidase purified from various snake venoms are

given in Table 4. 5`Nucleotidases are high molecular weight species having Mol. Wt

between 73 -100 kDa (Chen and Lo, 1968; Dieckhoff et al., 1985; Ouyang and Huang,

1983, 1986). In general, venom 5` nucleotidases are metalloenzymes, since metal chelators

are known to inhibit the enzyme activity (Iwanaga and Suzuki, 1979; Ouyang and Huang,

1983; Francis et al., 1992; Freitas et al., 1992; Rael, 1998). Fini et al., (1990) using flame

atomic absorption spectrometry, showed that the Zn/protein ratio was 1.85- 2 zinc atoms

per mol of protein. Further, zn2+ is also known to inhibit enzymatic activity (Lin and Lin-

Shiau, 1982; Ouyang and Huang, 1983). Thus it can be concluded that zn2+ containing site

may be the active site for its enzyme activity. Though there is no report claming the

existence of isoforms, the existence of multi-molecular forms is reported in venoms

(Mannherz and Magener, 1979; Ouyang and Huang, 1983; Dhananjaya et al., 2006).

Although there is no information on amino acid or full- length cDNA sequence for venom

5`Nucleotidase, ESTs generated from cDNA library of Bothrops Insularis, L. muta, and D.

acutus species are shown to have representatives for 5`Nucleotidase gene (Junqueirs-de-

Azevedo and Ho, 2002; Junqueirs-de-Azevedo et al., 2006; Oinghua et al., 2006). The

accession numbers of 5`Nucleotidase EST’s from the different snakes species are given in

Table 1.12.

Although 5`Nucleotidase is widely distributed among venomous snake taxa, there is

a lack of information about their biological activities. The Agkistrodon acutus and T.

gramineus 5`Nucleotidase is shown to inhibit platelet aggregation (Ouyang and Huang,

47

1983; Ouyang and Huang, 1986). T. gramineus 5`Nucleotidase inhibited platelet

aggregation induced by ADP, collagen, sodium arachidonate and ionophore-A-23187 in

Platelet Rich Plasma (PRP), and by thrombin in Platelet Poor Plasma (PPP) (Ouyang and

Huang, 1983). This protein also exhibited ADPase activity. However, A. acutus

5`Nucleotidase in addition to collagen, sodium arachidonate induced platelet aggregation,

inhibited ADP induced platelet aggregation by 36 % (Ouyang and Huang, 1986). This

protein did not possess ADPase activity or PDE activity. This inhibitory action of venom

5`Nucleotidases on platelet aggregation is correlated with the liberation of adenosine by its

enzymatic action. Therefore, the decreased inhibitory action of A. acutus 5`Nucleotidase

when compared with T. gramineus 5`Nucleotidase on platelet aggregation could be because

of the absence of associated ADPase activity in A. acutus 5`Nucleotidase. Boffa and Boffa,

(1974) while investigating on factors affecting blood coagulation and platelet function from

Vipera aspis, showed that a component displaying ADPase/5`Nucleotidase activity was the

most potent inhibitor of ADP-induced platelet aggregation. It was found that the inhibitory

effect was not dissociated from enzymatic activity. Thus, it can be implied that the anti-

platelet aggregatory effect of 5`Nucleotidase may be due to the liberation of inhibitory

AMP or adenosine by its action on ADP released upon initiation of aggregation. Venom

5`Nucleotidase is also known to synergistically act in vivo with other toxins like ADPases,

phospholipases and disintegrins to exert more pronounced anticoagulant effect (Jorge da

Silva and Aird, 2001). Recently, we have shown the involvement of 5`Nucleotidase in the

anticoagulant effect of Naja naja venom (Dhananjaya et al., 2006). It has been found that

N. naja 5`Nucleotidase interacts directly or indirectly with factors of intrinsic pathway to

cause anticoagulant effect. This study showed that the enzyme was capable of bringing

about the pharmacological action independent of catalytic activity. It is possible that during

envenomation 5`Nucleotidase synergistically acts with hemorrhagic proteases and

fibrinogenases found in the venom to affect normal hemostatic functions, leading to blood

loss and circulatory collapse in the prey/victim.

Plant 5`Nucleotidase

A membrane bound and cytosolic form of 5`Nucleotidase have been described in

plants. Characterization of the membrane bound form from peanut cotyledon (Sharma et al.,

1986; Mittal et al., 1988), Corn microsomes (Carter and Tipton, 1986) and soyabean root

nodules (Ostergaard et al., 1991) has shown it to have glycoiprotein membrane anchor and

48

the molecular mass to be 55 KDa with approximately 40 % carbohydrate content. This form

of the enzyme is highly specific for 5`AMP and has no metal ion requirements.

On the other hand, the soluble form can hydrolyse significantly 3`AMP (Polya,

1975; Carter and Tipton, 1986) or ADP and ATP (Chen and Kristopeit, 1981) eventhough

5`AMP is the preferred substrate. Characterization of this and other from from wheat germ

has shown to have molecular mass of 50-69 KDa, and 110 KDa respectively. However,

both forms show maximum activity between pH 5.0 – 6.0. 5`Nucleotidase from various

sources of higher plants varies considerably concerning functional properties. This can be

due to the species or tissue heterogeneity, as well as to the fact that preparations were only

partly purified.

Bacterial 5`Nucleotidase

Two forms of 5`Nucleotidase, a membrane bound and a cytosolic soluble form have

been identified in bacteria.

Intrinsic membrane bound 5`Nucleotidase is found in halophilic and marine bacteria

of genera vibrio and photo bacteria. This form of enzyme contains an N-terminal sequence

very similar to the sequence for the cleavage site of lipoproteins (Tamao et al., 1991).

However, the type of the enzyme anchor is not very clear. The apparent molecular weight is

found to be 70 KDa on SDS-PAGE. This enzyme hydrolyses substrates such as ATP, ADP

and AMP equally at an optimum pH of 8.0, in presence of Cl- and Mg2+ which may be

replaced by Co2+ or Mn2+. However Zn2+ has an inhibitory effect on this enzyme and

3`nucleotides are not substrates of this enzyme.

In Bacillus subtilis and Escherichia coli, 5`Nucleotidase is secreated into

periplasmic space and can be isolated as a soluble form called as periplasmic

5`Nucleotidase. This is also called as UDP-sugar hydrolyase as it can hydrolyze UDP

bound to the sugar such as UDP-D-Gluose, UDP-D-Galactose, UDP-N-acetly-D-

Glucosamine and UDP-N-acetly-D-Galactosamine to UMP and sugar or phosphorylated

sugar derivatives at an optimum pH of 7.0-8.0. Apart from these substrates, it can hydrolyse

all 5`ribo and 5`deoxyribonucleotides, their di and triphosphate forms with pre ference for

AMP Km value 1-130 mM, but at pH 6.0. This enzyme does not effect cyclic and 2`AMP.

The enzyme is a zinc metalloenzyme and is not inhibited by inorganic phosphate.

Two soluble cytosolic forms have been recognized in Bacillus subtilis based on

their requirement of Mg2+ for activation. The Mg2+ requiring forms has a molecular weight

of 23 KDa and can hydrolyze substrates such as nucleotide 5`monophosphates at an

49

optimum pH of 7.5. other divalent cations such as Zn2+, Cu2+ and Fe2+ strongly inhibit the

enzyme and it can not hydrolyse UDP bound sugars.

Yet another form of bacterial 5`Nucleotidase which does not hydrolyse sugar

phosphates has been isolated from Saccharomyces oviformis. This is capable of hydrolysie

not only ribo or deoxyribonucleotidase but also NAD, NADH, FAD, ATP and is said to

have nucleotide pyrophosphates activity. It converts NAD+, mto AMPand NMN, and

continues to hydrolyze AMP to adenosine and inorganic phosphate. Crystal structure are

known for cdN2 for E. coli periplasmic 5`Nucleotidase they share a DX-DX(V/T) motif

critical for catalysis and show structural similarity to the haloacid dehalogenase superfamily

of enzymes (Rinaldo-Matthis, 2002). IT is now known E. coli 5'-nucleotidase undergoes a

hinge-bending domain rotation resembling a ball-and-socket motion (Knofel and Strater,

2001) and this is necessary for the catalytic function of Escherichia coli 5'-nucleotidase

(Schultz-Heienbrok et al., 2005).

Mammalian 5`Nucleotidase

Mammalian 5`Nucleotidase has been classified into seven families Table. 1. 04

(Bianchi and Spychala, 2003). All of them have relatively broad substrate specificities. In

aggrement with the structural information on the active sites (Knofel and Strater, 1999;

Allegrini, 2001), all family members except eN are absolutely dependent on magnesium for

activity. Fig. 1.04 shows structure of active site of mdN. Table. 1.05 shows distinctive

features of mammalian 5`nucleotidase

Ecto-5` nucleotidase—eN, also known as CD73, is a glycosylated protein bound to the

outer surface of the plasma membrane by a glycosyl phosphatidylinositol anchor (Misumi

et al., 1990) and co-localizes with detergent-resistant and glycolipid-rich membrane

subdomains called lipid rafts. Up to 50% of the enzyme may be associated to intracellular

membranes (for review, see Zimmermann, 1992) and be released during homogenization.

Early reports of a soluble low Km nucleotidase (for review, see Zimmermann, 1992) were

because of this phenomenon (Piec amd Le Hir, 1991). Although eN has broad substrate

specificity, AMP is considered to be the major physiological substrate (for review, see

Zimmermann, 1992) (Yegutin et al., 2002; Resta et al., 1998; Spychala, 2000).

Independently of the enzymatic function, the protein acts as co-receptor in T cell activation

(for review, see Resta et al., 1998) and as cell adhesion molecule (for review, see Spychala,

2000). eN is variably expressed in a wide number of cell types under physiological and

50

pathological conditions (for review, see Zimmermann, 1992; Resta et al., 1998; Spychala,

2000). In neuronal cells eN expression is linked to developing or plastic states (for review,

see Spychala, 2000). The proximal promoter region of the gene contains a number of

tissue-specific elements (Hansen etal., 1995; Spychala et al., 1999).

Cytosolic 5` nucleotidase IA—cN-IA was named AMP-specific 5` nucleotidase for its high

specific activity with AMP at millimolar concentrations. Subsequent detailed kinetic studies

revealed high affinities toward deoxypyrimidine monophosphates (Garvey et al., 1998). It

is highly expressed in skeletal and heart muscle where it has a physiological function in the

generation of signaling adenosine during ischemia (Sala-Newby et al., 1999; Sala-Newby et

al., 2000). The high affinity for deoxynucleoside monophosphates suggests a role in the

regulation of deoxyribonucleotide pools. A homologous sequence related to human

autoimmune infertility gene (AIRP) and with highest expression in testis has been cloned

and designated cN-IB (Sala-Newby and Newby, 2001).

Cytosolic 5` nucleotidase II—cN-II is a 6-hydroxypurine-specific nucleotidase, most active

with (d)IMP (for review, see Itoch, 1993) and critically positioned to regulate ATP and

GTP pools. This tetrameric protein is stimulated by (d)ATP and GTP and regulated by

substrate and phosphate in a complex manner (for review, see Itoch, 1993) (Sala-Newbay et

al., 2000; Spychala et al., 1999; Gazziola et al., 1999), possibly involving subunit

association and dissociation (Spychala et al., 1988). Under physiological conditions cN-II

can catalyze phosphotransfer from a purine nucleotide donor to inosine or guanosine

(Worku and Newby, 1982; Pesi et al., 1994). This reaction is responsible for the activation

of several anti-viral and anti-cancer nucleoside analogs that are not substrates for cellular

nucleoside kinases (Johnson and Fridland, 1989; Keller et al., 1985).

Cytosolic 5` nucleotidase III—cN-III is highly expressed in red blood cells where it

participates in the degradation of RNA during erythrocyte maturation (for review, see Rees

et al., 2003). It prefers pyrimidine ribo- over deoxyribonucleotides with CMP being the best

substrate. It is inactive with purine nucleotides. The enzyme has a phosphotransferase

activity (Amici et al., 1997) less efficient than cN-II (Pesi et al., 1994). The sequence of

cN-III coincides with that of p36, an interferon induced protein of unknown function

(Amici et al., 2000). Alternative splicing of exon 2 gives rise to two proteins that are 286

51

and 297 amino acids long (Marinaki et al., 2001), with the shorter form corresponding to

cN-III. Fig. 1.06 and 1.07 shows structctre of human cytosolic 5`nucleotidase III.

Cytosolic 5`(3`)-Deoxynucleotidase—cdN is a ubiquitous enzyme, first purified to

homogeneity from human placenta (Hoglund and Reichard, 1990). It is the major

deoxynucleotidase activity in cultured human cells (Rampazzo et al., 2002; Gallinaro et al.,

2002). In contrast to cN-III, human cdN is not strictly pyrimidine-specific and works

efficiently with dIMP and dGMP. dAMP is a poor substrate and dCMP is inactive. The

enzyme is very active on 2_- and 3_-phosphates (Rampazzo et al., 2000; Hoglund and

Reichard, 1990). Neither the highly purified human placental cdN nor the recombinant

mouse and human enzymes showed phosphotransferase activity (Rampazzo et al., 2000;

Hoglund and Reichard, 1990), in contrast to what was reported for cdN purified from

human red blood cells (Amici et al., 1997).

Mitochondrial 5`(3`)-Deoxynucleotidase—mdN is highly homologous to cytosolic cdN

(52% amino acid identity). The two enzymes are coded by nuclear genes with identical

structure, probably derived by a gene duplication event (Rampazzo et al., 2000). With its

high preference for dUMP and dTMP, mdN shows remarkably narrow substrate specificity.

Similarly to cdN, mdN prefers deoxyover ribonucleotides and accepts 2_- and 3_-

nucleoside monophosphates (Rampazzo et al., 2000; Gallinaro et al., 2002). Its enzymatic

features suggest that mdN regulates mitochondrial dTTP and prevents accumulation of

mutagenic dUTP within mitochondria. Fig. 1.08 shows aligned conserved motifs of

intracellular human 5`nucleotidase.

Catalytic mechanism

The crystal structure of mdN and work on the active site of cN-II form the basis for

a reaction mechanism of intracellular 5` nucleotidase (Rinaldo-Matthis et al., 2002;

Allegrini et al., 2001). The reaction creates a phosphoenzyme intermediate involving the

first aspartate in the DXDX( V/T) motif (Allegrini et al., 2001). A detailed scheme of the

catalytic process derived from the crystal structure of mdN (Rinaldo-Matthis et al., 2002)

involved both aspartates in the motif (Fig. 1.04). The first (Asp-41) generates a

pentacovalent phosphorus intermediate with similar basic organization as the intermediate

detected in the structure of _-phosphoglucomutase (Lahiri et al., 2003). The x-ray structure

of cdN suggests a catalytic mechanism identical with that of mdN. Differences within the

52

active sites account for differences in substrate specificity (Rinaldo-Matthis et al., 2002).2

Using the structurally important residues the best alignment was between the two

eoxynucleotidases and cN-III (Rinaldo-Matthis et al., 2002). Two 5` nucleotidase, cN-II

and cNIII, exhibit phosphotransferase activity (for reviews see Itoh, 1993; Rees et al., 2003)

possibly because of higher stability of the phosphoenzyme intermediate or faster exchange

of the nucleoside product with the nucleoside acceptor. The active site of E. coli 5`

nucleotidase, the paradigm for eN, contains two zinc ions and the catalytic dyad Asp-His

(Knofel and Strater, 1999). No phosphoenzyme intermediate is formed during catalysis, but

a water molecule performs the nucleophilic attack on the phosphate (Knofel and Strater

2001)

FIG. 1.04. Structure of the active site of mdN with the pentavalent phos phorous intermediate produced

by nucleophilic attackof As p-41 on the phos phate (10). Asp-43 first promotes the protonation of the

leaving deoxyribonucleoside (R) and then activates the water nucleophile that releases the phosphate. Asp-41

and Asp-43 are the two aspartates in the mot if conserved in intracellu lar 5Nucleotidase

Table.1.04. Classification of mammalin 5`Nucleotidase

Protein Full name Unigene Aliases References

Nomenclature and gene symbol Cluster no.

eN Ecto-5`-nucleotidase, Hs. 153952 ECTO-5`-Nt; Misumi et al, 1990

NT5E low Km 5`Nt;

eNT; CD73

cN-IA Cytosolic 5`Nucleotidase IA, Hs.307006 AMP-specific 5`NT; Sala-Newby etal. 1999

NT5C1A cN-I Hunsucker et al., 2001

cN-IB Cytosolic 5`-nucleotidase IB Hs.120319 cN-IA homolog; AIRP Sala-Newby et al., 2001

NT5C1B

cN-II Cytosolic 5`-nucleotidase II, Hs. 138593 High Km 5`-NT; Oka et al., 1994

NT5C2 Purine 5`-NT;

GMP, IMP-specific 5`-NT

cN-III Cytosolic 5`-nucleotidase III, Hs.55189 PN-I; P5`N-I; UMPH Amici et al., 2000

NT5C3

cdN Cytosolic 5`(3`)-deoxynucleotidase, Hs.67201 dNT-1; PN-II Rampazzo et al., 2000a

NT5C Mazzon et al., 2003

mdN Mitochondrial 5`(3`)-deoxynucleotidase, Hs.16614 dNT-2 Rampazzo et alk., 2000b

NT5M

53

Table.1.05. Distinctive features of mammalian 5`Nucleotidase

Structure of 5`Nucleotidase

The primary structure of 5`Nucleotidase in rat liver (Misumi et al., 1990a), human

plancenta (Misumi et al., 1990b) and brian of electric ray (Volknandt et al., 1991) consists

of 548 aminoacids with molecular weight of 61 KDa. The C-terminus contains a stretch of

uncharged and hydrophobic amino acid residues which are presumably exchanged for a

glysyl phosphatidyl inositol (GPI) anchor bound to serine 523 (Ogata et al., 1990)The

enzymes are shown to contain 4 to 5 potential glycosylation sites. Studies on secondary

structure of 5`Nucleotidase from bull seminal plasma by fourier transform infrared

spectroscopy has revealed the presence of -sheet structure (54%), -helix (18%), -turns

(22%) and unordered structure (5%) (Fini et al., 1992). It is apparent from both

immunochemical and biochemical evidence that thecDNAs isolated so far correspond to the

membrane bound and surface located forms of the enzyme.

Ecto 5`nucelotidase is a -dimer with interchain disulphide bridge (Dieckhoff et

al., 1985). The intact disulphide bridges are essential for enzymatic activity and thiol

reagents inactivate the enzyme. In humans the enzyme is encoded on chromosome 6. The

GPI anchors of both rat liver and human placenta 5`Nucleotidase contain (Molar ratios in

parenthesis); ethanolamine (2), Mannose (3), glucosamine (1), inositol (1) and fatty acids

(2). Fatty acids include stearic acid, myristic acid and palmitic acid (Ogata et al., 1990;

Misumi et al., 1990b). of the two ethanolamines, one is presumably involved in linkage of

GPI to the a-carboxyl group of serine 523, and other could be phosphodiester- linked to a

mannose reside of the glycan core. This GPI moiety may play role as a signal for targeting

the attached protein selectively to the apical membrane surface (Lisanti and Rodriguez-

Boulan, 1990)

54

Fig.1.05. Tetrameric structure of cytosolic 5`Nucleotidase-II (cN-II). The active site,

The effector sites 1 and 2, and the subunit interfaces A and B are pointed out. Substrates (red and yellow), Magnesuim (Orange), and two adenosine (White) are shown. Polar atoms

are color coded as following: nitrogen in blue and oxygen in red.

Fig. 1.06. Structure of Human Cytosolic 5`Nucleotidase III (cN-III)

55

Fig.1.07. Structure of D41N variant of mdN with dUMP (orange) and Magnesium

(green) bound in the active site

Figure.1.08. Aligned conserved motifs of the six known intracellular human

5`Nucleotidases mdN, Cytosolic 5`(3`)-deoxyribonucleotides (cdN), Cytosolic

5`Nucleotidase IA (cN-IA), Cytosolic 5`-nucleotidase IB (cN-IB), cN-II and cN-III.

Completely conserved residues are shown on black background and partly conserved residues are boxed.

56

5`Nucleotidase Inhibitors

Endogenous inhibitors have been described for both bacteria and plants. A

5`Nucleotidase specific inhibitor present in the cytoplasm of a number of bacteria including

E.coli has been described by Dvork et al., (1966) and Neu (1967). This appered to be a

proteinaceous inhibitor destroyed by pronase. A proteinaceous inhibitor specific for

5`Nucleotidase has also been described for lympocytes by sun et al., (1983) which was later

proved to be an experimental artifact. The inhibitor turned out to be adenosine deaminase.

A peptide inhibitor was isolated a partially characterized from Ehrlich Ascites Tumour cells

(Amruthesh and D`Souza, 1986). The results of the studies by Rajput and D`Souza showed

the presence of inhibitor in tissues of rat except brain. In this study all tissues showed

presence of two types of protein inhibitors. However, presence of an inhibitor in each

tissue, which did not inhibit the 5`Nucleotidase from its own source but only from other

tissues was intriguing.

Con A inhibit Crotalus atrox and N. naja venom 5`Nucleotidase activity by

specifically interacting with a-D-glucose and a-D- manose residues in glycoprotein and

exert reversible inhibitory effect (Stefanovic et al., 1975; Ogawara, 1985; Mannherz 1979;

Dhananjaya et al., 2006). Citrate and EDTA inhibit venom 5`Nucleotidase activity by

metal- ion chelating effect (Francis et al., 1992). Nucleocidine, melanocidine A and

melanocidine B, are polysaccharide in nature where steric effects are known to play major

role in inhibition (Uchino et al., 1985). Polyphenolic inhibitors isolated from wine grape

“Koshu” and Areca catechu are known to posses tannic activity i.e., ability to precipitate

proteins (Toukairin et al., 1991). Several nucelside analogus have been shown to inhibit

5`Nucleotidase activity (Garvey et al., 1998; Garvey and Prus, 1999; Mazzon et al., 2003).

Physiological role of 5`Nucleotidases

One of the most important functions is in the catabolism of nucleotides

5`Nucleotidase plays an important role in Purine salvage pathway.

Due to its catalytic properties the release of purines from its substrate hydrolysis

acts as a signaling molecule via receptor mediated responses known as purinergic

signalling

57

Purine Receptors

Purines (Adenosine, ADP, ATP) are important s ignaling molecules that mediate

diverse biological effects via.., cell surfaced receptors termed Purine receptors. The concept

of purines as extracellular signaling molecules was instigated by Dury and Szent-Gyorgyi

in 1929. Many years later the term “Purinergic” was introduced by Burnstock in 1972. It is

now widely recognized that purinergic signaling is a primitive system involved in many

non-neuronal as well as neuronal mechanisms, including exocrine and endocrine

secreations, immune responses, inflammation, pain, platelet aggregation and endothelial

mediated vasodilation (Burnstock and Knight, 2004; Burnstock, 2006). Cell proliferation,

differentiation and death that occur in development and regeneration are also mediated by

purinergic receptors (Abbracchio and Burnstock, 1998; Burnstock, 2002).

There are two main families of Purine receptors, adenosine or P1 receptors, and P2

receptors (recognizing primarily ATP, ADP, UTP and UDP). Adenosine/P1 receptors have

been further subdivided, according to convergent molecular, biochemical and

pharmacological evidence into four subtypes, A1, A2A, A2B, and A3, all of which are couple

to G proteins. Based on differences in molecular structre and signal transduction

mechanisms, P2 receptors are naturally divided into two families of ligand-gated ion

channels and G protein-coupled receptors termed P2X and P2Y receptors, respectively. To

date seven mammalian P2X receptors (P2X1-7) and Five P2Y receptors (P2Y1, P2Y2, P2Y4,

P2Y6, P2Y11) have been cloned, characterized, and accepted as valid members of the P2

receptor family.

P1 receptors

In 1989, complementary DNAs (cDNAs) encoding two different P1 receptor subtypes (A1

and A2) were isolated (Libert et al., 1989), and, shortly after, the A3 subtype was identified.

Four different P1 receptor subtypes, A1, A2A, A2B and A3, have been cloned and

characterized (Fredholm et al., 2001; Olah and Stiles, 2000; Cobb and Clancy, 2003; Yar et

al., 2005). All are members of the rhodopsinlike family of G protein-coupled receptors.

Their N termini are relatively short (7 – 13 residues in length), as are their C-termini (32 –

120 residues). In the transmembrane domains (TMI-TMVII), human adenosine receptors

share 39 – 61% sequence identity with each other and 11 – 18% identity with P2Y

receptors. Each of the four human P1 receptor genes contains an intron within the coding

region, located immediately after the end of the third transmembrane domain.

Polymorphisms have been observed in the A1 and the A2A receptors. P1 receptors couple

58

principally to adenylate cyclase. A1 and A3 are negatively coupled to adenylate cyclase

through the Gi/o protein a-subunits, whereas A2A and A2B are positively coupled to

adenylate cyclase through Gs (Reshkin et al., 2000). The human A2B receptor has also been

observed to couple through Gq/11 to regulate phospholipase C activity, and the A3 receptor

may interact directly with Gs. A number of P1 subtype- selective agonists and antagonists

have been identified. All of the known P1 receptor agonists are closely related to adenosine

in structure, with few modifications permitted. The most selective agonist for the A1

subtype is CCPA (2-chloro-N6-cyclopentyladenosine). CGS 21680 is the most selective

A2A agonist, while NECA (5’-N-ethylcarboxamido adenosine) is the most potent A2B

receptor agonist. Cl-IB-MECA is 11-fold selective for the human and about 1400-fold

selective for the rat A3 receptor. Methylxanthines such as caffeine and theophylline are

weak P1 receptor antagonists. DPCPX(8-cyclopentyl-1,3-dipropylxanthine) is anA1

receptor antagonist with sub-nanomolar affinity. The most selective A2B receptor antagonist

is MRS1751. MRE 3008F20 is the most selective human A3 receptor antagonist. The

characteristics with diverse physiological functions of P1 receptors are as given in Table.

1.06. Fig. 1.09 shows schematic representation of P1 receptors.

The diverse physiological effects mediated by the different P1 receptor subtypes,

particularly modulation of the cardiovascular, immune and central nervous systems, have

been confirmed by transgenic knockout mice (Ledent et al., 1997; Sun et al., 2001). Null

mice have been generated for each of theA1, A2A and A3 receptors, and in all knockout

animals generated, the P1 receptors in question do not appear to play a critical role during

development. Knockout mice have yet to be described for the A2B receptor subtype. In

contrast to knockout studies, overexpression of either A1 or A3 subtypes in transgenic mice

has a cardioprotective effect (Lankford et al., 2006). P1 and P2Y receptors are frequently

expressed in the same cells.

59

Fig.1.09. Schematic representation of P1 family of receptors

Table.1.06. Characteristics of P1 receptors

60

P2 receptors

P2 receptors are naturally divided into two families of ligand-gated ion channels and

G protein-coupled receptors termed P2X and P2Y receptors, respectively. To date seven

mammalian P2X receptors (P2X1-7) and Five P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6,

P2Y11) have been cloned, characterized, and accepted as valid members of the P2 receptor

family (Burnstock, 2007). Fig. 1.10 shows schematic representation of P2X family of

receptors

P2X receptors

The first cDNAs encoding P2X receptor subunits were isolated in 1994. Members

of the family of ionotropic P2X1–7 receptors show a subunit topology of intracellular N-

and C-termini possessing consensus binding motifs for protein kinases; two

transmembranespanning regions (TM1 and TM2), the first involved with channel gating

and the second lining the ion pore; a large extracellular loop, with 10 conserved cysteine

residues forming a series of disulfide bridges; a hydrophobic H5 region close to the pore

vestibule, for possible receptor/channel modulation by cations and an ATP-binding site,

which may involve regions of the extracellular loop adjacent to TM1 and TM2. The P2X1–7

receptors show 30– 50% sequence identity at the peptide level (North, 2002; Egan et al.,

2006; Roberts et al., 2006). The stoichiometry of P2X1–7 receptors is thought to involve

three subunits, which form a stretched trimer or a hexamer of conjoined trimers (North,

2002). The Characteristics of P2X family subtypes are as given in Table.1.10. Fig. 1.10

shows schematic representation of P2X family of receptors

P2Y receptors

The first P2Y receptors were cloned in 1993 (Lustig et al., 1993; Webb et al., 1993).

Since then several other subtypes have been isolated by homology cloning and assigned a

subscript on the basis of cloning chronology (P2Y4, P2Y6, P2Y11). The long awaited Gi-

coupled ADP receptor (P2Y12) of platelets was finally isolated by expression cloning in

2001 (Hollopeter et al., 2001), while P2Y13 and P2Y14 receptors were characterizedlater

during a systematic study of orphan receptors (Chambers et al., 2000; Communi et al.,

2001). At present, there are eight accepted human P2Y receptors: P2Y1, P2Y2, P2Y4, P2Y6,

P2Y11, P2Y12, P2Y13 and P2Y14 [Abbracchio et al., 2006; Abbracchio et al., 2003] (see

Table 1). The missing numbers represent either non-mammalian orthologs, or receptors

61

having some sequence homology to P2Yreceptors, but for which there is no functional

evidence of responsiveness to nucleotides. In particular P2Y3 may be a chicken ortholog of

P2Y6 (Li et al., 1998), while P2Y8 and P2Y could be the Xenopus and turkey orthologs of

P2Y4, respectively. p2y7 is a leukotriene B4 receptor (Yokomizo et al., 1997). p2y5 and

p2y10 are considered orphan receptors. A p2y8 receptor was cloned from the frog embryo,

which appears to be involved in the development of the neural plate (Bogdanov et al.,

1997). p2y9 was reported to be a novel receptor for lysophosphatidic acid, distant from the

Edg family (Noguchi et al., 2003). P2Y15 was recently introduced to designate the orphan

receptor on the basis that it would be a receptor for AMP (Inbe et al., 2004). But it is now

firmly established that it is actually a receptor for a-ketoglutarate, as underlined in a report

by the IUPHAR P2Y Subcommittee (Abbracchio et al., 2005). The Characteristics of P2X

family subtypes are as given in Table.1.10. Fig. 1.11 shows schematic representation of

P2Y family of receptors

Second messenger systems and ion channels

P2Y1, P2Y2, P2Y4 and P2Y6 receptors couple to G proteins to increase inositol

triphosphate (IP3) and cytosolic calcium. Activation of the P2Y11 receptor by ATP leads to

a rise in both cAMP and in IP3, whereas activation by UTP produces calcium mobilization

without IP3 or cAMP increase. The P2Y13 receptor can simultaneously couple to G16, Gi

and, at high concentrations of ADP, to Gs. The activation of several P2Y receptors is

commonly associated with stimulation of several mitogen-activated protein kinases

(MAPKs), membrane through certain actions of activated G proteins. Such actions are now

well-established in closing (or in certain cases opening or potentiating) various classes of

K+ channels and voltage-gated Ca2+ channels (Dolphin et al., 2003). There have been

several demonstrations of ion channel responses upon activation of native P2Y receptors in

brain neurons (Zhang et al., 2003; Bowser and Khakh, 2004). For exampleATP (or UTP, or

their products ADP or UDP) present at synapses, plusATP diffusing fromastrocytes,

activates P2Y receptors on distinct subsets of brain neurons, regulating their activities by

the coupling of those receptors to specific ion channels. While ion channel coup lings of

P2Y receptors are primarily of importance in neurons, they have in a few cases been

detected also in various other tissues, e.g., in cardiac muscle cells (Vassort, 2001). Among

the channels with which the SCG cell membrane is well endowed are two types of voltage-

gated channels, which are important in receptor- based regulation of neuronal activity, the

Ca2+ channel of the N-type and the M-current K+ channel (Selyanko et al., 2001).

62

Fig.1.10. Schematic representation of P2X family of receptors

Fig.1.11. Schematic representation of P2Y family of receptors

63

Table.1.10. Characteristics of Purine mediated receptors

64

Adenosine liberation due to the action of Nucleases / Nucleotidases /

phosphomonoesterases

The synergistic action of nucleases, nucleotidases and phosphatases results in the

generation of purine and pyrimidine nucleotides (Aird, 2002). Among these nucleotides,

adenosine generation is pharmacologically important as it exhibits several snake

envenomation related symptoms (Ralevic and Burnstock, 1998; Aird, 2002; Burnstock,

2006; Sawynok, 2007).

Generation of adenosine by synergistic actions of venom enzymes can takes place

by different pathways. Enzymes like nucleotidase and PDE act immediately upon

envenomation on available ATP molecules to release adenosine (Figure 1.12 shows

schematic representation of adenosine generation by venom enzymes from ATP

hydrolysis). DNases, RNases and PDE liberate purine and pyrimidine nucleotides from the

cell genome. The liberation of adenosine by the action of these enzymes requires cell

necrosis brought about by proteases/hemorrhagins, phospholipases, myotoxins, cardio

toxins, and cytolytic peptides (Figure 1.13 represents cell necrosiss brought about by

venom enzymes) (Ownby et al., 1978; Bernheimer and Rudy, 1986; Nunes et al., 2001; Ma

et al., 2002). Once the cell is ruptured the venom PDE and DNases/RNases acts on preys

DNA/RNA, releases nucleotide 5’mono phosphates (NMP`s). 5`Nucleotidase specifically

or non-specific phosphomonoesterases acting on these 5`NMP`s liberate adenosine (Fig

1.14 shows schematic representation of adenosine generation by venom enzymes from

DNA/RNA hydrolysis). There is always a possibility that the released adenosine in vivo is

converted to inosine by the action of adenosine deaminase of the prey/victim. However, this

is also important since inosine is also responsible for inducing many pharmacological

actions. Some of the pharmacological actions exhibited by adenosine and inosine are

summarized in Table 1.15 and 1.16. Some of their pharmacological actions in relation to

snake envenomation are described here (for more details see Aird, 2002).

Liberated adenosine could help in the diffusion of toxins into prey’s tissues by

inducing increased vascular permeability through vasodialation (Hargraves et al., 1991;

Sobrevia et al., 1997) and/or inhibition of platelet aggregation (Seligmann et al., 1998).

Thus adenosine could act as potent spreading agent helping in spreading of toxins. Along

with vascular permeability effects adenosine induced edema (Ramkumar et al., 1993) can

help in potentiating venom-induced hypertension (Aird, 2002). In addition adenosine is also

known to cause paralysis, by inhibiting neurotransmitter release at both central and

65

peripheral nerve termini (Ralevic and Burnstock, 1998; Redman and Silinsky, 1993) thus

potentiating venom-induced paralysis (Aird, 2002). Further, along with hemolytic, myolytic

and cardiolytic toxins of snake venom, adenosine may also be involved in venom induced

renal failure and cardiac arrest (Olsson and Pearson, 1990; Aird, 2002; Castrop, 2007).

Other common behavioral disturbances such as nociceptive, locomotar alterations and pain

percieved upon envenomation may also be brought about by adenosine (Dunwiddie and

Worth, 1982; Barraco et al., 1983; Winsky and Harvey, 1986; Palmour et al., 1989;

Nikodijevic et al. 1991; Jain et al., 1995; Sawynok et al., 1997; Sawynok, 1998; Aird,

2002). Therefore, it seems that adenosine plays a central role in envenomation strategies of

prey immobilization (Aird, 2002, 2005). Although, experimental data in deciphering these

actions by purified enzymes are lacking, there is enough evidence for direct involvement of

adenosine and adenosine signaling in snake envenomation (Lumnsden et al., 2004; Aird,

2005).

66

Fig 1.12. Schematic representation of adenosine generation by venom enzymes from

ATP hydrolysis. Venom constituents are outlined with ovals and bold letters

indicates end products released upon enzymes action.

Fig 1.13. Cell necrosis brought about venom enzymes. Venom constituents are outlined

with ovals and bold letters indicates end products released upon enzymes action.

67

Fig 1.14. Schematic representation of adenosine generation by venom enzymes from

DNA/RNA hydrolysis. Venom constituents are outlined with ovals and bold

letters indicates end products released upon enzymes action.

Apart from endogenous liberation of adenosine that can bring about various

pharmacological actions, it is also possible that these enzymes can interfere in many

physiological process of an organism directly. Since they are hydrolytic enzymes, their

pharmacological actions need not only be based on their catalytic activity, but could also

perform additional pharmacological activities, since venom enzymes have evolved to

interfere in diverse physiological process (Kochva, 1987; Fry, 2005). Hence it is likely that

nucleases, nucleotidase and phoshatases also posses distinct pharmacological activities like

those of venom PLA2 and proteases (Kini, 1997; Gutierrez and Rucavado, 2000). Our

recent work on anticoagulant effect of N. naja 5`Nucleotidase confirms that the

pharmacological effect is independent of catalytic activity (Dhananjaya et al., 2006).

Although nucleases, nucleotidases and phosphomonoesterases are near ubiquitous in

distribution, little progress has been made towards understanding these enzymes with a

toxinological perspective. As already discussed, characterization of individual nucleases,

nucleotidases and phosphatases has not been clearly established since they hydrolyze

68

similar substrate and share similar biochemical properties. Future research on complete

biophysical characterization of the purified enzymes could reveal the existence of unique

venom proteins or proteins having multiple domains to perform different catalytic

functions. Determination of complete cDNA and/or amino acid sequence will also enable

evaluation of the degree of homology of these enzymes from various species and families

of snakes. However a renewed interest in these enzymes in recent years is based on their

involvement in generation of adenosine, a multitoxic component, but the direct involvement

of these enzymes in the generation of adenosine in vivo, has to be established. Apart from

these, nucleases, nucleotidases and phosphomonoesterases could also possess distinct

pharmacological properties that are independent of catalytic activity, which has to be

verified by carrying out pharmacological studies with purified proteins. Venom

nucleotidases (ATPases, ADPases and 5`Nucleotidase) along with other hemostatically

active component can lead to the formation of uncoagulable blood. This could help in

diffusion of other venom toxins to their site of action. An analogous mechanism is found in

blood feeding organisms where apyrases (ATP diphosphohydrolase), 5`Nucleotidases and

other enzymes provide the redundant “anti-hemostatic barrier” to prevent host defenses

triggered by blood feeding. Further research is needed to isolate and biologically

characterize these enzymes in snake venoms, such that their role in venom is clearly

established.

Treatment of snake envenomation

Snake envenomation is considered a medical emergency because of the rapidity

with which some of the venom components exits in toxic and lethal effec ts simultaneously,

sequentially and disjunctively on blood, cardiovascular, respiratory and nervous system.

The rules for preventing snakebite and the first aid measures to be taken have been

formulated by Moore (1980). The accepted treatment for snakebite is antivenom therapy.

The best known medicine made out of snake venom is antivenom. In 1894 Mr. Albert

Calmette found a way to hyper- immunize animals against snake venom, by injecting them

with small quantities of venom and increasing the dose slowly. He also showed that he

could save animals with his antivenom. The findings from Calmette are even now still used

as a base to produce antivenom. Different species of animals are used in the production of

antivenom. Although horses, rabbits, sheep, goats and chickens are used most.

Botanically cure is one of the most widely used to some extent effective practice

among folk treatments. This involves application of various plant concoctions prepared

69

usually from roots or other plant parts. The extracts are either applied to the bite region or

are administered orally. A large number of plants (Duke, 1985; Mors et al., 2000) and their

components are claimed to antagonize the action of snake venoms. Indian plants used in the

treatment include Acalypha indica, Capsicum annum, Achyranthus superba, Achyranthus

aspera, Capsicum annuum, Datura fastuosa, Strychnos colubrine, Rauwolfia serpentina,

Hemidesmus indicus, Aritolochia radix, Mimosa pudica and Withania somnifera (Chopra et

al., 1958; Nadkarni, 1976; Sathyavathi et al., 1976; Gowda, 1997; Alam and Gomes, 1998;

Mahanta and Mukherjee, 2001; Deepa and Gowda, 2002; Girish et al., 2004).

The medically prescribed treatment for envenomation is immunotherapy. The

mortality due to snake venom poisoning is markedly reduced by the use of antivenins or

antivenoms. Administering antivenins is the best method currently in use against snakebite

(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). Both monovalent antivenins, which

prepared against a single species of snake and polyvalent antivenins, prepared against a

mixture of related snake species are available (Theakston and Warrell, 1991). Many

improved techniques of immunization that result in high titers of antibodies have been

developed by extensive research. Though the mortality due to snake venom poisoning is

reduced markedly by the use of antivenoms, 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 antivenom

increases the potential risk of serum sickness, which leads to arthritis, vasculitis and

nephritis.

70

Table 1.11 Properties of purified endonucleases from snake venoms

Snake venoms Action on Molecular pI References

Substrate weight (Da)

DNase

Bothrops atrox DNA, RNA - 5.0 Georgatsos and Laskowski, 1968

Poly-AU

RNase

Naja naja oxiana RNA ~15,900 - Vasilenko and Bubkina, 1965

Naja naja Polyribocytidine ~14,000 - Mahalakshmi and Pandit, 1997

rRNA Mahalakshmi et al., 2001

Note; Phosphodiesterase which also exhibit endonuclease activity has been described elsewhere.

Abbreviations: -; Data not available.

71

Table 1.12 Properties of purified phosphodiesterases from snake venoms

Snake venoms Action on Molecular pI Carbohydrate isoenzyme Inhibitors References

substrate weight(Da)

Bothrops atrox Bis-pNPP 130,000 9.2 - yes (2) EDTA Philipps, 1976

PolyadenylicAcid

Bothops alternatus Bis-pNPP 105,000 8-9.8 No - EDTA Valerio etal.,2003

Cerastes cerastes Bis-pNPP 110,000 9.0 No - EDTA Halim et al., 1987

Cysteine

AMP,ADP

Crotalus adamentus Bis-pNPP 115,000 9.0 yes - ND Philipps, 1975;

140,000 Stoynov etal., 1997

Crotalus mitchilli cAMP, ATP 110,000 8.5 - - EDTA Perron et al., 1993

Pyrrhus ADP

Crotalus rubber Native DNA/ 98,000 8.5 - yes EDTA, Mori et al., 1987

Rubber RNA, cAMP TGA, PCMB

Crotalus viridis Native DNA/ 114,000 - - - EDTA Mackessy, 1989

oreganus RNA, cAMP

Trimeresures Bis-pNPP - - yes yes (4) EDTA Kini andGowda,1984

Flavoviridis

Trimeresures DNA/RNA 140,000 - No - EDTA Sugihara et l.,1986

Mucrosquamatus PCMB

Abbreviations: EDTA, ethylenediaminetetraacetic acid; AMP, Adenosine monophosphate; ADP, Adenosine

diphosphate; DTT, dithiothreitol; PCMB, p-chloromercuribenzoate; Bis-pNPP , bis-p-nitrophenyl

phosphate; camp,Cyclic adenosine monophosphate; -; Data not available.

72

Table 1.13 The accession number of Phosphodiesterase EST`s from various snakes species.

Snakes species Accession number References

Deinagkistrodon acutus DV561486, DV563305 Oinghua et al., 2006

Lachesis muta DY403207,DY403416 Junqueirs-de-Azevedo et al., 2006

Sistrurus catenatus edwardsii DY587965.1 Pahari et al., 2006

73

Table 1.14 Properties of purified nucleotidases from snake venoms

Snake venoms Action on Molecular protein Carbohydrate Inhibitors Biologcial

References

substrate weight(Da) nature propertries

5`Nucleotidases

Agistrodon acutus AMP 82,000 Acidic - - Platelet Ouyang and Hung, 1986

Aggregation

Inhibition

Trimeresures gramineous AMP, 74,000 Basic yes EDTA Platelet Ouyang and Hung, 1983

ADP Aggregation

Inhibition

ADPases

Agistrodon acutus ADP 94,000 Basic No - Platelet Ouyang and Hung, 1986

Aggregation

Inhibition

Abbreviations: ND, Not Determined; EDTA, ethylenediaminetetraacetic acid; AMP, Adenosine monophosphate; ADP,

Adenosine diphosphate.-; Data not available.

74

Table 1.15 The accession number of nucleotidase EST`s from various snakes species.

Snakes species Accession number References

Bothrops insularis BM401810 Junqueirs-de-Azevedo and Ho, 2002

Deinagkistrodon acutus DV564501, DV557329, Oinghua et al., 2006

DV558168

Lachesis muta DY403632, DY403686, Junqueirs-de-Azevedo et al., 2006

DY403766

75

Table 1.16 Pharmacological effects of Adenosine related to snake envenomation

Pharmacological Mediation References

Effect

Vasodilatation Vascular A2A receptors Hargraves et al., 1991

Vascular A2B receptors Sobrevia et al., 1997

Cardiac block Cardiac adenosine A1 receptor Olsson and Person, 1990

Vascular permeability Mast cell A3 receptors Tilley et al., 2000

Inhibition of neurotrans Adenosine A1 receptors

mitter release in central and Ralevic and Burnstock, 1998

Peripheral neurons Redman and Silinsky, 1993

Edema Mast cell A3 receptors Ramkumar et al., 1993

Anti-platelet agrregation A1 and A2 receptors Seligmann et al., 1998

Renal failure Renal adenosine A1 receptor Castrop, 2007

Behavioral effects

Sedative effects Central neuronal A1 receptors Barraco et al., 1983

Anxiolytic activity Central neuronal A1 receptors Jain et al., 1995

Anticonvulsant effect Central neuronal A1 receptors Dunwiddie and Worth, 1982

Aggression inhibition Central neuronal A1 receptors Palmour et al. 1989

Alterations of cognitive

Functioning Central neuronal A1 receptors Winsky and Harvey, 1986

Locomotor depression Central A1 and A2 receptors Nikodijevic et al. 1991

Nociceptive actions

Analgesia Adenosine A1 receptor Sawynok, 1998

Pain Adenosine A2 receptor Sawynok, 1998

Mast cell A3 receptors Sawynok et al., 1997

76

Table 1.17 Pharmacological effects of Inosine related to snake envenomation

Pharmacological Mediation References

Effect

Vascular permeability Mast cell A3 receptors Tilley et al., 2000

Inflammation Mast cell A3 receptors Tilley et al., 2000

77

Aim and scope of the Study

Snake venoms are complex mixture of biologically active enzymatic and non-

enzymatic proteins, peptides and small organic compounds. Snakebite is considered as a

subcutaneous/intramuscular injection of venom into the prey/human victims. The

pathophysiology of envenomation includes both local and systemic effects. Which includes,

neurotoxicity (pre/postsynaptic), myotoxicity, cardiotoxicity, coagulant (pro/anti), hemostatic

(activating/inhibiting), hemorrhagic, hemolytic and edema forming activities. The systemic

toxins acting on vital organs are extensively studied as a matter of fact that some of them

serve as prototypes for drug designing and some as tools to uncover many unsolved

physiological events. Due to the catalytic activity some of the ubiquitously present enzymes

are known to endogenously liberate purines a multi toxin, thus involving in snake

envenomation stratergies during accusion and digestion of prey. 5`Nucleotidase is known to

be ubiquitously present in snake venoms and known to liberate purines. Apart from this it is

known to inhibit platelet aggregation through release of adenosine from ADP. Apart from this

not much is known about the enzyme role in snake envenomation. A tharough investigation of

components involved in snake envenomation is nessosory to neutralize the pharmacological

effects of snake envenomation and thus help in management of snakebite. Therefore, the

present study is undertaken to systamatically evaluate and address the role of 5`Nucleotidase

enzyme in Indian cobra (Naja naja) and Russells viper (Daiboia russellii) venom induced

toxicity.