CHAPTER I GENERAL INTRODUCTION

Transcription

1 CHAPTER I GENERAL INTRODUCTION

2 Introduction Snakes and the venom of snakes have fascinated the mankind, since time immemorial. Throughout the course of the history of civilization, venomous snakes have held a imique and particular fascination for humans. Snakes have provoked deep interest, curiosity and analogies in many paths of human life: inspiring symbols in religion (the evil and original sin); science (pharmacy and medicine symbols) and others since ancient times (Ramos and Araujo, 2005). Venomous snakes are highly evolved reptiles, belonging to the phylum: Chordata, order: Squamata and sub order: Serpentes. They are widely distributed throughout the world except in Arctic, New Zealand and Ireland (Deoras, 1965). They are common in tropical and subtropical regions, but are rarely found at high altitudes while their number increases in humid regions (Russell, 1980). Nearly, 3500 species of snakes have been identified all over the world. Among them, 400 species of snakes are known to be venomous (Russell and Brodie, 1974; Philip, 1994). Based on their morphological characteristics, like arrangement of scales, dentition, osteology and sensory organs, these snakes are classified into different families. As per the recent update of classification, venomous snakes have been grouped into three families under the order: Serpentes (Wuster 1996, 1997, 1998). 1. Viperidae, which includes Russell's viper. Saw scaled viper. Puff adder. Water moccasins, Copper head. Pit vipers of Asia, European vipers, Gaboon vipers. Horned viper of Sahara. The family Viperidae is further classified into two-sub families, Viperinae and Crotalinae. The subfamily of Viperinae includes Puff adder, Gaboon vipers, Russell's viper. Homed viper of Sahara, Saw scaled viper, European vipers. The subfamily of Crotalinae includes Rattlesnakes, Copper head. Water moccasins and Pit vipers of Asia. 2. Elapidae, which includes Cobras, Coral snakes, Mambas and Kraits. 3. Colubroidae, which includes all sea snakes. In 1965, Deoras has listed about 216 species of snakes that are found in India out of which 52 are poisonous. However, "Romulus Whitker and Ashok Captain (2004) have provided a comprehensive list of 275 snakes recorded in the various parts of Indian subcontinent. Among venomous snakes only four pose threat to human beings as they are

3 found in the vicinity of human settlement, especially in rural areas, which are agricultural and have rats in abundance. The four venomous snakes are called 'Big Four'-the Spectacled Cobra, Common Krait, Russell's viper and Saw scaled viper. The distribution of these big four snakes along with King Cobra in the Indian subcontinent is given in Table Globally venomous snakebite is estimated to affect greater than 2.5 million humans annually, of which more than 100,000 will die (Chippaux, 1998). The burden of morbidity and mortality is greatest in the rural tropics (Lalloo et al., 1995; Laing et al., 1995; Warrell et al., 1999) but snake bite is not confined to poorer rural tropical areas. There is evidence that some of the most dangerous venomous snakes are invading urban areas, putting new groups of humans at significant risk (Melgarejo and Aguiar, 1995; Revault, 1995). Snake envenomation Snake envenomation is basically a subcutaneous or intramuscular injection of venom into the prey/human victims. It employs three well integrated strategies. Two of these are prey immobilization strategies and may denominated 'hypotensive' and 'paralytic' strategies. Both serve to limit prey flight, in the case of snake taxa which strike release and then track their prey (most viperids), or to overcome pre resistance, in the case of snakes that seize and bulldog their prey (many el^ids and all colubrids). The third strategy is digestive and commences degradation of prey tissues internally, even before the prey has been engulfed. Normally, all three strategies operate simultaneously and individual venom constituentsfi-equentlyparticipate in more than one of them. Each of these strategies contains interchangeable mechanisms, elements or sub strategies. Different venomous snake taxa employ different combinations of mechanisms and no single species employs them all (Aird, 2002). Snake venom Snake venoms contain a multitude of biologically active toxins that work together for the capture of prey. Their effects include blood coagulation (pro/anti), neurotoxicity (pre/post synaptic), myotoxicity, nephrotoxicity, cardiotoxicity and necrotoxicity (local tissue damage), hemorrhagic, edema inducing and possible direct action on vital organs.

4 The proportionate mixing of these biological activities varies considerable from venom to venom. The variability in venom composition is considered at several levels. The intergenus, interspecies, intersubspecies, intraspecies, geographical distribution, seasonal and age dependent change and variation due to sexual dimorphism (Chippaux et al., 1991). In addition to protein/peptide toxins, snake venom is also composed of organic and inorganic components such as metal ions like Ca^"^, Cu^^, Fe^"^, Mg^"^, Na^"^, Zn^"^ (Markland, 1998) and citrate, non-proteinaceous components include carbohydrates, lipids, bioactive amines like serotonin, nucleotides and amino acids (Freitas et al., 1992). Bioactive amines are predominantly found in viperid venoms (Hider et al., 1991; Freitas et al., 1992). Citrate was identified as the major constituent found in many types of venom. It is foimd in greater than 5% of dry weight of venom of Crotalous atrox and Bothrops asper (Freitas et al., 1992). Snake venoms have several enzymes that depend on metal ions for activity. For example, PLA2 requires Ca and metallo-proteases and hemorrhagins requires Zn. These enzymes are kept in an inactive form by the chelating effect of citrate. Other than this, citrate can also act as a buffering agent and also serve as a negative counter ion for basic proteins and polyamines (Odell et al., 1999). The biologically active protein and peptide toxins in snake venoms can be either enzymatic or non-enzymatic in property. Earlier investigators tried to explain all the biological activities of snake venoms based on the presence of enzymes or combination of enzymes. However, the initial contributions of several researchers (Weiland and Konz, 1936; Slotta and Frankel-Conrat, 1938; Ghosh et ah, 1941), becomes evident that there are several non-enzymatic proteins in snake venoms, which possess important biological activities and cannot be ignored. They are known to induce neurotoxicity (Larsen and wolf, 1968; Sato et al, 1969), myotoxicity (Ownby et al, 1976; Chang, 1979; Lomonte and Gutierrez, 1989), cardiotoxicity and platelet aggregation (Kini et al, 1988). Nerve growth factors (Oda et al, 1989; Kostiza and Meier, 1996) and bradykinin potentiating peptides are also reported from snake venoms (Ferreira et al, 1970; Ondetti et al, 1971; Aird, 2002). Snake venom enzymes Snake venoms contain several different enzymes. As many as 26 enzymes have been identified in snake venoms (Iwanaga and Suzuki, 1979). Most of these have been

5 isolated and characterized in detail. A list of enzymes found in snake venom and their general properties are given in Table 1.02 and 1.03 respectively. The distribution of the enzymes varies from one snake species to another. Some enzymes like L-amino acid oxidase, phospholipases, phosphodiesterases are foimd in almost all snake venoms (Rosenberg, 1979). The remaining enzymes are usually confined to certain taxonomic groups of snake (Russell, 1980; Iwanaga and Suzuki, 1979). For example viperid venom contains proteolytic enzymes like endo-peptidases, arginine ester hydrolases, thrombin like enzymes, kininogenases and procoagulant enzymes, which are not commonly found in el^id venoms (Zeller, 1948). Proteolytic and peptidase activities have been identified in some of the Elapid venoms like Naja nigricollis (Evans, 1984) and Naja atra (Boumrah, 1993). The enzymes of snake venoms generally act in the following ways. a) PLA2 cause neuromuscular blockages resulting in neurotoxicity. Proteinases, arginine ester hydrolases, hyaluronidases and some PLA2 cause tissue necrosis and local capillary damage (Gutierrez and Ownby, 2003; Girish et al, 2004; Petan et al., 2005) b) Proteinases and phospholipases are procoagulant or anticoagulant (Jagadeesha et al., 2002; White, 2005; Lu etal., 2005) c) Kininogenase release bioactive peptides, which cause acute hypotension (Markland, 1998). Most of the snake venoms contain an interguing variety of enzymes. All snake venoms contain L-amino acid oxidase (Zeller and Maritz, 1944), phosphodiesterase (Pareira et al., 1971), PLA2 (Rosenberg, 1979). Certain enzymes are characteristic of only a few species (Iwanaga and Suzuki, 1979; Russell, 1980). Acetylcholine esterase is characteristic of elapid venoms, which is never found in viperid and crotalid venoms. Arginine ester hydrolases, endopeptidase, thrombin like enzymes, kininogenase and procoagulant enzymes are distributed in viperid and crotalid venoms but have not detected in elapid venoms (Deutsch and Diniz, 1955). The enzymes mainly involved in various pharmacological activities are L-amino acid oxidase, PLA2, hyaluronidases and proteases (metallo/serine) listed in the Table 1.04.

6 PhosphoUpase A2 PLA2 (E.C ) enzymes specifically catalyzes the hydrolysis of fatty acid ester bond at position 2 of 1, 2, diacyl-sn-3 phosphoglycerides liberating free fatty acids and lysophospholipids (Van deewen and Dehaas, 1963). These enzymes are ubiquitous in nature and are found extra-cellularly and intra-cellularly. Extra-cellular PLA2S are found in mammalian pancreatic tissue, as well as in snake, bee and scorpion venoms (Van eijk et al., 1983). High levels of extra-cellular PLA2 enzymes have also been found at inflamed sites as exudates in some experimental animals and himians with diseases (Hara et al., 1991). PLA2 enzymes are usually small and stable as compared to most other proteins. They withstand harsh conditions of extreme temperature and ph. The stability is probably due to the presence of large number of (6-7) intramolecular disulfide bridges. The venom PLA2 display molecular masses from kda and consists of amino acid residues (Scott and Sigler, 1994). PLA2 have become a super family of distinct enzymes that have pivotal role in various biological activities. Among the enzymatic toxins, PLA2 constitutes the major part of the venom toxin pool and is being attributed to be involved in abnost all the pharmacological effects of snake venom. They are known to induce various pharmacological effects like neurotoxicity, myotoxicity, cardiotoxicity, cytotoxicity edema inducing activity, anti coagulant activity etc. (Kini, 1997). To date, ~280 PLA2S (enzymes/analogs) primary amino acid sequences have been determined (Danse et al., 1997; Tan et al., 2003). They share any where between % homology in their primary amino acid sequence. Three-dimensional structures of more than 20 PLAas from snake venoms have been determined by X-ray crystallographic studies. They share similar three-dimensional folding but exhibit wide differences in their pharmacological properties (Scott, 1997). PLA2S have been sub-classified based on a number of parameters. A commonly used and recently updated classification has been made by Six and Dennis (2000) which divides PLA2 into ten main type or group based on molecular weight, amino acid sequence, calcium dependent and cellular origin. It is however convenient to divide PLA2 into two large classes based on size, Ca^"^ requirement and catalytic mechanism. The first class consists of secretory enzymes that are small extensively disulfide cross-linked secreted molecules which require millimolar concentration of Ca^"^ for activity. The

7 second class consists of mechanistically distinct large molecular weight enzymes, which are Ca^"*" independent or require micromolar concentration of Ca^"^ for activity. The PLA2S, so far isolated from snake venoms, all come imder the type I and II. More than 300 PLA2S have been sequenced. Type I and IIPLA2S are highly homologous and have similar kinetic behaviour. Both Type I and Type II PLA2's are kda proteins with similar tertiary structure (Renetseder et al., 1985). In addition, venom PLA2 enzymes also exhibit species specificity (Harris, 2003). The neuromuscular blockages induced by crotoxin from Crotalus durissus terrificus, taipoxin from Oxyuranus scutellatus scutellatus and p-bungarotoxin from Bungarus multicinctus venom have similar characteristics but their potencies are species dependent. Taipoxin is three times more potent than P-bungarotoxin and five times more potent than crotoxin in blocking mouse phrenic nerve diaphragm transmission (Chang et al., 1977; Howard and Gundersen, 1980). VRV-PL-VI from Daboia russelii (north) causes hemorrhage in the intraperitonial cavity, pituitary and thyroid glands as well as liver and kidney (Vishwanath et al, 1988a), on the other hand VRV-PL-VIII (south) causes hemorrhage in the lungs (Uma and Gowda 2000). The most toxic VRV-PL-V from the same venom is a presynaptic neurotoxin (Kasturi and Gowda, 1989b). Hyaluronidase Hyaluronidase, (E.C ) an endoglycosidase, has been considered an invariant factor in the venom of snakes and is frequently referred to as "Spreading factor". Spreading activity has been evident from its ability to promote the local hemorrhagic effect of a toxin from Trimeresums flavoviridis venom (Tu and Hendon, 1983). Distortion in the integrity of ECM of local tissue(s) due to degradation of hyalxironan; dissemination of target specific toxins is presumed to be the critical event in the enzyme mediated spreading process. Despite the key role played in local effect(s) of envenomation, the enzyme has been explored exhaustively. It is evident from the limited number of studies carried out on this enzyme (Xu et al, 1982; Pukrittayakamee et al, 1983; Kudo and Tu, 2001). Recently, Girish et al, (2004) demonsfrated the degradation of extra-cellular matrix in humans and other tissue samples. This property of the hyaluronidase is attributed for the fast diffusion of the lethal toxins. The action of

8 hyaluronidase in the degradation of extra-cellular matrix is considered an important step for assisting toxins inducing local tissue damage. Its toxic effect is due to the synergetic action associated with the other venom toxins (Girish et al., 2004). Regulation of this enzyme is highly beneficial in mitigating the venom toxicities. In addition, information on the existence of isoforms of hyaluronidase in snake venoms is limited. Although, there are reports on the isolation and characterization of this enzyme from the venom of snakes such as Agkistrodon acutus (Xu et al, 1982), Daboia russelii (Pukrittayakamee et al., 1983) Agkistrodon contortrix contortrix (Kudo and Tu, 2001) and the presence of isoenzymes of hyaluronidase in Naja naja venom (Girish et al., 2004). Proteolytic enzymes Proteases (E.C ) form the major toxin pool in the venom of viperid family snakes. These proteases are esterases that hydrolase primarily proteins and thus also serve as a digestive role. Further, they are responsible for most of the local tissue damage following envenomation. The important proteolytic enzymes reported fi-om the venom of snakes are: endo-peptidases, peptidases, arginine ester hydrolases, kininogenases and some of them also possess pro/anticoagulant activities. Proteases with variety of activities such as coagulant effects (Meume, 1966), hemorrhagic effect (Kini and Evans, 1992; Matsue et al., 2000) and local tissue necrosis (Markland, 1998; Lu et al., 2005) have been reported fi'om snake venoms. Snake venom proteases are heterogeneous group of proteins with a wide range of molecular masses between kda (Kini and Evans, 1992). Some of them are single chain proteins (Evans, 1984) and several others are multi-subunits proteins (Zaganelli et al, 1996; Fry, 1999). More than 150 different proteases have been isolated and about one third of them structurally characterized. The complete amino acid sequence of about 40 of them have been determined either by protein sequencing or deduced from the nucleotide sequence of the cdna.

9 Arginine ester hydrolases Deutsch and Diniz (1955) first reported the arginine ester hydrolase in snake venom. Later, several investigators reported the presence of arginine ester hydrolase activity fi-om both crotalid (White and Gate, 1982; Schwartz et al., 1984 & Silva et al, 1985) and viperid (Samel et al, 1987) venoms. The venoms of Elapidae and Hydrophidae generally do not show any arginine ester hydrolase activities. The venom of Ophiophagus hannah and Noja melanoleuca are exceptions, which showed weak activity towards arginine esters such as N-a-benzoyl-L-arginine ethyl ester (BAEE), /?-tosyl-larginine-methyl ester (TAME), L- a-acetyl tyrosine ethyl ester (ATEE) and N- a- benzoyl-l-arginine /7-nitroanilide (BAPNA). The substrate specificities of arginine ester hydrolases are strictly directed towards the hydrolysis of ester or peptide linkage to which an arginine residue contributes to the carbonyl group (Iwanaga and Suzuki, 1979). Kininogenases All crotalid and viperid venoms contain kininogenase (bradykinin releasing enzyme) activity. This enzyme specifically acts on plasma kininogen and liberates a physiologically active (vasoactive) nanopeptide, bradykinin. The bradykinin lowers the blood pressure (Iwanaga and Suzuki, 1979). It also hydrolyzes the synthetic arginine ester substrates (BAEE, SAME and TAME). Kininogenase has been isolated and purified fi-om the venom of Bothrops jararaca, Crotalus atrox, Agkistrodon halys blomhoffii, Echis coloratus, Bitis gabonica (Iwanaga and Suzuki, 1979). Vipera labetina (Siigur et al, 1982) and Cratalus atrox (Bjamason et al, 1983). Coagulant activity (Pro/anticoagulant) Blood coagulation is the result of a series of zymogen activation. Over 30 different substances that affect blood coagulation have been foxmd in the blood and tissues. Some promote coagulation are called procoagulants and others that inhibit coagulation are called anticoagulants; whether or not the blood will coagulate depends on the degree of balance between these two groups of substances. Normally, the anticoagulants predominate and thus the blood does not coagulate, but at the site of trauma the activity of procoagulants become much greater than that of anticoagulants,

10 and then clot does develop. Snake venoms contain many biologically active proteins, which intervene at different steps in blood coagulation (Teng et al, 1985). Snake venom proteins such as PLA2S, proteases, thrombin-like enzymes, pro-thrombin activators, factor-v activators, factor-x activators, fibrin(ogen)olytic and factor-x binding proteins and some non-enzymatic polypeptides are known to interfere in the blood coagulation processes (Evans et al., 1980; Ouyang et al, 1992; Markland, 1998; Huang et al, 1999; Matsui et al., 2000; Xu et al, 2001; Koh et al, 2001; Samel et al, 2002). There are six sites in the blood coagulation cascade where the venom procoagulants can interact. 1. Indirect factor-x activator 2. Activation of factor-x 3. Indirect pro-thrombin activator 4. Activator of factor-v 5. Conversion of pro-thrombin to thrombin 6. Direct conversion of fibrinogen to fibrin (thrombin like activity) Factor-X activators are either metallo-proteinases or serine proteinases (Tans and Rosing, 2001). The presence of factor-x activating enzymes have been reported from both crotalid and viperid venoms (Williams and Esnouf, 1962, Denson et al, 1972; Amphlett et al, 1982; Hofinann et al, 1983; Teng et al, 1984a; Hemker et al, 1984; Jayanthi, 1987). The mechanism of activation of factor-x by the purified procoagulant protein fi"om Daboia russelii has been shown to be calcium dependent (Fujikawa et al, 1972; Lindquist et al, 1978; Hofinann et al, 1983), Factor-X activators fi-om Ophiophagus Hannah and Bungarus fasciatus have been reported to be serine proteinases. Factor-V activating enzyme has been identified in the venoms of Vipera aspis (Boffa and Boffa, 1974). The physiological activator of pro-thrombin is thought to be a complex of factor-xa, factor-v, phospholipids and calcium. In addition it can also be activated by certain snake venoms of the family elapid and viperid (Walker et al, 1980; Chester and Crawford, 1982). Several factor-v activators have been described fi"om Bothrops atrox, Vipera russelii, Vipera labetina, Vipera ursine, Naja naja oxiana and Naja nigricollis venom (Rosing et al, 2001).

11 Prothombin (also known as factor II) is a single chain glycoprotein with a molecular weight of 72 kda (Rosing et al., 1988; Rosing and Tans, 1991, 1992). A large number of snake venoms contain prothrombin activators, which convert prothrombin into meizothrombin or thrombin (Rosing and Tans, 1992). Based on their structure, functional characteristic and cofactor requirements, they are classified into four groups. Group A prothrombin activators are metallo-proteinases and activate prothrombin efficiently without cofactors, such as phospholipids (PLs) or cofactor Va. Group B prothrombin activators are Ca^"^ dependent. They contain two subunits linked non-covalently: a metallo-proteinase and a C-type lectin like disulfide linked dimmer. Group C prothrombin activators are serine proteases foimd in Australian Elapids requiring Ca^^, PLs or factor-va for maximal activity. Oscutarin from Oxyuranus scutellatus also activates factor VII. Group D prothrombin activators are serine proteases and are strongly dependent on Ca, negatively charged PL and factor-va, Thrombin is a proteinase that activates or inactivates many different factors. It can also initiates aggregation of blood platelets. Thrombin-like enzymes are widely distributed in the venoms of the snakes belonging to Crotalidae and Viperidae. These are glycoproteins with a molecular weight ranging from kda. They are similar to thrombin in their physical and chemical properties but are not sensitive to thrombin inhibitors such as heparin, anti-thrombin-iii (Aronson, 1976) and soyabean trypsin inhibitor. Unlike thrombin, thrombin-like enzymes do not activate factor-xiii (Stocker and Barlow, 1976; Markland and Pirkle, 1977), which stabilizes the fibrin clot dimng coagulation. The mechanism of fibrinogen clot formation by the venom thrombin-like enzymes are different from that provoked by thrombin. The venom enzymes preferentially release only fibrinopeptide A (or B) (Markland and Pirkle, 1977). Endo-peptidases Endo-peptidases are mainly found in viperid venoms. A common feature of venom endo-peptidase is that they are metallo-proteases, capable of hydrolyzing peptide bonds with amino groups contributed by leucine and phenylalanine residues. Endopeptidases can easily be inactivated by EDTA and reducing agent such as cysteine (Iwanaga and Suzuki, 1979). Venom endo-peptidase catalyzes the hydrolysis of peptide 10

12 bonds of a variety of natural and synthetic substrates, including casein, hemoglobin, gelatin, elastin, collagen, fibrinogen, insulin, glucagon and bradykinin (Liu and Huang, 1997; Gutierrez et al., 2005). Endo-peptidases, which exhibit hemorrhagic activity, have been isolated fi-om several snake venoms such as Trimeresurus gramineus (Ouyang and Shiau, 1970), Agkistrodon acutus (Xu et al., 1981), Crotalus horridus (Civello et al., 1983), Bothrops neuwiedi (Mandelbaum et al., 1984), Crotalus atrox (Hagihara et al., 1985), Crotalus ruber ruber (Mori et al., 1987), Bothrops jararacussu (Mazzi et at., 2004), Bothrops lanceolatus (Stroka et al., 2005) and Trimeresurus malabaricus (Raghavendra et al., 2006). The hemorrhagic effect is attributed to enzymatic disruption of the basement membrane with loss of integrity of the vessel wall (Hati et al., 1999; Gutierrez and Rucavado, 2000). However, still it has to be established whether the hemorrhagic activity is due to direct action on basement membrane or indirectly by the release of a tissue factors which can be responsible for the disruption. The venom proteases are generally classified by their structure into: (1) Serine proteases and (2) Metallo-proteinases. There is only a weak or indirect evidence for the presence of thiol and aspartic proteases in the venoms. Some of them are seen to degrade mammalian tissue proteins at the site of bites in a non-specific manner to immobilize the victims. A number of them, however, cleave some of plasma proteins of the victims in a relatively specific manner to give potent effects, as either the activators or the inhibitors, on their hemostasis and thrombosis, such as blood coagulation, fibrinolysis and platelet aggregation (Ouyang et al., 1990; Pirkle and Theodor, 1990; Pirkle and Stocker, 1991; Markland Jr., 1991; Tu, 1996; Pirkle, 1998; Markland Jr., 1998). Serine proteases Some of the serine proteases have both fibrinogenolytic and fibrinolytic activities. But a number of them have only fibrinogenolytic activity and are also called 'thrombinlike' proteases if they show 'fibrinogen clotting activity' (Pirkle and Theodor, 1990; Pirkle, 1990; Pirkle, 1991; Markland Jr., 1991, 1998). However, their actions toward fibrinogen as well as the other substrates of thrombin are not exactly identical to those of 11

13 thrombin. Instead of fibrin(ogen)olytic activity for releasing bradykinin from kininogen is like mammalian kallikrein (or kininogenase) [Iwanaga, et al., 1976; Bjamason et al., 1983] and are also called 'kallikrein-like' proteases (Bjamason et al., 1983). In addition, there have been some reports on the snake venom serine proteases (S VSPs) with a unique activity, such as the activation of factor-v (Tokunaga et al., 1988), protein C (Kisiel et al, 1987), plasminogen (Zhang et al, 1995,1997) or platelets (Serrano et al., 1995). Most SVSPs are glycoprotein showing a variable number of N- or O- glycosylation sites in sequence. The type and site of glycosylation vary in SVSPs. They contain twelve cysteine residues, ten of which form five disulfide bonds, based on the homology with trypsin (Itoh et ah, 1987), the remaining two cysteines form a unique and conserved bridge among SVSPs, involving Cys245 (chymotrypsinogen numbering), found in the C-terminal extension (Parry et al., 1998). Metallo-proteinases The snake venoms contain a variety of metallo-proteases that are highly toxic resulting in severe bleeding by interfering with the blood coagulation and hemostatic plug formation or by degrading the basement membrane or extra-cellular matrix components of the victims (Iwanaga and Takeya, 1993; Bjamason and Fox, 1994; Marsh, 1994). More than 100 metallo-proteinases, including the isozymes from the same species, have been isolated and the amino acid sequences of about 20 enzymes have been determined. They are all Zn^"^ metallo-proteinases with a Zn^* binding motif of HEXXHXXGXXH and belong to the metzincin family as well as matrixins such as mammalian matrix metallo-proteinases (Stocker et al., 1995). Snake venom metallo-proteinases (SVMPs) are involved in the pathogenesis of local tissue damage, myonecrosis, edema and other reactions associated with inflammation (Gutierrez et al, 1995b; Rucavado et al, 1998, 2002; Clissa et al, 2001; Costa et al, 2002; Laing et al, 2003). The multiple roles of SVMPs in the pathogenesis of local tissue damage are summarized in the Fig Snake Venom Hemorrhagins Hemorrhage is a serious manifestation of snake bite, causing prolonged and sometimes permanent disability. Hemorrhage is principally caused by metalloproteinases, enzymes that are responsible for degrading proteins of extra-cellular matrix. 12

14 they also have cytotoxic effect on endothelial cells and act on components of the hemostatic system (Kamiguti et al., 1996). Hemorrhage is a common phenomenon in the victims of crotalidae and viperidae envenomation (Arnold, 1982). Hemorrhagins (the term was introduced by Grotto, 1967) act directly on the capillary basement membrane and the endothelial cells to cause internal hemorrhage. In mild envenomation, 'their action is limited to the site of the bite' however, in severe cases hemorrhage can be widespread involving the whole extremity concerned and even organs distant from the site of the bite, such as heart, limg, kidney, intestine and brain. Venom induced hemorrhage has been shown to be caused primarily by zmc dependent proteolytic action of hemorrhagic toxins, capable of degrading extra-cellular matrix proteins and blood clotting factors (Baramova er a/., 1989,1990; Markland, 1998; Gutierrez and Rucavado, 2000). Hemorrhagic activity has been associated with proteolytic activity. Chelation of the zinc atom abolishes both proteolytic and hemorrhagic effects (Bjamason and Fox, 1988; 1994). Of the 65 hemorrhagic toxins, 12 have been analyzed for their metal content, all of them have been foimd to contain zinc and many more are inhibited by metal chelators. Ten of the twelve toxins contained approximately 1 mole of zinc per mole of toxin (Bjamason and Fox, 1994). Therefore, that venom induced hemorrhage is primarily caused by metal dependent, proteolytic activities of the hemorrhagic toxins, probably acting on connective tissue and basement membrane components. Although, hemorrhagins are the main causative agents of hemorrhage, several other components residing in the crude venom can act as secondary factors to augment the process. Components that cause fibrinogenolysis render blood almost completely incoagulable. Anticoagulant factors directly block the clotting phenomenon. There are platelet aggregation inhibitors and enzymes that release kinin from kininogen. In the absence of blood coagulation and platelet aggregation, the two principle phenomena that occur following damage to blood vessels, hemorrhage initiated by hemorrhagins can go on imchecked with massive extravasation of RBCs into surrounding tissues, giving rise to swelling, blistering and edema (Bjamason and Fox, 1994). Some hemorrhagins also induce pharmacological effects such as myonecrosis (bilitoxin and ba HI), fibrinogenolysis (Atrolysin f, jarahagin), inhibiton of platelet aggregation (atrolysin a) etc 13

15 (Nikai et al., 1984; Ownby et al, 1990; Kamiguti et al. 1994; Gutierrez et ai, 1995; Jia e/a/., 1997). Structure and classification of hemorrhagins More than 65 hemorrhagins from 24 different species of snakes have been purified and characterized. Most of these have been found to be metallo-proteinases (Table 1.05). Snake venom hemorrhagic toxins are zinc dependent metallo-proteinases which belong to the family of 'metzincins', together with astacins, serralysins, matrix metallo-proteinases (MMPs) and ADAMs (enzymes with a disintegrin and 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-tum (Bode et al., 1993). On the basis of the domain structure, SVMPs have been classified into four main groups according to the domain constitution: (i) P-I class SVMPs has only a metalloproteinase domain apart from the pre- and pro-sequences. Their molecular masses vary from kda. They exhibit low hemorrhagic activity but with strong direct acting fibrinogenolytic activity. These are mostly weakly acidic proteins; (ii) P-II class SVMPs include enzymes presenting the metallo-proteinase domain followed by a disintegrin-like domain. The molecular mass is kda and their hemorrhagic potency is also low; (iii) P-III class SVMPs contain a cysteine-rich domain in addition to metallo-proteinase and disintegrin like domain. The molecular mass is kda, they possess strong hemorrhagic potency; (iv) P-IV class SVMPs are comprised by enzymes with two subunits, one constituted by the three domains characteristic of P-III enzymes and another being a C-type lectin protein, linked through disulfide bridges to the first one. The molecular mass ranges from kda and they possess very low hemorrhagic potency (Bjamason and Fox, 1994; Hite et al., 1994). Fig shows the schematic structures of snake venom metallo-proteinases. Mechanism of hemorrhage In hemorrhagic action of metallo-proteinases the enzymatic fiinction is given primary emphasis that helps in elucidating the factor(s) responsible for the leakage of 14

16 blood from the vessels. Subsequently, it became evident that the enzymatic disruption of the basement membrane (BM) xmderlying the endothelial cells of the capillaries (which have been found to be prime target of the hemorrhagins) is main factor responsible for hemorrhage. However, in-depth studies reveal certain other factors they may facilitate the process hemorrhage. A. Enzymatic disruption of basement membrane Both in vitro biochemical studies as well as in vivo microscopic observations have confirmed that hemorrhagins cause local hemorrhage by proteolytic digestion of the BM proteins. Basement membranes are extra-cellular sheets consisting of certain proteins such as type-iv collagen, laminin, nidogen (entactin), fibronectin and heparan sulfate proteoglycans (Inoue, 1989; Yurchenco et ai, 1990). BMs, also known as basal lamina, are placed beneath the epithelia (under capillary endothelium also). The chief constituent is type-iv collagen, the structxire of which is more flexible when compared with the fibrillar form. The specialized orientation pattern of these molecules result in the formation of a basic frame like mesh work to which the other constituents bind by means of specific associations. Laminin is a flexible complex of three long polypeptide chains and short arms of laminin can also bind to collagen (Martin, 1987). The molecules of nidogen are of special interest regarding the assembly and degradation of BM. Thus, it is thought to act as a bridge between the collagen type- IV and laminin networks. Secondly, nidogen has been found to be highly influenced by Zn and also highly susceptible to proteolytic degradation, which can allow rapid disruption of the BM structure. In fact, hemorrhagins can effectively degrade both type- IV collagen and nidogen. In addition, they can hydrolyze laminin and fibronectin but not the proteoglycans. These capabilities have made them very effective toxins mediating disruption of BMs to cause the hemorrhage. In hemorrhagins, such as sfrolysins the relationship between the hemorrhagic and general proteolytic potency is not always parallel and may even be inverse. All these observations have necessitated a search for mechanisms of hemorrhage other than BM degradation. The hemorrhagic potency of the toxin is related to the action on specific substrates such as BM proteins rather than general non-specific substrates (Baramova etal., 1989). 15

17 B. Enzymatic disruption of Capillary endothelial cells Capillaries, with a single cell thick wall, are the main targets of the hemorrhagic toxins. Exposure of capillaries to these toxins induces a disturbance in the endothelial cells (ECs), the degree of which varies from a simple fall-off from the substratum (BM) to complete lysis. Once this was established, investigations turned to explore whether the extravasation is hyper rhexis (through the cell by disrupting the plasma membrane and the integrity of the cell) or per diapedesis (through the gaps between the cells, keeping them viable and intact) mechanism. Interestingly, hemorrhagins have adopted both of them, some through the lysis of the cells (per rhexis), and the others through the formation of gaps {per diapedesis) between the cells. Mechanism of extravasation by some of the hemorrhagins are listed in the Table 1.06 (Hati et al, 1999) Hemorrhage/; r rhcms The pathogenesis of local hemorrhage has been investigated with a number of purified hemorrhage causing metallo-proteases at the ultra-structural level. In the majority of the cases,/?er rhexis mechanism has been described in which endothelial cells of capillary blood vessels become affected rapidly after metallo-proteinases injection. This is characterized by an initial swelling of cells, followed by formation of blebs from the luminal plasma membrane that occurs within a short interval of time. Transmission electron microscopic observations frequently depict swollen mitochondria, but intercellular junctions remain unaltered. The cells get detached from the substratum with subsequent to, prior rupture of the plasma membrane, allowing the blood to pass through the damaged cells into the surrounding tissue space. Capillary basement membrane, at the same time, gets disorganized and is often wholly or partially absent. Many larger vessels in most cases have been foimd to be congested with erythrocytes and platelets. Persistent hemorrhage occurs in capillaries in the form of extravasated and hemolyzed erythrocytes. Capillaries are also congested with platelets. With advancement of time they become very obscure due to extensive damage to the cells. In some capillaries platelets appear outside the lumen, suggesting direct damage to 16

18 the plasma membrane. A large amoimt of intravascular as well as extravascular fibrin is also present. The sequence in which endothelial cell damage and BM degradation occurs or whether both of them occur concomitantly has not yet been determined conclusively. Apart fi-om direct mechanism of cell damage, some indirect mechanisms have also been suggested. Rucavado et al. (1995) after studying BaHl and BaPl (Bothrops asper) in detail, have suggested that extracellular degeneration in vivo is only a secondary event resulting fi-om disturbance in the interaction between these cells and the surrounding basement membrane. Hemorrhage p^r diapedesis In contrast, the mechanism described earlier suggest that erythrocytes escape through widened intercellular junctions instead of gaps in endothelial cell cytoplasm (Ohsaka et al, 1975; Ohsaka., 1979). This apparent discrepancy in the process of extravasation might be due to actual differences in the mechanism of action of hemorrhagic toxins. Although it is more likely a consequence of variations in the methodologies and the types of micro-vessels examined. Conflicting results have been also reported concerning the cytotoxic activity of hemorrhagic metallo-proteinases on endothelial cells. Despite, observations of endothelial cell pathology in vivo after injection of Bothrops asper venom metalloproteinases BaHl and BaPl (Moreira et al, 1994; Lomonte et al, 1994a), these toxins are shown to be devoid of cytotoxicity on endothelial cells in culture, as judged by the lack of release of intra-cellular enzymes (Obrig et al, 1993; Lomonte et al, 1994a). The only effect observed in vitro was a dose dependent detachment of these cells from their substratum, probably due to proteolytic degradation of extra-cellular matrix components. Such effects were abolished when metallo-proteinases were incubated with chelating agents that inhibit enzymatic activity (Borkow et al, 1995). Simultaneously, hemorrhagic toxins devoid of proteolytic activities have also been reported (Omori et al, 1964; Toom et al, 1969). Vishwanath et al, 1987b purified and characterized a PLA2 (VRV-PL-VI) from Vipera russelii (North) venom, which 17

19 induced necrosis, hemorrhage in the liver, kidney, pituitary and thyroid glands, hemorrhage and bleeding in the peritoneal cavity and edema in the footpad of mice. TF- PL-Ia and TF-PL-Ib from Trimeresurus flavoviridis venom was non-proteolytic but induced hemorrhagic spot on the iimer surface of the ventrolateral side of the skin at the site of injection (Vishwanath et al., 1987). VRV-PLV-III from Vipera russelii showed lung hemorrhage (Kasturi and Gowda, 1989). While metallo-proteases which are devoid of hemorrhagic activity also reported from Russell's viper {Vipera russelii) and Indian cobra (Naja naja) venom (Jayanthi et al., 1990; Jagadeesha et al., 2002). To complicate the situation, hemorrhagins have often been found to exert additional toxic effects, such as edema, myotoxicity, platelet aggregation and fibrinogen depletion, which considered as local and systemic manifestations. Local manifestations Changes at the site of envenomation are the earliest manifestations of snakebite (Reid, 1979). Features are noted within 6-8 min but may have onset up to 30 min (Reddy, 1980; Reid and Theakston, 1983). Localized pain with radiation, tenderness at the site of bite and the development of small reddish wheal, occur at first. This is followed by edema (Paul, 1993) and swelling which can progress quite rapidly and extensively, even involving the trunk (Saini et al., 1984). Tingling and numbness over the tongue, mouth, scalp and paraesthesias around the wound occur, mostly in viper bites (Reddy, 1980). Local bleeding including ptechial and/or piupuric rash is also seen, most conmionly at the site of bite with this family of snakes. Crotalid and Viperid venoms are known to cause local effects, which frequently include pain, swelling, echymoses and local hemorrhage, are usually apparent within minutes of the bite. Such signs are sometimes followed by liquefaction of the area surrounding the bite. The local area of bite may become devascularized with features of necrosis, predisposing to onset of gangrenous changes. Secondary infections including tetanus and gas gangrene may also result (Tu, 1991; Philip, 1994). 18

20 Edema inducing activity Swelling and edema are often the chief early symptoms of snake venom poisoning at the affected part of the victim. Snakebite leads to increase in the capillary permeability, which may cause loss of blood and plasma volume into the extra-cellular space. Accumulation of fluid in the interstitial space is responsible for edema. The edema induced by Bothrops jararaca venom is mediated by cyclooxygenase and lipoxygenase eicosanoid products, and by the action of Li and L2 adrenergic receptors (Trebien and Calixto, 1989). Pretreatment with indomethacin, a well known inhibitor of the cyclooxygenase pathway reduced the edema induced by Bothrops asper and Bothrops jararaca venoms. This suggested the role of eicosanoids formed in the edema induction phenomena (Trebian and Calixto, 1989; Campbell, 1990). The venoms of Trimeresurus flavoviridis (Vishwanath et al., 1987; Yamaguchi et al., 2001), Trimeresurus mucro squamatus (Teng et ah, 1989; Chiu et al., 1989), Naja naja naja (Bhat and Gowda, 1989; Basavarajappa and Gowda, 1992), Echis carinatus (Kemparaju et al., 1994), Bothrops asper (Lomonte et al., 1993; Chaves et al., 1995) and Bothrops lanceolatus (de Faria et al, 2001) are reported to induce edema. Systemic manifestations The systemic manifestations depend on the pathophysiological changes induced by the venom of that particular species. Elapid venoms produce symptoms as early as 5 minutes (Paul, 1993) or as late as 10 h (Reid, 1979) after bite, whereas vipers take slightly longer time, the mean duration of onset being 20 min. (Paul, 1993). However, symptoms may be delayed for several hours. Sea snake bites invariably produce myotoxic features within 2 h so that they are reliably excluded if no symptoms are evident within this period (Paul, 1993). The magnitude of systemic toxicity induced by toxins are directly relay on the concentration, efficiency and rate of diffusion of target specific toxins. Based on the predominant constituents of venoms of a particular species, snakes were loosely classified as neurotoxic (notably Cobras and Kraits), hemorrhagic (vipers) and myotoxic (sea snakes). However, it is now well recognized that such a strict categorization is not valid as each species can induce any kind of manifestations (Estevao-Costa et al., 2000; Moura-da-silva et al., 2003). 19

21 Myotoxicity Myotoxicity is one of the common and a serious consequence of snake venom poisoning. Local hemorrhage and necrosis affecting the skin and muscle tissues are the chief manifestations of myotoxicity. Myonecrosis may be due to the vascular degeneration and ischemia caused by venom metallo-proteinases, or it may result from the direct action of myotoxins upon the plasma membrane of muscle cells, which is evident from the rapid release of cytoplasmic markers creatine kinase (CK), creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) accompanied by the prominent increase in total muscle calcium ion (Rucavado and Lomonte, 1996; Gutierrez and Lomonte, 1997; Gopalakrishnakone et al., 1997; Salvini et al., 2001; Souza et al., 2002). The increased influx of calcium ion leads to the cell death (Mebs and Samejima, 1980). Myotoxicity is associated with many pre-synaptically acting neurotoxins (Gopalakrishnakone et al., 1980; Ziolkowske and Bieber, 1992). In addition, several myonecrotic polypeptides and myotoxic PLAa enzymes have been isolated and characterized from various snake venoms (Fohlman and Eaker, 1977; Harris and Maltin, 1982; Mebs, 1986; Mebs and Samejima, 1986; Kasturi and Gowda, 1989; Weinstein et al., 1992; Geh et al., 1992; Lomonte et al., 1994a, b; Thwin et al., 1995; Ownby tt al., 1997; Radis-Baptista etal, 1999; Nunez et al., 2001). Intramuscular injection of many hemorrhagic metallo-proteinases results in acute muscle cell damage, i.e., myonecrosis (Gutierrez et al., 1995; Franceschi et al., 2000). The mechanism by which venom metallo-proteinases induce muscle damage has not been fully elucidated. However, Gutierrez et al., (1995), investigating the action of hemorrhagic metallo-proteinases BaGl from Bothrops asper venom, suggested that muscle damage was secondary to the ischemia that ensue in skeletal muscle as a consequence of bleeding. Platelet aggregation Platelet aggregation means platelets sticking to each other rather to a different surface. In the circulating blood, discoidal platelets are considered to be in the resting state and in this state; they do not readily adhere to any surface. They become sticky when there is any damage to the vascular endotheliimi. They aggregate into a mass at the 20

22 site of vascular injury and form hemostatic plug, which seals off the break in the blood vessel. Platelets become sticky upon stimulation by diverse agonists which includes small molecular weight compounds such as AD?, arachidonate, serotonin and epinephrine; enzymes such as thrombin and trypsin; particulate materials, such as collagen and antigen-antibody complexes; lipids, such as platelet activating factor (PAF-acether) and ionophores, such as A23187 (Zucker, 1989). Stimulation by these diverse agonists initiates a series of cellular responses such as adhesion, change in platelets shape from disc to sphere and release of various substances (Kini and Evans, 1990). Enzymatic and non-enzymatic platelet aggregating factors have been isolated from so many different snake venoms (Kini and Evans, 1990 and references therein; Ouyang et al., 1992). Snake venom components such as PLA2S, hemorrhagic metallo-proteinases, RGD containing disintegrins, GPI6-binding proteins, lectin-like proteins and antiplatelet polypeptides (Kamiguti et al., 1998; Kemparaju et al., 1999; Siigur and Siigur, 2000; Jagadeesha et al., 2002; Wei et al., 2002) are known to interfere in platelet function. Fibrinogenolytic proteinases Fibrinogenolytic activity has been described in the venoms of members of the Viperidae and Elapidae families (Markland Jr., 1991). The substrates for the fibrinogenolytic enzymes is fibrinogen, appears as large trinodular protein by electro microscopy. The protein contains two symmetric half-molecules which are disulfidelinked. Each half contains three chains designated as A-a, B-P and y with molecular weights of 63.5, 56 and 47 kda respectively. The fibrinogen molecule has a molecular weight of 340 kda (Bauer and Rosenberg, 1987). Fibrinogen contains long stretches of amino acids, which are exposed to proteolytic enzymes including the snake venom proteinases. Fibrin, however, has a cross-linked structure and is much less susceptible to proteolysis. Most of the enzymes characterized are zinc metallo-proteinases and degrade A-a chain of fibrinogen preferentially. Serine proteases have specificity toward the B-p chain 21

23 of fibrinogen. However, there are exceptions to these generalizations and specificity for Aa or BP chains, as there is substantial degradation of alternate chain with time. Most of the metallo-proteinases are fibrinolytic and many of the serine proteinases are both fibrinogenolytic and fibrinolytic (Brand et al., 2000). Fibrinogenolytic metallo-proteinase cleave amino-terminal to hydrophobic amino acids, while fibrinogenolytic serine protease cleave carboxy-terminal to basic amino acids. Non-enzymatic venom components Most of the deleterious pharmacological effects of snake venoms are attributed to enzymes present in the venom. However, the polypeptides without enzyme activity contribute significantly towards the lethal potency and also elaborate several pharmacological properties of the venom. Several polypeptides, possessing important pharmacological properties have been identified, purified and characterized fi-om different snake venoms (Tablel.07). So far more than 100 non-enzymatic proteins have been characterized, and these protein toxins are grouped into well recognized families as follows: 1) three-finger toxins (includes neurotoxins and cardiotoxins), 2) serine protease inhibitors, 3) lectins, 4) sarafatoxins, 5) nerve growth factors, 6) atrial natriuretic peptides, 7) bradykinin-potentiating peptides, 8) disintegrins, and 9) helveprins/crisp (Mebs and Claus,1991; Kini, R. M ; McLane, et al, 1998). In addition to independently acting toxic peptides, there are reports of nonenzymatic non-toxic peptides that interact synergistically with PLA2S or other venom proteins and thereby enhance the toxicity of interacting protein (Jayanthi and Gowda, 1990). Other important non-enzymatic components are inhibitors of acetylcholine esterase, phospholipase and proteases. Enzyme inhibitors from snake venoms Inhibitors of enzymes such as acetylcholine esterase, phospholipase and proteases have been reportedfi-omseveral snake venoms. Naturally occurring phospholipase-inhibitor complexes have been shown to be present in the venom of both Elapidae and Crotalidae (Braganca et al., 1970; Vidal and 22

24 Stoppani, 1971; Breithaupt, 1976; Simon and Bdolah, 1980). Rudrammaji, 1994 has reported the isolation and characterization of PLA2 inhibitors from the venom of Indian Cobra, Naja naja naja. These inhibitors are small peptides and appear to have ionic interaction with phospholipases. Potent inhibitors of serine proteases have been detected in venoms such as Vipera russelii (Takahashi et al., 1974), Naja nivea, Haemachatus haemachatus (Hokoma et al., 1976), Bungarus fasciatus (Liu et al, 1983). These inhibitors are low molecular weight basic polypeptides and are homologous to bovine basic pancreatic trypsin inhibitor (BPTT). Inhibitors of carboxypeptidase (angiotension converting enzyme or bradykinin) have been reported from Bothrops jararaca venom. These inhibitors inhibit the bradykininase activity and potentiate the activity of the peptide hormone bradykinin. Hence they are named as Bradykinin potentiating factors (BPF) [Greene, 1974]. The inhibitors of thrombin like enzyme factor-x activating enzyme and prothrombin activator have been reported from the venoms of Agkistrodon halys blomhoffii, Bothrops jararaca and Ophiophagus scutellatus scutellatus respectively (Ohshima et al., 1969; Walker et al., 1980) Inhibitors of hemorrhagins (antihemorrhagins) foimd in Crotalus atrox were characterized as acidic glycoproteins with a molecular weight ranging from 65 to 80 kda (Weissenberg et al., 1991). Omori-Satoh et al., 1972 isolated an anti hemorrhagic factor from the serum of the habu snake, Trimeresurus flavoviridis. The purified serum factor inhibited two immonologically distinct hemorrhagic principles, HRi and HR2 in the venom. Application of snake venom components The wide range of activities of snake venom proteins on human physiological system has provoked researchers to look for potential use of venom components. Snake venom proteins are used for the treatment of pathological conditions such as leprosy, epilepsy, chronic pains of the nervous system, cancer, neuritis, migraine, neuralgia, arthralgia, tuberculosis etc. Some components are used as therapeutic agents while others 23

25 have served as research tools to understand some of the physiological functions. Snake venoms are rich source of enzymes, many of which like phosphodiesterases, LAO, thrombin like enzymes are purified and sold commercially. Post-synaptic neurotoxins act as antagonists to acetyl choline. They have been used to quantitative determination of acetyl choline receptors (ACHR) in neuromuscular junctions. Cobra venom factor (CVF) has found application during organ transplantation due to its anti-compliment factors. Nerve growth factor (NGF) stimulates the growth of nerve fibers in vitro. Fibrinogenolytic enzymes of venoms are used to dissolve blood clots without causing hemorrhage. PLA2 is also being used for preparation of lyso-phospholipids, as catalysts to synthesize phospholipids and in studies on membrane asymmetry. Treatment of snake bite Snake venom poisoning is a serious medical, social and economic problem in many tropical countries especially in Africa, South America and Southeast Asia including India. Snake venom poisoning can simultaneously, sequentially and disjimctively exert toxic and lethal effects on local tissues, blood, cardiovascular, respiratory and nervous system (Ohsaka, 1979); hence it is a medical emergency. There are impressive number of stories about the treatment of snakebite in mythology and folklore. All of them display similar themes in different cultures over the span of centuries (Russell, 1980). Though, these methods have historical value none of them are effective in curing snake venom poisoning. Botanical cure is the most popular and widely used of all folklore remedies for snakebite. The qjplication of various plants, most often the root extract, either in the form of poultice to the bitten region or orally is still in practice. A large number of plants (Morton, 1981; Duke, 1985; Mors et al., 2000) and its components are claimed to antagonize the action of snake venoms. In India, the plants that have been used for the snakebite include Acalypha indica, Achyranthus aspera, Achyranthus superba. Capsicum annuum. Datura fastuosa, Strychnos colubrine, Rauwolfia serpentina, Hemidesmus indicus, Aristolochia radix. Mimosa pudica, Withania somnifera and Tamarindus indica (Chopra e/a/., 1958; Nadkami, 1976; Sathyavathi etal, 1976; Gowda, 1997; Alam and 24

26 Gomes, 1998; Mahanta and Mukherjee, 2001; Deepa and Gowda, 2002; Ushanandini et al., 2006). Even now attempts are continuously being made in this direction for neutralization of venom toxicity using active principles isolated from various plants. The mortality due to snake venom poisoning is reduced markedly by the use of antivenoms, which are the most useful and best remedy available until today for snakebite treatment (Gutierrez et al., 1985; Lomonte et al., 1996; Leon et al., 1997; Gutierrez et al., 1998; Leon et al., 1999; Leon et al., 2000; Rucavado et al., 2000). Since Calmette prepared cobra antivenom in 1884 (See, Grasset, 1957), antisera against various kinds of snake venoms have been prepared and their effectiveness in treatment of snake venom poisoning has been widely accepted. Monovalent (prepared against single species of snake venom) and polyvalent (prepared against mixture of selected species of snake venoms) antivenoms are produced commercially by several laboratories all over the world (Theakston and Warrell, 1991). Though the mortality due to snake venom poisoning is reduced markedly by the use of anti-venoms, there are several inherent drawbacks associated with it, some of them are; their limited availability, specificity, storage, dosage, solubility and sensitivity of individuals towards antivenoms. Excess infusion of anti-venom increases the potential risk of serum sickness, which can lead to arthritis, vasculitis and nephritis. 25

27 Introduction. Common name Scientific name Family Distribution Indian Spectacled Cobra King Cobra Naja naja naja Ophiophagus hannah Elapidae Elapidae Throughout India, sea level up to 4000 m (in the Himalayas) Confined to the dense forests of the Western Ghats and the Northern hill forests. Himalayan foot hills (up to 2000 m). Forests of Assam, Orissa, Bihar, West Bengal and the Andamans. Common icrait Bungarus caeruleus Elapidae Throughout India, sea level up to 1700 m. Russell's viper Vipera russelii Viperidae Hills and plains throughout India Up to 3,000 m. Saw-scaled viper Echis carinatus Viperidae Table 1.01 Distribution of Big Four snakes in India Throughout India, sea level up to 2000 m. Enzymes found in all venoms Phospholipase A2 Phosphodiesterase Phosphomonoesterase L-amino acid oxidase 5' Nucleotidase Enzymes found mainly in Viperid venoms Endopeptidase Arginine ester hydrolase Factor X activator Enzymes found mainly in Elapid venoms Acetylcholinesterase Glycerophosphatase Enzymes found in some venoms Glutamate-pyruvate transaminase Amylase Heparin like enzyme Deoxyribonuclease Adenosine triphosphatase NAD nucleotidase Ribonuclease Hyaluronidase Kininogenase Thrombin like enzyme Prothrombin activator Phospholipase B Catalase Lactate dehydrogenase Table 1.02 Enzymatic proteins found in snake venoms 26

28 Introduction. Trivial name Phospholipase A2 Trivial name Phosphatidylcholine M.Wt (Daltons) Characteristics Simple protein, histidine at active site L- amino acid oxidase Phosphodiesterase 5'-Nucleotidase Phosphomonoesteras e Deoxyribonuclease Ribonuclease Hyaluronidase NAD-nucleosidase Arylamidase Endopeptidase Arginine ester hydrolase Kininogenase Thromobin-like enzyme Factor X activator Prothrombin activator Factor V activator Acetylcholineesterase Phospholipase B L-amino acid Oligonucleotides 5'- Mononucleotides p-nitrophenylphosphate DNA RNA Hyaluronan NAD L-Leucine napthylamide Casein, Hemoglobin BAEE, TAME Plasma kininogen, BAEE, Fibrinogen, BAEE Factor X Prothrombin Factor V, BAEE Acetylcholine Lysolecithin Glycoprotein, 2 moles FAD per mole enzyme, heat labile Heat labile, EDTA sensitive, acid unstable, optimum at ph 9 Heat labile, EDTA sensitive, acid unstable, optimum at ph Heat labile, EDTA sensitive, acid unstable, optimum at ph Optimum at ph 7-9, specific towards pyrimidine nucleotides Heat labile. Optimum at ph 5-6, resembles testicular enzyme Heat labile, optimum at ph 7.5, nicotinamide sensitive Heat labile, SH-enzyme, PCMB sensitive, optimimi at ph 8.5 Glycoprotein, metal (Ca^*, Zn "^ protease, EDTA sensitive, heat labile, optimum at ph 8-9 Glycoprotein, heat stable, DFP sensitive, optimum at ph 8-9 Heat stable, DFP sensitive, specific towards kininogen Glycoprotein, heat stable DFP sensitive Glycoprotein, heat labile, DFP insensitive, EDTA sensitive, activates Factor DC. Glycoprotein, heat labile, DFP insensitive, EDTA sensitive. DFP sensitive, heat stable Heat labile, DFP sensitive, optimum at ph Heat stable, optimum at ph 10 Table 1.03 Some of the properties of enzymes found in snake venoms 27

29 Introduction. Characteristics Features Pharmacological activities PLA2 Catalyzes the Ca ^^ dependent hydrolysis of 2-acyl ester bond in 3-Sn-phosphoglycerides releasing lysophosphatide and free fatty acids. Neurotoxicity (post/pre), myotoxicity. Hemorrhage, hypotensive, cardiotoxic, platelet aggregation, edema inducing and convulsant. LAO Glycoprotein, 2 moles FAD per mole enzyme, heat unstable, releases hydrogen peroxide Exact role is not known, but it possesses antibacterial, apoptosis, anti-platelet activities. Serine protease Most SVSPs are glycoproteins, the kind and site of glycosylation differ from one SVSPs to other. They contain twelve cysteine residues They act on specific factors of the coagulation cascade systems to cause imbalance of the haemostatic system of the prey. Metallo-proteinase They belongs to Reprolysin subfamily, They are zinc-dependent metallo-proteinases They are known to induce hemorrhage, myonecrosis and inflammatory reactions Hyaluronidase Endoglycosidase, hydrolytic and ubiquitous enzyme. It catalyzes the cleavage of internal glycosidic bonds of ceratin acid mucopolysaccharides They are spreading factors, which promote local tissue damage Table 1.04 Major snake venom proteins and their function 28

30 Introduction. Class I Toxin M.Wt (kda) MHD (fig) References A.acutus EP Ouyang and Huang, 1976, A.acutus Ac Mori et al., 1984; Nikai et al., 1977 A.acutus Ac Mori etal., 1984 A.acutus Ac Mori etal., 1984 A.acutus AaH-I Xue/a/., 1981 A.acutus AaH-II XueM/., 1981 A.acutus AaH-III Xuetal., 1981 Bitis arietans HT-1 ND - Mebs and Panholzer, 1980 Bothrops jararaca HF-1 ND 0.1 Assakura er a/., 1986 Bothrops moojeni MP-A 22.5 ND Assakura et al., 1985 C adamanteus Protease I 24.6 ND Kurecki and Kress, 1985 C.adamanteus Protease II 23.7 ND Kurecki and Kress, 1985 C.atrox HT-b 24 3 Bjamason and Tu, 1978; Bjamason et al., 1983 C.atrox HT-c 24 8 Bjamason and Tu, 1978; Shannon et al., 1989 C.atrox HT-d Bjamason and Tu, 1978; Shannon et al., 1989 C.atrox HT-e Bjamason and Tu, 1978; Kite et al., 1992a C.b. basiliscus B-1 27 <10 Molina ef a/., 1990 C.b. basiliscus B <10 Molina e/a/., 1990 C. ruber ruber HT Mori et al., 1987; Takeya et al., 1990b C.ruber ruber HT Mori et al., 1987; Takeya et al., 1990b C.s.scutulatus P Martinez et al., 1990 Lachesis muta muta LHF-II Sanchez e^ a/., 1991 T.flavoviridis HR-2a Takahashi and Ohsaka, 1980; Nikai et al., 1987 T.flavoviridis HR-2b Takahashi and Ohsaka, 1980; Nikai et al., 1987; T.flavoviridis HR-2a 24 - Yonaha etal., 1991 (Okinawa) T.flavoviridis HR-2b 19 - Yonahae/a/., 1991 (Okinawa) T.gramineus T. mucrosquamatus HR-1 HR-a Ouyang and Shiau, 1970 Nikai e/fl/., 1985b T. mucrosquamatus HR-b mkai etal., 1985b 29

31 Introduction. V.labetina Lebetase Siigur and Siigur, 1991 Cerastes cetastes Class II Cerastase F Daoudefa/., 1986a,b A.acutus A.acutus A.bilineatus B.jararaca B.jararacussu B.neuwiedi B.neuwiedi Calloselasma rhodostoma Ac-3 Ac-4 Bilitoxin Bothropsin Bjussu MP-I NHF-a NFF-b HP-I _ Moii etal., 1984 Mode/a/., 1984 Imai et al., 1989 Mandelbaum e/a/., 1982 Mazzi et al., 2004 Mandelbaum e/a/., 1984 Mandelbaum et al., 1984 Bandoe/a/., 1991 C.atrox C.atrox C.h.horridus C.r.ruber V.a.ammodytes V.a.ammodytes V.a.ammodytes V.berus berus V.palaestinae V.palaestinae V.palaestinae T.flavoviridis Atractaspis engaddesis HT-f HT-g HP-rv HT-1 HT-1 HT-2 HT-3 HMP HR-1 HR-2 HR-3 HR-1 HT Nikai et al., 1984 Nikai et al., 1984 CivelloeM/., 1983a, b Mon etal., 1987 Samel and Siigur, 1990 Ovadia, 1978a Ovadia, 1978a Ovadia, 1978a Yonahdi etal., 1991 Ovadia, 1987 Class III A.halys blomhoffti B.asper Bitis arietans HR-1 BaH4 BHRa Oshimae/a/., 1972 Franceschi et al., 2000 Omori-Satoh e/a/., 1995 B.jararaca B.jararaca B.jararaca C.adamanteus C.atrox BHRb HF-2 HF-3 Jarahagin Proteinase H HT-a Omori-Satoh e/a/., 1995 Omori etal., 1964; Oshima etal, 1972 Assakura et al., 1986 Assakura et al., 1986 Paine e/a/., 1992 Kurecki and Kress, 1985 Bjamason and Tu, 1978; Bjamason et al

32 V.aspis aspis HT T.flavoviridis T.flavoviridis HR-IA HR-IB Omori-Satoh and Sadahiro, 1979 Omori-Satoh and Sadahiro, 1979; Takeya et al., 1990 T.gramineus HR Huang e? a/., 1984 Ophiophagus Hannah Hannahtoxin Tan and Saifliddin, 1990 V.a.ammodytes VaHl 70 >1 Leonard! era/., 2001 VaH2 70 >1 Leonard! era/., 2001 Class IV A.h.blomhoffii HR-II Omori et al., 1964; Satake et al., 1963; Iwanaga etal., 1965; Oshima etal., 1968, 1972 L.muta muta T. mucrosquamatus LHF-I Mucrotoxin Sanchez er a/., 1987 Sugihara er a/., 1983 Trimeresurus purpureomaculatus Hemorrhagin 72 - Khow et al., 2002 Tablet.05 List and proposed Classification of known Hemorrhagic Toxins isolated from snake venom SNAKE VENOM METALLOPROTEINASES Oogndaiion of MoodclotUng ( ctort ExtractHular nurtifx dtgndition Figure 1.01 Summary of the multiple roles of snake venom metallo-proteinases in the pathogenesis of local tissue damages (Gutierrez and Rucavado, 2000) 31

33 m^^!^ Introduction. idii^ntigfih P4 MBMlopratBlVHW?'\\ EXiNioitn pnrareor R6& Nil tigtiitmihiiirmais p-rv 0-t P8l«*ln j-bs-' mohlofirdmrmae whhi Figure 1.02 Schematic structures of snake venom metallo-proteinases Toxin Species Mechanism References ACI-l Bulitoxin Agkistrodon contortrix laticincttts Agkistrodon bilineatus bilineatus Per rhexis Per rhexis Ovmby et al., 1997 Ownbyero/., 1990 HT-1 and 2 Crotalus ruber ruber Per rhexis Obng etal., 1993 Atrolysin a Crotalus atrox Per rhexis Obrigera/., 1993 Proteinase IV Proteinase H Crotalus horridus horridus Crotalus adamenteus Per rhexis Per rhexis Ownby and Geren, 1978 Anderson et al., 1977 HR-l,-2a,-2b Trimeresurus flavoviridis Per diapedesis Ohasaka, 1976 BaHl Bothrops asper Per diapedesis Borkowe/o/., 1995 Table 1.06 Mechanism of Extravasation by hemorrhagins (Hati etal., 1999) 32

34 Introduction. Non enzymatic toxins M.Wt (IcDa) Snalce species References Elapidae Neurotoxin (toxin a) 6,787 Naja nigricollis Karlssone/a/., (1966) Cobramine A and Cobramine B Dendrotoxin (DTX) a-neurotoxin, B.F.III Cardiotoxin Muscarinic toxin (MTxs) Phospholipase Inhibitor (NN-I3) Nawaprin Viperidae: Crotalinae Crotamine Myotoxin a Peptide C Myotoxin CAM-toxin Wagleri toxin Lethal peptide I Viperinae Neurotoxin Trypsin Inhibitor (Tj) Ammodytin L (AMDL) Hydropliidae 6,400 7,077 6,500 7,000 7,500 6,500 4,900 4,400 4,932 5,035 5,132 8,900 2,504 11,600 6,900 14,000 Naja naja Dendroaspls angusticeps Bungarus fasciatus Naja nigricollis Dendroaspis angusticeps Naja naja naja Naja nigricollis Crotalus durissus terrificus Crotalus viridis viridis Crotalus viridis helleri Crotalus viridis concolor Crotalus adamanteus Trimeresurus wagleri Trimeresurus wagleri Vipera palaestinae Vipera russelii Vipera ammodytes Larsen and Wolff (1968) Harvey and Karlsson (1980) Jie/a/.,(1983) Kini era/., (1989) Ademefa/., (1988) Rudrammaji and Gowda (1998) Torres, e/a/., (2003) Laure, (1975) Ownbyefa/.,(1976) Maedae/a/.,(1978) Biebere/a/., (1983) Samejimae/a/., (1988) Tan and Tan, (1989) Weinsteine/a/., (1991) Morazefa/., (1967). Jayanthi and Gowda (1990). Krizaje/a/.,(1991). Erubutoxin a 6,760 Laticauda semifasciata Tamiyae/a/.,(1967) Erubutoxin b 6,780 Laticauda semifasciata Tamiya et al., (1967) Neurotoxic peptide 6,520 Laticauda laticaudata Laticauda colubrine Sato ef a/., (1969) Table 1.07 Non-enzymatic toxic proteins/ peptides found in snake venoms 33

35 AIM AND SCOPE OF THE INVESTIGATION Snake venom is a complex mixture of protein and peptide toxins with diverse pharmacological activities. Snake bite is a subcutaneous/intramuscular injection into the prey/human victim. The pathophysiology of envenomation includes both local and systemic effects, which include hemorrhage, edema, myotoxicity, neurotoxicity, cardiotoxicity, coagulant (pro/anti), hemostatic (activating/inhibiting) effects (Kini, 1997, Shashidharamurthy et al., 2002, Aird, 2002). With some exceptions, snakes of the viperidae family induce envenomation characterized by hemorrhage, in severe cases the systemic hemorrhage (Tan and Ponnudurai, 1990; Mebs and Langeluddeke, 1992). Venom enzymes such as hemorrhagic metallo-proteases, myotoxins (enzymatic/non-enzymatic) and hyaluronidases are found to be the key toxins which are responsible for causing local tissue damage and systemic toxicity as well (Gutierrez and Lomonte, 1989; 1997; Gopalakrishnakone et al., 1997; Escalante et al., 2000; Anai et al., 2002; Girish et al., 2002). However, snake venom do vary greatly with respect to the relative abundance of these agents viz., myotoxic PLA2 and cardiotoxins are predominant in cobra venom, while hemorrhagic metallo-proteases are rich in viper venoms (Yingprasertchai et al., 2003). Venom hemorrhagic metallo-proteinases provoke rapid spreading of venom components from the injected area into systemic circulation, as well as causing local tissue damage. Although the precise mechanism remains unclear, it is likely that the venom components are easily differed to the tissue and absorbed into vessels by the degradation of extra cellular matrix and vascular basement membrane by venom hemorrhagic metallo-proteinases. Most of the snake venom hemorrhagic metalloproteinases are Zn^"^ dependent metallo-proteinases, which is a characteristic of viperid snake venoms. These hemorrhagic metallo-proteinases may play key roles in the pathogenesis of both local and systemic complications resulting from snake envenomation and therefore, may become the target molecule for study (Franceschi et al., 2000). Among the viperidae family Russell's viper {Daboia russelii) is one of the most conmion poisonous snakes of India responsible for several thousand deaths and also 34

36 perhaps, more people suffer from myonecrosis and hemorrhage due to its bite. A striking geographical variation is observed in the composition of Russell's viper venom as well as in the clinical manifestation by its bite. Variation in the composition of Vipera russelii venom samples obtained from northern, eastern, western and southern parts of India has been reported (Prasad et al., 1999a). Venom samples from eastern region is characterized by high lethal potency, hemorrhagic and proteolytic activities but less PLA2 activity compare to other regions. The bite by Russell's viper in this region induces spontaneous hemorrhage (Chakrabarty et al., 2000; Madhu kumar and Gowda, 2006). Hemorrhagic metallo-proteases are the main contributors towards hemorrhagic activity of the Russell's viper venom. Protease hemorrhagins like RWX and AE-II (Jayanthi, 1987) and VRH-I (Chakrabarty et al., 1993) were purified from southern and eastern regional Vipera russelii venoms respectively. No reports are available except Uma, 1999 (Ph.D Thesis) for the presence of hemorrhagic complex in the eastern Daboia russelii venom. Even the presence of weakly acidic/neutral PLA2 complex and its isoforms are not reported in the same venom till now. In view of the above facts, following study was undertaken i) Isolation and characterization of hemorrhagic complex (HC) from the eastern region D. russelii venom ii) Neufralization of pharmacological properties of HC and D.russelii venom against antibody/antiserum raised (HC-IgG). iii) Purification and characterization of weakly acidic/neutral PLA2 complex (VRV-PL-II) and its three iso-forms VRV-PL-IIa, VRV-PL-IIb, VRV-PL-IIc. D. russelii (east) venom is not abundant in systemic toxins but contains high concenfration of hydrolytic enzymes, peptides and PLA2 enzymes. These enzymes are responsible for many pathological activities of the snake bite. Nowadays many purified snake venom toxins are used for therapeutic purposes. Thus the contribution of hemorrhagins and PLA2 enzymes to the lethal potency and pharmacological properties of the whole venom poisoning is not well known. Hence in the present study an attempt is 35

37 made to isolate and characterize the HC and PLA2 iso-forms from D.russelii (east) venom with a view to understand 1) the contribution of HC to the venom toxicity 2) to study the effect of polyclonal antibodies/antiserum prepared against HC and whole venom on the biological properties 3) the contribution of PLA2 enzymes to the lethal potency and pharmacological properties of the whole venom. 36