Toxicon 54 (2009) 949957

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Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Biochemistry and toxicology of toxins puried from the venom of the snake Bothrops asper
Yamileth Angulo a, b, Bruno Lomonte a, *
a b

a, Universidad de Costa Rica, San Jose , Costa Rica Instituto Clodomiro Picado, Facultad de Microbiolog mica, Escuela de Medicina, Universidad de Costa Rica, San Jose , Costa Rica Departamento de Bioqu

a r t i c l e i n f o
Article history: Received 11 November 2008 Accepted 9 December 2008 Available online 16 December 2008 Keywords: Bothrops asper Snake Venom Toxin

a b s t r a c t
The isolation and study of individual snake venom components paves the way for a deeper understanding of the pathophysiology of envenomings thus potentially contributing to improved therapeutic modalities in the clinical setting and also opens possibilities for the discovery of novel toxins that might be useful as tools for dissecting cellular and molecular processes of biomedical importance. This review provides a summary of the different toxins that have been isolated and characterized from the venom of Bothrops asper, the snake species causing the majority of human envenomings in Central America. This venom contains proteins belonging to at least eight families: metalloproteinase, serine proteinase, C-type lectin-like, L-amino acid oxidase, disintegrin, DC-fragment, cystein-rich secretory protein, and phospholipase A2. Some 25 venom proteins within these families have been isolated and characterized. Their main biochemical properties and toxic actions are described, including, in some cases, their possible relationships to the pathologic effects induced by B. asper venom. 2008 Published by Elsevier Ltd.

1. Introduction The harmful and even fatal consequences of snakebites have been noted by mankind since the times of ancient civilizations. Venomous snakes can inspire both fear and fascination, and have been linked to mystical and religious concepts in many cultures throughout history. It is not surprising that with the advent of modern science, snake venoms became the subject of intense studies aimed at understanding their biochemical composition, and the modes by which they cause harmful effects. Snake venoms are toxic secretions produced by a pair of specialized exocrine glands connected to the fangs by ducts (Kochva et al., 1980; Mackessy and Baxter, 2006). Such secretions are complex mixtures of molecules of different biochemical nature, with a predominance of proteins, many of which are endowed with enzymatic activities

* Corresponding author. E-mail address: blomonte@cariari.ucr.ac.cr (B. Lomonte). 0041-0101/$ see front matter 2008 Published by Elsevier Ltd. doi:10.1016/j.toxicon.2008.12.014

nez-Porras, 1970; Tu, 1977). This heterogeneous (Jime nature of venom composition was evidenced since the earliest analytical studies, and hence associated with the wide variety of bioactivities, both in vitro and in vivo, that were observed clinically or experimentally. Thus, it became widely established that specic activities of a snake venom could be attributed to particular components or toxins. While this general principle has been very useful and important in the study of venoms, it is not always valid, as there may be effects that are caused by two or more toxins acting in combination, i.e. synergistically. Moreover, a given toxin may have more than one specic activity, and therefore, it may play multiple roles in the overall effects of envenoming. Notwithstanding these considerations, the isolation and characterization of individual venom components constitutes the mainstay of toxinology, as a key strategy to dissect and to analyze the complex series of events involved in envenomings. Thus, steered by the development and renement of chromatographic techniques, early studies of snake venoms using whole, unfractionated secretions rapidly evolved into detailed

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analyses of their individual components. The purpose of this review is to provide an updated summary of the different toxins that have been isolated and characterized from the venom of Bothrops asper, the snake species causing the majority of human envenomings in Central rrez, 1995). America (Gutie 2. Toxins isolated from B. asper venom The rst biochemical analyses of B. asper venom attempting to dissect its constituents and to establish their correlation with different enzymatic activities are probably nez-Porras (1964), who utilized starch gel those of Jime electrophoresis to separate at least 14 fractions from the venom of this species (at the time classied as Bothrops atrox). Differences in the venom composition of B. asper from the Pacic and the Caribbean (Atlantic) regions of Costa Rica were noticed since early studies on its nez-Porras, biochemical and toxicological properties (Jime ek, 1981). rrez et al., 1980; Arago n and Gubens 1964; Gutie Variations in the frequency of occurrence of some electrophoretic bands, as well as in the activity levels of several enzymes and pharmacological effects were described. For

example, B. asper specimens inhabiting the Caribbean versant of Costa Rica were shown to produce venom that is more hemorrhagic and myotoxic than that of specimens from the Pacic versant, which display higher proteolytic rrez et al., 1980). activity (Gutie Proteomic analyses have now revealed the presence of proteins belonging to at least eight families in B. asper venom: metalloproteinase (4144%), phospholipase A2 (2945%), serine proteinase (418%), L-amino acid oxidase (59%), disintegrin (12%), C-type lectin-like (0.5%), cystein-rich secretory protein (CRISP) (0.1%), and DC-frag n et al., 2008). Representative ment (<0.1%) (Alape-Giro toxins of some of these protein families have been isolated, as listed in Table 1. A few of them have been characterized biochemically and structurally, and have been the subject of a number of studies aimed at understanding their mechanisms of action. Other toxins have only been described with partial biochemical and functional characterizations. The following sections summarize the main properties and actions of the toxins listed in Table 1, and in cases where such information has been obtained, their relationships to the pathologic effects induced by B. asper venom.

Table 1 Proteins isolated from the venom of the snake Bothrops asper. Protein family Phospholipase A2 D49 Toxin name PLA I PLA II PLA2 1 PLA2 2 PLA2 3 myotoxin I myotoxic PLA2 myotoxin III myotoxin II myotoxin IV Properties acidic, 32 kDa acidic, 16 kDa acidic, 11 kDa acidic, 11 kDa acidic, 29 kDa basic, 15 kDa basic, 15 kDa basic, pI 8.7, 15 kDa basic, pI 9.1, 15 kDa basic, 15 kDa Sequence, PDBa P20474 P24605, 1CLP Reference ek (1978) Ferlan and Gubens ek (1978) Ferlan and Gubens n et al. (1980) Alago n et al. (1980) Alago n et al. (1980) Alago rrez et al. (1984) Gutie Mebs and Samejima (1986) Kaiser et al. (1990) rrez (1989) Lomonte and Gutie az et al. (1995) D

K49

Metalloproteinase P-I P-III

proteinase G BaP1 BaH1 BH2 BH3 BaH4 basparin A

neutral, pI 7.1, 18 kDa basic, pI 8.2, 23 kDa acidic, acidic, acidic, acidic, acidic, pI 4.5, 64 kDa pI 5.2, 26 kDa pI 5.0, 55 kDa pI 5.3, 69 kDa 70 kDa

P83512, 1ND1

ek (1987) n and Gubens Arago rrez et al. (1995a) Gutie Borkow et al. (1993) Borkow et al. (1993) Borkow et al. (1993) Franceschi et al. (2000) a et al. (2003) Lor

Serine proteinase asperase cozyme thrombin-like

L-amino

acidic, 30 kDa acidic, 25 kDa acidic, 27 kDa

Q072L6

ek (1978) n-Ortiz and Gubens Arago tova et al. (1990) For rez et al. (2008) Pe

acid oxidase Laao 1 Laao 2a Laao 2b acidic, 125 kDa acidic, 125 kDa acidic, 125 kDa a (1982a) Uman a (1982a) Uman a (1982a) Uman

C-type lectin-like aspercetin Disintegrin bothrasperin

a

acidic, 30 kDa

Rucavado et al. (2001)

acidic, 8 kDa

Pinto et al. (2003)

Amino acid sequence entry code in UniProtKB/TrEMBL, and Protein Data Bank (PDB) entry code.

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2.1. Myotoxic phospholipases A2 (PLA2) and PLA2 homologues In similarity with other species of viperid snakes rcio et al., 2006; Calvete et al., (Serrano et al., 2005; Gue 2007; Sanz et al., 2008; Angulo et al., 2008; Lomonte et al., rrez et al., 2008b), the venom of B. asper 2008; Gutie contains a signicant proportion of PLA2 enzymes (Alape n et al., 2008), both acidic and basic. Giro ek (1978) isolated two PLA2 enzymes Ferlan and Gubens of this venom, named PLA I and PLA II, with approximate molecular masses of 32 kDa and 16 kDa, respectively. On the basis of their amino acid compositions, these should be acidic. Their intravenous lethal activities for mice were estimated at 2 and 10 mg/g body weight, respectively. These lethal potencies are markedly lower than those corresponding to the highly neurotoxic PLA2s present in the venoms of many elapid snake species (Rosenberg, 1990), and such nding would be in agreement with the lack of neurotoxic symptoms in patients bitten by B. asper rrez, 1995), since no PLA2s with high lethal/neuro(Gutie n et al. toxic potency have been found in this venom. Alago (1980) also reported the isolation and biochemical characterization of acidic PLA2s from B. asper venom, named PLA2 1, PLA2 2, and PLA2 3 (Table 1). However, their possible toxic activities were not determined. On the basis of reports on acidic PLA2s isolated from the venoms of other Bothrops o-Escarso et al., 2002; species (Serrano et al., 1999; Andria Roberto et al., 2004; Modesto et al., 2006), it is likely that acidic enzymes of B. asper may display effects upon platelet aggregation, but other activities such as neuromuscular blockade, myotoxicity, and hypotensive effect have also o-Escarso et al., been reported for these enzymes (Andria 2002; Cogo et al., 2006; Rodrigues et al., 2007). The possible role(s) of the acidic PLA2 isozymes in the pathophysiology of envenomings by B. asper needs to be evaluated in future studies. On the other hand, basic PLA2s are more abundant than n their acidic counterparts in B. asper venom (Alape-Giro et al., 2008) and several isoforms have been isolated (Table 1). All of them have been shown to damage skeletal muscle tissue within minutes of their intramuscular injection in mice, reproducing the drastic myonecrotic picture induced rrez and Lomonte, 1989, 1995, by the whole venom (Gutie 1997, 2003). Thus, this group of proteins has been commonly referred to as PLA2 myotoxins. Selective neutralization of these basic PLA2s by the use of specic anti-myotoxin antibodies nearly abrogates the muscle damage induced by the whole venom of B. asper (Lomonte et al., 1987, 1990, 1992), therefore indicating that these toxins are the main components responsible for myonecrosis in envenomings by this species. If administered systemically, i.e. by intravenous route, the PLA2 myotoxins do not increase the plasma levels of creatine kinase, an enzyme marker of skeletal muscle damage, indicating that they behave as locally acting rrez and Ownby, 2003; Gutie rrez et al., myotoxins (Gutie 2008a). Biochemical characterization and amino acid sequence determination have shown that these proteins, all classied within the group IIA of secreted PLA2s, belong to two distinct subgroups. The Asp49 subgroup includes isoforms that are catalytically active, whereas the subgroup

of Lys49 PLA2 homologues does not display such enzymatic activity (Lomonte et al., 2003; Chioato and Ward, 2003). B. asper myotoxins I, III, as well as the PLA2 reported by Mebs and Samejima (1986) are Asp49 isoforms, whereas myotoxins II and IV are members of the Lys49 PLA2 homologues subgroup (Table 1). Noteworthy, B. asper presents an ontogenetic regulation in the expression of all these basic PLA2 myotoxins in its venom: they are absent in newborns, and appear as snakes become juveniles, reaching high proportions in the venom composition of adults rrez et al., 1980; Lomonte and Carmona, 1992; Saravia (Gutie n et al., 2008). et al., 2001; Alape-Giro The basic PLA2s of B. asper are made of 121122 amino acid residues, containing 14 Cys residues that engage in seven disulde bridges, and have pI values in the range of 8.59.5 (Kaiser et al., 1990; Francis et al., 1991). These proteins are not glycosylated, and form stable, noncovalently associated homodimers in their native state rrez, 1989; Francis et al., 1991). In the (Lomonte and Gutie case of myotoxin II, it was shown that under acidic pH conditions (<5.0) the dimers dissociate into monomers with a concomitant decrease in toxic potency, both in vitro and in vivo (Angulo et al., 2005). Thus, it is possible that the dimeric structure of these PLA2s may lead to a higher avidity for their target(s) by means of a divalent interaction, in comparison to the monovalent binding of the dissociated monomers. A similar hypothesis has been presented by Montecucco and Rossetto (2008) to explain the oligomeric structure of the highly potent neurotoxic PLA2s from snake venoms. Among the basic PLA2s of B. asper, myotoxin II (Lomonte rrez, 1989) is the most thoroughly studied toxin, and Gutie structurally and functionally. The three-dimensional structure of this Lys49 protein was determined by Arni et al. (1995) and Murakami et al. (2005) at resolutions of 2.8 and 1.7 , respectively, allowing a deeper understanding of its structurefunction relationships. The mechanism of action of myotoxin II involves the interaction of its highly cationic C-terminal region with anionic moieties on target membranes, which subsequently lose permeability control and undergo a series of degenerative events related to Ca2 inux, ultimately leading to necrotic cell death (Lomonte rrez and Ownby, 2003). Although et al., 1994b, 2003; Gutie the main region responsible for toxicity on this protein has been identied (Fig. 1A), knowledge on the nature and identity of its acceptor molecule(s) on the target cells has been elusive. In contrast to their Lys49 counterparts, the Asp49 PLA2 myotoxins utilize their catalytic activity upon membrane phospholipids as part of the muscle-damaging mechanism, but the detailed events occurring at the molecular and cellular level are largely unknown. The mechanisms of action of both types of PLA2 myotoxins, i.e. Asp49 and Lys49 proteins, have been recently reviewed rrez and Ownby, 2003; Lomonte et al., 2003; Soares (Gutie et al., 2004; Montecucco et al., 2008). Besides their prominent myotoxic effect in vivo, other activities described for the group of basic PLA2s of B. asper, include the induction of rrez et al., 1986; Lomonte and Gutie rrez, 1989; edema (Gutie rrez Chaves et al., 1998), in vitro anticoagulant action (Gutie et al., 1986), induction of cytokines (Lomonte et al., 1993; Rucavado et al., 2002; Chacur et al., 2004), cytotoxic action (Lomonte et al., 1994a,b, 1999; Angulo and Lomonte, 2005),

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Fig. 1. Ribbon representations of the three-dimensional structures of (A) Bothrops asper myotoxin II (1CLP; Arni et al., 1995), a Lys49 PLA2 homologue, and (B) B. asper BaP1 (1ND1; Watanabe et al., 2003), a P-I metalloproteinase with weak hemorrhagic activity. In (A), a myotoxin II homodimer is represented highlighting its C-terminus region 115129 (black), in the approximate orientation that approaches membrane bilayers (bottom), drastically altering their permeability. The toxin interacts with anionic sites (AS) of unknown identity in skeletal muscle, which may be phospholipids or proteins. N- and C-termini are labelled as N and C, respectively. In (B), the metalloproteinase BaP1 is represented with its Zn2 atom (sphere) in the catalytic site. By hydrolyzing protein components of the extracellular matrix (ECM) and of the basal membrane of capillaries, this enzyme alters dermo-epidermal junctions and disrupts the integrity of microvessels and their endothelial cells (End), ultimately causing local dermonecrosis and hemorrhage. Images are not drawn to scale.

ramo et al., 1998; Santamar a et al., bactericidal effect (Pa 2005), induction of hyperalgesia (Chacur et al., 2003, 2004), activation of macrophages (Zuliani et al., 2005), apoptotic effects (Mora et al., 2005, 2006), KDR/VEGF receptorbinding (Fujisawa et al., 2008), and lympahtic vessel contraction (Mora et al., 2008). 2.2. Metalloproteinases Zn2-dependent metalloproteinases are abundantly found in snake venoms, and classied into four structural groups, from P-I to P-IV, on the basis of their domain composition (Bjarnason and Fox, 1994; Fox and Serrano, 2005, 2008; Ramos and Selistre-de-Araujo, 2006). In B. asper venom, the presence of metalloproteinases of the rst three groups has been demonstrated by proteomic n et al., 2008), although only repretechniques (Alape-Giro sentatives of the P-I and P-III groups have been isolated. The rst protein of this family isolated from B. asper was ek, 1987), a gly n and Gubens metalloproteinase G (Arago cosylated enzyme of 18 kDa, which probably belongs to the P-I group. This enzyme hydrolyzes a number of protein substrates in vitro such as casein, hemoglobin, gelatin and brinogen (particularly its alpha-chain). Despite this activity, the enzyme was reported to lack hemorrhagic and coagulant actions. Borkow et al. (1993) puried three toxins, named BaH1, BH2 and BH3, showing strong hemorrhagic activity. Their inhibition by ortho-phenanthroline and by the chelating agent EDTA identied these proteins as metalloproteinases. On the basis of their molecular masses (Table 1), BaH1 and BH3 should correspond to group P-III enzymes, whereas

BH2 is likely a P-I metalloproteinase. Interestingly, a clear synergistic effect was observed in the hemorrhagic action of these toxins in mice: one-quarter MHD (minimal hemorrhagic dose) of each isolated toxin caused only a vague hemorrhagic spot, whereas a mixture containing one-eight MHD of each toxin resulted in hemorrhagic spot of one MHD (Borkow et al., 1993). BaH1 was the most potent of the three enzymes in inducing this effect, with a minimum hemorrhagic dose of 0.18 mg. The hemorrhagic action of BaH1 has been studied in vivo at the ultrastructural level and using endothelial cell cultures. Pathological alterations induced by this toxin in vivo occur in endothelial cells and in their basal membrane, which appears to be proteolytically degraded (Moreira et al., 1994). However, BaH1 is not cytotoxic to cultured endothelial cells, which detach from their substrate but still retain viability (Lomonte et al., 1994a,b; Borkow et al., 1995). A mild myonecrotic effect of slow onset has been described for BaH1, probably secondary to ischemic conditions caused by hemorrhage and blood ow impair rrez et al., 1995b). The pathogenesis of ment (Gutie hemorrhage by BaH1 and other hemorrhagic toxins has rrez and Rucavado, 2000; been recently reviewed (Gutie rrez et al., 2005). Gutie rrez et al. (1995a) isolated BaP1, an abundant P-I Gutie metalloproteinase of 23 kDa in the venom of B. asper from the Pacic region of Costa Rica. This protein is highly active in the proteolysis of general substrates, but it is only weakly hemorrhagic, having a minimum hemorrhagic dose of 20 mg. It lacks coagulant, debrinating, and platelet aggregation-inhibitory effects, but is capable of inducing a mild rrez et al., 1995a; Escalante et al., 2004). myonecrosis (Gutie

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Other activities displayed by this toxin include dermonec nez et al., rosis and blistering (Rucavado et al., 1998; Jime 2008), complement activation (Farsky et al., 2000), and induction of an inammatory response including leukocyte recruitment, hypernociception, and synthesis of matrix metalloproteinases and cytokines (Rucavado et al., 2002; Fernandes et al., 2006, 2007). In similarity with BaH1, BaP1 is not cytotoxic to endothelial cells in culture (Rucavado et al., 1995a). However, BaP1 induces anoikis in these cells, a particular form of apoptosis related to their detachment az et al., 2005). This in vitro effect was from substrate (D also reported for the P-III hemorrhagic metalloproteinase jararhagin (Tanjoni et al., 2005). Nevertheless, apoptosis of endothelial cells does not seem to be detected in vivo nez et al., 2008). The degenerative changes observed (Jime after BaP1 injection include damage to capillary basement membrane (Escalante et al., 2006) and endothelial cells, the rrez et al., latter effect requiring an intact blood ow (Gutie 2006). The hemorrhagic action of this toxin is exerted locally, but not systemically, probably due to its inactivation by the plasmatic protease inhibitor a2-macroglobulin (Escalante et al., 2004). BaP1 is antigenically distinct from BaH1, since no cross-reactivities were recorded by using rabbit antibodies raised against each one (Rucavado et al., 1995b). Watanabe et al. (2003) determined the primary and three-dimensional structures of BaP1, which consists of 202 amino acid residues, including three disulde bridges, and contains the characteristic signature of the zincbinding region of the metzincin superfamily of metal th, 2003). The crystal structure of loproteinases (Gomis-Ru BaP1 (Fig. 1B) will be most valuable to analyze the subtle relationships between structure and function among metalloproteinases with markedly different hemorrhagic activities. BaH4 is another P-III metalloproteinase of B. asper venom, with hemorrhagic activity (Franceschi et al., 2000). This toxin is antigenically related to the previously described BaH1 (Borkow et al., 1993), generating a pattern of partial identity with the latter by double immunodiffusion analysis. BaH4 differs from BaH1 by having a slightly higher isoelectric point (pI 5.3), and a lower hemorrhagic potency, with a minimum hemorrhagic dose of 2 mg (Franceschi et al., 2000). Its median lethal dose (LD50) by intravenous injection in mice was estimated at 0.37 mg/g body weight, and this route of administration resulted in prominent pulmonary hemorrhage. These functional characteristics of BaH4 suggest that it may play a relevant role in the systemic toxicity induced by B. asper venom. a et al., 2003) is a single-chain, glycoBasparin A (Lor sylated, P-III metalloproteinase of 70 kDa and pI of 5.4. In addition to its metalloproteinase domain, it presents disintegrin-like and high-cystein domains. This toxin displays potent prothrombin-activating and platelet aggregationinhibitory activities in vitro, causing debrin(ogen)ation and thrombosis in mice. In contrast to other venom metalloproteinases, basparin A does not degrade the typical components of the extracellular matrix. Its ability to inhibit collagen-induced platelet aggregation is not dependent on its proteolytic activity. The plasma proteinase inhibitors a2-macroglobulin and murinoglobulin do not prevent its a et al., 2003). The ability to clot human plasma (Lor

debrination syndrome observed after the intravenous or intramuscular administration of this enzyme, together with its ability to induce formation of pulmonary thrombi after intravenous injection, suggest that basparin A is likely to play a relevant role in the coagulopathy observed during envenomings by B. asper. 2.3. Serine proteinases In B. asper venom, serine proteinases are abundant constituents that account for 518% of the proteins, depending on age and geographic region variations (Alape n et al., 2008). Of these enzymes, those with thrombinGiro like clotting activity have been isolated and studied. Asperase, the rst serine proteinase with thrombin-like activity isolated from B. asper venom, was described by ek (1976) and Arago ek n-Ortiz and Gubens Ortiz and Gubens (1978). This enzyme is glycosylated, with an estimated molecular mass of 30 kDa. By intravenous route, asperase had an LD50 of 0.18 mg/g body weight in mice. It is likely that tova et al., 1990), such enzyme is the same as cozyme (For rez and the one recently characterized in more detail by Pe et al. (2008), a 27 kDa serine proteinase with in vitro thrombin-like activity, which promotes debrin(ogen)ation in mice after intravenous injection. In addition to its ability to induce coagulopathy, this toxic enzyme also causes behavioral alterations such as loss of the righting reex, opisthotonus, and intermittent rotations over the rez et al., 2008). This feature long axis of the body (Pe resembles that of gyroxin, also a thrombin-like enzyme, from the venom of Crotalus durissus terricus (Alexander et al., 1988). Its N-terminal amino acid sequence (VIGGDECNIN EHRSLVVLFX SSGFLCAGTL VQDEWVLTAA NCDSKNFQ) is identical to a serine proteinase cDNA cloned from B. asper venom gland (UniProtKB/TrEMBL entry Q072L6). The proportion of this thrombin-like enzyme in the venom is very low, nearly 0.13%. Considering its in vitro clotting potency for plasma (minimum coagulant dose 4.1 mg) and for brinogen (4.2 mg), and its in vivo debrin(ogen)ating effect (minimum debrin(ogen)ating rez et al. (2008) concluded that its dose 1.0 mg) in mice, Pe contribution to the coagulopathy induced by B. asper venom is probably of lower signicance than that provided by prothrombin-activating metalloproteinases such as a et al., 2003). basparin A (Lor 2.4. L-Amino acid oxidase a (1982a,b) isolated three isoforms of the enzyme Uman acid oxidase from B. asper venom, with a similar molecular mass of 125 kDa, but slight variations in their electrophoretic mobility. Under reducing and denaturing conditions, subunits of 60 kDa and 75 kDa were obtained from these puried enzymes. However, no data for possible bioactivities or toxicity proles of these venom components were described.
L-amino

2.5. C-type lectin-like toxins A disulde-linked heterodimeric protein, with a molecular mass of 29,759 and a pI of 4.5, was isolated from

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B. asper venom and named aspercetin (Rucavado et al., 2001). Its N-terminal amino acid sequence identied it as a member of the C-type lectin-like family of proteins, with a high homology to botrocetin from the venom of Bothrops jararaca (Andrews et al., 1989). Aspercetin is a potent platelet-aggregating agent only in the presence of plasma or von Willebrand factor, since its activity results from the interaction of this factor with platelet receptor GPIb (Rucavado et al., 2001). In contrast to other representatives of the C-type lectin family of snake venoms, aspercetin lacked anticoagulant and hemagglutinating activities. In mice, this protein induced a rapid drop in circulating platelet numbers, prolonged the bleeding time, and enhanced the hemorrhagic activity of a puried metalloproteinase. All these observations strongly suggest that aspercetin contributes signicantly to the prominent hemorrhagic effects manifested in envenomings by B. asper. 2.6. Disintegrins Disintegrins are non-enzymatic polypeptides broadly distributed in the venoms of viperid snakes, which antagonize the adhesive functions of several types of integrin receptors (Calvete, 2005; Calvete et al., 2005). A mediumsized disintegrin, named bothrasperin, was isolated from B. asper venom (Pinto et al., 2003) and partially characterized. This acidic protein showed an estimated molecular mass of 8 kDa, and its N-terminal amino acid sequence (EAGEEX DXGTE) evidenced homology with disintegrins isolated from the venoms of B. jararaca and other pitvipers (Scarborough et al., 1993). In vitro, bothrasperin inhibited ADP-induced human platelet aggregation, with an estimated IC50 of 50 nM. Recent proteomic analyses of B. asper venom disclosed the presence of several isoforms of this disintegrin, with slight n et al., 2008). Further differences in mass (Alape-Giro biochemical and functional characterization of bothrasperin and its variants is currently underway. 3. Concluding remarks The isolation and study of individual snake venom components paves the way for a deeper understanding of the pathophysiology of envenomings thus potentially contributing to improved therapeutic modalities in the clinical setting and also opens possibilities for the discovery of novel toxins that might be useful as tools for dissecting cellular and molecular processes of biomedical importance. The venom of B. asper has provided a source of varied and interesting components, some of which have been well characterized, most notably the Lys49 PLA2 homologue myotoxin II and the P-I metalloproteinase BaP1 (Fig. 1). The intensive study of these two toxins has allowed to gain signicant insights into the pathological phenomena of myotoxicity and hemorrhage, respectively. However, many more components of this venom await to be isolated, or to be characterized in greater detail. With the recently established proteomic database on the composition of B. asper venom, and the ongoing work to dene its transcriptome, this task should be greatly facilitated, opening a new era in the study of this venom. Within this framework, venom components that are scarce or difcult

to isolate could be cloned and expressed in recombinant form. As pointed out before, however, it should be kept in mind that although the study of isolated toxins constitutes an invaluable approach to dissect and analyze venom actions in great detail, the effects induced by whole, unfractionated venom should always be considered as an important reference frame to envisage the relative contributions of the isolated components within its context. A number of clinically relevant toxins of B. asper remain to be isolated and studied. One of the important consequences of envenomings by this species is kidney damage, and too little attention has been paid to this effect as well as to the toxins responsible for it. Likewise, the acidic PLA2s need to be studied further to assess their possible contributions to the toxic actions of this venom. A number of abundant serine proteinases and metalloproteinases, which are likely to have signicant roles in the pathophysiology of envenomings, await to be isolated and characterized. Components such as members of the CRISP family of proteins are virtually unknown, and the group of medium-sized disintegrins should be also focused in more depth to dene their role(s) in envenomings. It is hoped that in addition to the fascinating basic knowledge to be gained from the study of this venom and its toxins, strategies to improve the clinical treatment of snakebite victims may also emerge. Acknowledgements We thank all colleagues and collaborators that have contributed to the study of B. asper venom at our Institute a de Investigacio n, as well as abroad, the Vicerrector Mar a University of Costa Rica for support, and Dr Jose rrez for critical reading of the manuscript. We would Gutie ger like to dedicate this review to the memory of Drs Ro os and Luis Cerdas, former Directors of the Instituto Bolan Clodomiro Picado, for their vision and their encouraging support to new generations of researchers in the eld of toxinology. Conicts of interest The authors declare that there are no conicts of interest. References
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