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Toxicon 54 (2009) 949–957

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Toxicon

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

Biochemistry and toxicology of toxins purified from the venom ofthe snake Bothrops asper

Yamileth Angulo a,b, Bruno Lomonte a,*

a Instituto Clodomiro Picado, Facultad de Microbiologıa, Universidad de Costa Rica, San Jose, Costa Ricab Departamento de Bioquımica, Escuela de Medicina, Universidad de Costa Rica, San Jose, Costa Rica

a r t i c l e i n f o

Article history:Received 11 November 2008Accepted 9 December 2008Available online 16 December 2008

Keywords:Bothrops asperSnakeVenomToxin

* Corresponding author.E-mail address: blomonte@cariari.ucr.ac.cr (B. Lo

0041-0101/$ – see front matter � 2008 Published bdoi:10.1016/j.toxicon.2008.12.014

a b s t r a c t

The isolation and study of individual snake venom components paves the way for a deeperunderstanding of the pathophysiology of envenomings – thus potentially contributing toimproved therapeutic modalities in the clinical setting – and also opens possibilities forthe discovery of novel toxins that might be useful as tools for dissecting cellular andmolecular processes of biomedical importance. This review provides a summary of thedifferent toxins that have been isolated and characterized from the venom of Bothropsasper, the snake species causing the majority of human envenomings in Central America.This venom contains proteins belonging to at least eight families: metalloproteinase, serineproteinase, C-type lectin-like, L-amino acid oxidase, disintegrin, DC-fragment, cystein-richsecretory protein, and phospholipase A2. Some 25 venom proteins within these familieshave been isolated and characterized. Their main biochemical properties and toxic actionsare described, including, in some cases, their possible relationships to the pathologiceffects induced by B. asper venom.

� 2008 Published by Elsevier Ltd.

1. Introduction

The harmful and even fatal consequences of snakebiteshave been noted by mankind since the times of ancientcivilizations. Venomous snakes can inspire both fear andfascination, and have been linked to mystical and religiousconcepts in many cultures throughout history. It is notsurprising that with the advent of modern science, snakevenoms became the subject of intense studies aimed atunderstanding their biochemical composition, and themodes by which they cause harmful effects.

Snake venoms are toxic secretions produced by a pair ofspecialized exocrine glands connected to the fangs by ducts(Kochva et al., 1980; Mackessy and Baxter, 2006). Suchsecretions are complex mixtures of molecules of differentbiochemical nature, with a predominance of proteins,many of which are endowed with enzymatic activities

monte).

y Elsevier Ltd.

(Jimenez-Porras, 1970; Tu, 1977). This heterogeneousnature of venom composition was evidenced since theearliest analytical studies, and hence associated with thewide variety of bioactivities, both in vitro and in vivo, thatwere observed clinically or experimentally. Thus, it becamewidely established that specific activities of a snake venomcould be attributed to particular components or toxins.While this general principle has been very useful andimportant in the study of venoms, it is not always valid, asthere may be effects that are caused by two or more toxinsacting in combination, i.e. synergistically. Moreover, a giventoxin may have more than one specific activity, andtherefore, it may play multiple roles in the overall effects ofenvenoming. Notwithstanding these considerations, theisolation and characterization of individual venomcomponents constitutes the mainstay of toxinology, asa key strategy to dissect and to analyze the complex seriesof events involved in envenomings. Thus, steered by thedevelopment and refinement of chromatographic tech-niques, early studies of snake venoms using whole,unfractionated secretions rapidly evolved into detailed

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analyses of their individual components. The purpose ofthis review is to provide an updated summary of thedifferent toxins that have been isolated and characterizedfrom the venom of Bothrops asper, the snake speciescausing the majority of human envenomings in CentralAmerica (Gutierrez, 1995).

2. Toxins isolated from B. asper venom

The first biochemical analyses of B. asper venomattempting to dissect its constituents and to establish theircorrelation with different enzymatic activities are probablythose of Jimenez-Porras (1964), who utilized starch gelelectrophoresis to separate at least 14 fractions from thevenom of this species (at the time classified as Bothropsatrox). Differences in the venom composition of B. asperfrom the Pacific and the Caribbean (Atlantic) regions ofCosta Rica were noticed since early studies on itsbiochemical and toxicological properties (Jimenez-Porras,1964; Gutierrez et al., 1980; Aragon and Gubensek, 1981).Variations in the frequency of occurrence of some electro-phoretic bands, as well as in the activity levels of severalenzymes and pharmacological effects were described. For

Table 1Proteins isolated from the venom of the snake Bothrops asper.

Protein family Toxin name Properties

Phospholipase A2

D49 PLA I acidic, 32 kDaPLA II acidic, 16 kDaPLA2 1 acidic, 11 kDaPLA2 2 acidic, 11 kDaPLA2 3 acidic, 29 kDamyotoxin I basic, 15 kDamyotoxic PLA2 basic, 15 kDamyotoxin III basic, pI 8.7, 15 kDa

K49 myotoxin II basic, pI 9.1, 15 kDamyotoxin IV basic, 15 kDa

MetalloproteinaseP-I proteinase G neutral, pI 7.1, 18 kDa

BaP1 basic, pI 8.2, 23 kDa

P-III BaH1 acidic, pI 4.5, 64 kDaBH2 acidic, pI 5.2, 26 kDaBH3 acidic, pI 5.0, 55 kDaBaH4 acidic, pI 5.3, 69 kDabasparin A acidic, 70 kDa

Serine proteinaseasperase acidic, 30 kDaficozyme acidic, 25 kDathrombin-like acidic, 27 kDa

L-amino acid oxidaseLaao 1 acidic, 125 kDaLaao 2a acidic, 125 kDaLaao 2b acidic, 125 kDa

C-type lectin-likeaspercetin acidic, 30 kDa

Disintegrinbothrasperin acidic, 8 kDa

a Amino acid sequence entry code in UniProtKB/TrEMBL, and Protein Data Ban

example, B. asper specimens inhabiting the Caribbeanversant of Costa Rica were shown to produce venom that ismore hemorrhagic and myotoxic than that of specimensfrom the Pacific versant, which display higher proteolyticactivity (Gutierrez et al., 1980).

Proteomic analyses have now revealed the presence ofproteins belonging to at least eight families in B. aspervenom: metalloproteinase (41–44%), phospholipase A2

(29–45%), serine proteinase (4–18%), L-amino acid oxidase(5–9%), disintegrin (1–2%), C-type lectin-like (0.5%), cys-tein-rich secretory protein (CRISP) (0.1%), and DC-frag-ment (<0.1%) (Alape-Giron et al., 2008). Representativetoxins of some of these protein families have been iso-lated, as listed in Table 1. A few of them have beencharacterized biochemically and structurally, and havebeen the subject of a number of studies aimed atunderstanding their mechanisms of action. Other toxinshave only been described with partial biochemical andfunctional characterizations. The following sectionssummarize the main properties and actions of the toxinslisted in Table 1, and in cases where such information hasbeen obtained, their relationships to the pathologiceffects induced by B. asper venom.

Sequence, PDBa Reference

– Ferlan and Gubensek (1978)– Ferlan and Gubensek (1978)– Alagon et al. (1980)– Alagon et al. (1980)– Alagon et al. (1980)– Gutierrez et al. (1984)– Mebs and Samejima (1986)P20474 Kaiser et al. (1990)

P24605, 1CLP Lomonte and Gutierrez (1989)– Dıaz et al. (1995)

– Aragon and Gubensek (1987)P83512, 1ND1 Gutierrez et al. (1995a)

– Borkow et al. (1993)– Borkow et al. (1993)– Borkow et al. (1993)– Franceschi et al. (2000)– Lorıa et al. (2003)

– Aragon-Ortiz and Gubensek (1978)– Fortova et al. (1990)Q072L6 Perez et al. (2008)

– Umana (1982a)– Umana (1982a)– Umana (1982a)

– Rucavado et al. (2001)

– Pinto et al. (2003)

k (PDB) entry code.

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2.1. Myotoxic phospholipases A2 (PLA2) and PLA2 homologues

In similarity with other species of viperid snakes(Serrano et al., 2005; Guercio et al., 2006; Calvete et al.,2007; Sanz et al., 2008; Angulo et al., 2008; Lomonte et al.,2008; Gutierrez et al., 2008b), the venom of B. aspercontains a significant proportion of PLA2 enzymes (Alape-Giron et al., 2008), both acidic and basic.

Ferlan and Gubensek (1978) isolated two PLA2 enzymesof this venom, named PLA I and PLA II, with approximatemolecular masses of 32 kDa and 16 kDa, respectively. Onthe basis of their amino acid compositions, these should beacidic. Their intravenous lethal activities for mice wereestimated at 2 and 10 mg/g body weight, respectively. Theselethal potencies are markedly lower than those corre-sponding to the highly neurotoxic PLA2s present in thevenoms of many elapid snake species (Rosenberg, 1990),and such finding would be in agreement with the lack ofneurotoxic symptoms in patients bitten by B. asper(Gutierrez, 1995), since no PLA2s with high lethal/neuro-toxic potency have been found in this venom. Alagon et al.(1980) also reported the isolation and biochemical char-acterization of acidic PLA2s from B. asper venom, namedPLA2 1, PLA2 2, and PLA2 3 (Table 1). However, their possibletoxic activities were not determined. On the basis of reportson acidic PLA2s isolated from the venoms of other Bothropsspecies (Serrano et al., 1999; Andriao-Escarso et al., 2002;Roberto et al., 2004; Modesto et al., 2006), it is likely thatacidic enzymes of B. asper may display effects upon plateletaggregation, but other activities such as neuromuscularblockade, myotoxicity, and hypotensive effect have alsobeen reported for these enzymes (Andriao-Escarso et al.,2002; Cogo et al., 2006; Rodrigues et al., 2007). Thepossible role(s) of the acidic PLA2 isozymes in the patho-physiology of envenomings by B. asper needs to be evalu-ated in future studies.

On the other hand, basic PLA2s are more abundant thantheir acidic counterparts in B. asper venom (Alape-Gironet al., 2008) and several isoforms have been isolated (Table 1).All of them have been shown to damage skeletal muscletissue within minutes of their intramuscular injection inmice, reproducing the drastic myonecrotic picture inducedby the whole venom (Gutierrez and Lomonte, 1989, 1995,1997, 2003). Thus, this group of proteins has been commonlyreferred to as PLA2 myotoxins. Selective neutralization ofthese basic PLA2s by the use of specific anti-myotoxinantibodies nearly abrogates the muscle damage induced bythe whole venom of B. asper (Lomonte et al., 1987, 1990,1992), therefore indicating that these toxins are the maincomponents responsible for myonecrosis in envenomings bythis species.

If administered systemically, i.e. by intravenous route,the PLA2 myotoxins do not increase the plasma levels ofcreatine kinase, an enzyme marker of skeletal muscledamage, indicating that they behave as locally actingmyotoxins (Gutierrez and Ownby, 2003; Gutierrez et al.,2008a). Biochemical characterization and amino acidsequence determination have shown that these proteins,all classified within the group IIA of secreted PLA2s, belongto two distinct subgroups. The Asp49 subgroup includesisoforms that are catalytically active, whereas the subgroup

of Lys49 PLA2 homologues does not display such enzymaticactivity (Lomonte et al., 2003; Chioato and Ward, 2003).B. asper myotoxins I, III, as well as the PLA2 reported byMebs and Samejima (1986) are Asp49 isoforms, whereasmyotoxins II and IV are members of the Lys49 PLA2

homologues subgroup (Table 1). Noteworthy, B. asperpresents an ontogenetic regulation in the expression of allthese basic PLA2 myotoxins in its venom: they are absent innewborns, and appear as snakes become juveniles, reach-ing high proportions in the venom composition of adults(Gutierrez et al.,1980; Lomonte and Carmona,1992; Saraviaet al., 2001; Alape-Giron et al., 2008).

The basic PLA2s of B. asper are made of 121–122 aminoacid residues, containing 14 Cys residues that engage inseven disulfide bridges, and have pI values in the range of8.5–9.5 (Kaiser et al., 1990; Francis et al., 1991). Theseproteins are not glycosylated, and form stable, non-covalently associated homodimers in their native state(Lomonte and Gutierrez, 1989; Francis et al., 1991). In thecase of myotoxin II, it was shown that under acidic pHconditions (<5.0) the dimers dissociate into monomers witha concomitant decrease in toxic potency, both in vitro and invivo (Angulo et al., 2005). Thus, it is possible that the dimericstructure of these PLA2s may lead to a higher avidity for theirtarget(s) by means of a divalent interaction, in comparisonto the monovalent binding of the dissociated monomers.A similar hypothesis has been presented by Montecucco andRossetto (2008) to explain the oligomeric structure of thehighly potent neurotoxic PLA2s from snake venoms.

Among the basic PLA2s of B. asper, myotoxin II (Lomonteand Gutierrez, 1989) is the most thoroughly studied toxin,structurally and functionally. The three-dimensionalstructure of this Lys49 protein was determined by Arni et al.(1995) and Murakami et al. (2005) at resolutions of 2.8 Åand 1.7 Å, respectively, allowing a deeper understanding ofits structure–function relationships. The mechanism ofaction of myotoxin II involves the interaction of its highlycationic C-terminal region with anionic moieties on targetmembranes, which subsequently lose permeability controland undergo a series of degenerative events related to Ca2þ

influx, ultimately leading to necrotic cell death (Lomonteet al., 1994b, 2003; Gutierrez and Ownby, 2003). Althoughthe main region responsible for toxicity on this protein hasbeen identified (Fig. 1A), knowledge on the nature andidentity of its acceptor molecule(s) on the target cells hasbeen elusive. In contrast to their Lys49 counterparts, theAsp49 PLA2 myotoxins utilize their catalytic activity uponmembrane phospholipids as part of the muscle-damagingmechanism, but the detailed events occurring at themolecular and cellular level are largely unknown. Themechanisms of action of both types of PLA2 myotoxins, i.e.Asp49 and Lys49 proteins, have been recently reviewed(Gutierrez and Ownby, 2003; Lomonte et al., 2003; Soareset al., 2004; Montecucco et al., 2008). Besides their prom-inent myotoxic effect in vivo, other activities described forthe group of basic PLA2s of B. asper, include the induction ofedema (Gutierrez et al., 1986; Lomonte and Gutierrez,1989;Chaves et al., 1998), in vitro anticoagulant action (Gutierrezet 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),

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 representedhighlighting its C-terminus region 115–129 (black), in the approximate orientation that approaches membrane bilayers (bottom), drastically altering theirpermeability. The toxin interacts with anionic sites (AS) of unknown identity in skeletal muscle, which may be phospholipids or proteins. N- and C-termini arelabelled as N and C, respectively. In (B), the metalloproteinase BaP1 is represented with its Zn2þ atom (sphere) in the catalytic site. By hydrolyzing proteincomponents of the extracellular matrix (ECM) and of the basal membrane of capillaries, this enzyme alters dermo-epidermal junctions and disrupts the integrityof microvessels and their endothelial cells (End), ultimately causing local dermonecrosis and hemorrhage. Images are not drawn to scale.

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bactericidal effect (Paramo et al., 1998; Santamarıa et al.,2005), induction of hyperalgesia (Chacur et al., 2003, 2004),activation of macrophages (Zuliani et al., 2005), apoptoticeffects (Mora et al., 2005, 2006), KDR/VEGF receptor-binding (Fujisawa et al., 2008), and lympahtic vesselcontraction (Mora et al., 2008).

2.2. Metalloproteinases

Zn2þ-dependent metalloproteinases are abundantlyfound in snake venoms, and classified into four structuralgroups, from P-I to P-IV, on the basis of their domaincomposition (Bjarnason and Fox, 1994; Fox and Serrano,2005, 2008; Ramos and Selistre-de-Araujo, 2006). InB. asper venom, the presence of metalloproteinases of thefirst three groups has been demonstrated by proteomictechniques (Alape-Giron et al., 2008), although only repre-sentatives of the P-I and P-III groups have been isolated.

The first protein of this family isolated from B. asper wasmetalloproteinase G (Aragon and Gubensek, 1987), a gly-cosylated enzyme of 18 kDa, which probably belongs to theP-I group. This enzyme hydrolyzes a number of proteinsubstrates in vitro such as casein, hemoglobin, gelatin andfibrinogen (particularly its alpha-chain). Despite thisactivity, the enzyme was reported to lack hemorrhagic andcoagulant actions.

Borkow et al. (1993) purified three toxins, named BaH1,BH2 and BH3, showing strong hemorrhagic activity. Theirinhibition by ortho-phenanthroline and by the chelatingagent EDTA identified these proteins as metalloproteinases.On the basis of their molecular masses (Table 1), BaH1 andBH3 should correspond to group P-III enzymes, whereas

BH2 is likely a P-I metalloproteinase. Interestingly, a clearsynergistic effect was observed in the hemorrhagic actionof these toxins in mice: one-quarter MHD (minimalhemorrhagic dose) of each isolated toxin caused onlya vague hemorrhagic spot, whereas a mixture containingone-eight MHD of each toxin resulted in hemorrhagic spotof one MHD (Borkow et al., 1993). BaH1 was the mostpotent of the three enzymes in inducing this effect, witha minimum hemorrhagic dose of 0.18 mg.

The hemorrhagic action of BaH1 has been studied invivo at the ultrastructural level and using endothelial cellcultures. Pathological alterations induced by this toxin invivo occur in endothelial cells and in their basal membrane,which appears to be proteolytically degraded (Moreiraet al., 1994). However, BaH1 is not cytotoxic to culturedendothelial cells, which detach from their substrate but stillretain viability (Lomonte et al., 1994a,b; Borkow et al.,1995). A mild myonecrotic effect of slow onset has beendescribed for BaH1, probably secondary to ischemicconditions caused by hemorrhage and blood flow impair-ment (Gutierrez et al., 1995b). The pathogenesis ofhemorrhage by BaH1 and other hemorrhagic toxins hasbeen recently reviewed (Gutierrez and Rucavado, 2000;Gutierrez et al., 2005).

Gutierrez et al. (1995a) isolated BaP1, an abundant P-Imetalloproteinase of 23 kDa in the venom of B. asper fromthe Pacific region of Costa Rica. This protein is highly activein the proteolysis of general substrates, but it is only weaklyhemorrhagic, having a minimum hemorrhagic dose of20 mg. It lacks coagulant, defibrinating, and platelet aggre-gation-inhibitory effects, but is capable of inducing a mildmyonecrosis (Gutierrez et al., 1995a; Escalante et al., 2004).

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Other activities displayed by this toxin include dermonec-rosis and blistering (Rucavado et al., 1998; Jimenez et al.,2008), complement activation (Farsky et al., 2000), andinduction of an inflammatory response including leukocyterecruitment, hypernociception, and synthesis of matrixmetalloproteinases and cytokines (Rucavado et al., 2002;Fernandes et al., 2006, 2007). In similarity with BaH1, BaP1is not cytotoxic to endothelial cells in culture (Rucavadoet al., 1995a). However, BaP1 induces anoikis in these cells,a particular form of apoptosis related to their detachmentfrom substrate (Dıaz et al., 2005). This in vitro effect wasalso reported for the P-III hemorrhagic metalloproteinasejararhagin (Tanjoni et al., 2005). Nevertheless, apoptosis ofendothelial cells does not seem to be detected in vivo(Jimenez et al., 2008). The degenerative changes observedafter BaP1 injection include damage to capillary basementmembrane (Escalante et al., 2006) and endothelial cells, thelatter effect requiring an intact blood flow (Gutierrez et al.,2006). The hemorrhagic action of this toxin is exertedlocally, but not systemically, probably due to its inactivationby the plasmatic protease inhibitor a2-macroglobulin(Escalante et al., 2004). BaP1 is antigenically distinct fromBaH1, since no cross-reactivities were recorded by usingrabbit antibodies raised against each one (Rucavado et al.,1995b). Watanabe et al. (2003) determined the primaryand three-dimensional structures of BaP1, which consists of202 amino acid residues, including three disulfide bridges,and contains the characteristic signature of the zinc-binding region of the metzincin superfamily of metal-loproteinases (Gomis-Ruth, 2003). The crystal structure ofBaP1 (Fig. 1B) will be most valuable to analyze the subtlerelationships between structure and function among met-alloproteinases with markedly different hemorrhagicactivities.

BaH4 is another P-III metalloproteinase of B. aspervenom, with hemorrhagic activity (Franceschi et al., 2000).This toxin is antigenically related to the previouslydescribed BaH1 (Borkow et al., 1993), generating a patternof partial identity with the latter by double immunodiffu-sion analysis. BaH4 differs from BaH1 by having a slightlyhigher isoelectric point (pI 5.3), and a lower hemorrhagicpotency, with a minimum hemorrhagic dose of 2 mg(Franceschi et al., 2000). Its median lethal dose (LD50) byintravenous injection in mice was estimated at 0.37 mg/gbody weight, and this route of administration resulted inprominent pulmonary hemorrhage. These functionalcharacteristics of BaH4 suggest that it may play a relevantrole in the systemic toxicity induced by B. asper venom.

Basparin A (Lorıa et al., 2003) is a single-chain, glyco-sylated, P-III metalloproteinase of 70 kDa and pI of 5.4. Inaddition to its metalloproteinase domain, it presents dis-integrin-like and high-cystein domains. This toxin displayspotent prothrombin-activating and platelet aggregation-inhibitory activities in vitro, causing defibrin(ogen)ationand thrombosis in mice. In contrast to other venom met-alloproteinases, basparin A does not degrade the typicalcomponents of the extracellular matrix. Its ability to inhibitcollagen-induced platelet aggregation is not dependent onits proteolytic activity. The plasma proteinase inhibitorsa2-macroglobulin and murinoglobulin do not prevent itsability to clot human plasma (Lorıa et al., 2003). The

defibrination syndrome observed after the intravenous orintramuscular administration of this enzyme, together withits ability to induce formation of pulmonary thrombi afterintravenous injection, suggest that basparin A is likely toplay a relevant role in the coagulopathy observed duringenvenomings by B. asper.

2.3. Serine proteinases

In B. asper venom, serine proteinases are abundantconstituents that account for 5–18% of the proteins,depending on age and geographic region variations (Alape-Giron et al., 2008). Of these enzymes, those with thrombin-like clotting activity have been isolated and studied.

Asperase, the first serine proteinase with thrombin-likeactivity isolated from B. asper venom, was described byOrtiz and Gubensek (1976) and Aragon-Ortiz and Gubensek(1978). This enzyme is glycosylated, with an estimatedmolecular mass of 30 kDa. By intravenous route, asperasehad an LD50 of 0.18 mg/g body weight in mice. It is likely thatsuch enzyme is the same as ficozyme (Fortova et al., 1990),and the one recently characterized in more detail by Perezet al. (2008), a 27 kDa serine proteinase with in vitrothrombin-like activity, which promotes defibrin(ogen)a-tion in mice after intravenous injection. In addition to itsability to induce coagulopathy, this toxic enzyme alsocauses behavioral alterations such as loss of the rightingreflex, opisthotonus, and intermittent rotations over thelong axis of the body (Perez et al., 2008). This featureresembles that of gyroxin, also a thrombin-like enzyme,from the venom of Crotalus durissus terrificus (Alexanderet al., 1988). Its N-terminal amino acid sequence (VIG-GDECNIN EHRSLVVLFX SSGFLCAGTL VQDEWVLTAANCDSKNFQ) is identical to a serine proteinase cDNA clonedfrom B. asper venom gland (UniProtKB/TrEMBL entryQ072L6). The proportion of this thrombin-like enzyme inthe venom is very low, nearly 0.13%. Considering its in vitroclotting potency for plasma (minimum coagulantdose ¼ 4.1 mg) and for fibrinogen (4.2 mg), and its in vivodefibrin(ogen)ating effect (minimum defibrin(ogen)atingdose¼ 1.0 mg) in mice, Perez et al. (2008) concluded that itscontribution to the coagulopathy induced by B. aspervenom is probably of lower significance than that providedby prothrombin-activating metalloproteinases such asbasparin A (Lorıa et al., 2003).

2.4. L-Amino acid oxidase

Umana (1982a,b) isolated three isoforms of the enzymeL-amino acid oxidase from B. asper venom, with a similarmolecular mass of 125 kDa, but slight variations in theirelectrophoretic mobility. Under reducing and denaturingconditions, subunits of 60 kDa and 75 kDa were obtainedfrom these purified enzymes. However, no data for possiblebioactivities or toxicity profiles of these venom compo-nents were described.

2.5. C-type lectin-like toxins

A disulfide-linked heterodimeric protein, with a molec-ular 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 identified it asa member of the C-type lectin-like family of proteins, witha high homology to botrocetin from the venom of Bothropsjararaca (Andrews et al., 1989). Aspercetin is a potentplatelet-aggregating agent only in the presence of plasmaor von Willebrand factor, since its activity results from theinteraction of this factor with platelet receptor GPIb(Rucavado et al., 2001). In contrast to other representativesof the C-type lectin family of snake venoms, aspercetinlacked anticoagulant and hemagglutinating activities. Inmice, this protein induced a rapid drop in circulatingplatelet numbers, prolonged the bleeding time, andenhanced the hemorrhagic activity of a purified metal-loproteinase. All these observations strongly suggest thataspercetin contributes significantly to the prominenthemorrhagic effects manifested in envenomings by B. asper.

2.6. Disintegrins

Disintegrins are non-enzymatic polypeptides broadlydistributed in the venoms of viperid snakes, which antag-onize the adhesive functions of several types of integrinreceptors (Calvete, 2005; Calvete et al., 2005). A medium-sized disintegrin, named bothrasperin, was isolated fromB. asper venom (Pinto et al., 2003) and partially characterized.This acidic protein showed an estimated molecular mass of8 kDa, and its N-terminal amino acid sequence (EAGEEXDXGTE) evidenced homology with disintegrins isolated fromthe venoms of B. jararaca and other pitvipers (Scarboroughet al., 1993). In vitro, bothrasperin inhibited ADP-inducedhuman platelet aggregation, with an estimated IC50 of 50 nM.Recent proteomic analyses of B. asper venom disclosed thepresence of several isoforms of this disintegrin, with slightdifferences in mass (Alape-Giron et al., 2008). Furtherbiochemical and functional characterization of bothrasperinand its variants is currently underway.

3. Concluding remarks

The isolation and study of individual snake venomcomponents paves the way for a deeper understanding ofthe pathophysiology of envenomings – thus potentiallycontributing to improved therapeutic modalities in theclinical setting – and also opens possibilities for thediscovery of novel toxins that might be useful as tools fordissecting cellular and molecular processes of biomedicalimportance. The venom of B. asper has provided a source ofvaried and interesting components, some of which havebeen 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 hasallowed to gain significant insights into the pathologicalphenomena of myotoxicity and hemorrhage, respectively.However, many more components of this venom await to beisolated, or to be characterized in greater detail. With therecently established proteomic database on the composi-tion of B. asper venom, and the ongoing work to define itstranscriptome, this task should be greatly facilitated,opening a new era in the study of this venom. Within thisframework, venom components that are scarce or difficult

to isolate could be cloned and expressed in recombinantform. As pointed out before, however, it should be kept inmind that although the study of isolated toxins constitutesan invaluable approach to dissect and analyze venomactions in great detail, the effects induced by whole,unfractionated venom should always be considered as animportant reference frame to envisage the relative contri-butions of the isolated components within its context.

A number of clinically relevant toxins of B. asper remainto be isolated and studied. One of the important conse-quences of envenomings by this species is kidney damage,and too little attention has been paid to this effect as well asto the toxins responsible for it. Likewise, the acidic PLA2sneed to be studied further to assess their possible contri-butions to the toxic actions of this venom. A number ofabundant serine proteinases and metalloproteinases,which are likely to have significant roles in the patho-physiology of envenomings, await to be isolated and char-acterized. Components such as members of the CRISPfamily of proteins are virtually unknown, and the group ofmedium-sized disintegrins should be also focused in moredepth to define their role(s) in envenomings. It is hopedthat in addition to the fascinating basic knowledge to begained from the study of this venom and its toxins, strat-egies to improve the clinical treatment of snakebite victimsmay also emerge.

Acknowledgements

We thank all colleagues and collaborators that havecontributed to the study of B. asper venom at our Instituteas well as abroad, the Vicerrectorıa de Investigacion,University of Costa Rica for support, and Dr Jose MarıaGutierrez for critical reading of the manuscript. We wouldlike to dedicate this review to the memory of Drs RogerBolanos and Luis Cerdas, former Directors of the InstitutoClodomiro Picado, for their vision and their encouragingsupport to new generations of researchers in the field oftoxinology.

Conflicts of interest

The authors declare that there are no conflicts ofinterest.

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