BJ-PI2, A non-hemorrhagic metalloproteinase from Bothrops jararaca snake venom

Background

Envenoming by Bothrops jararaca can result in local pain, edema, hemorrhage and necrosis, partially mediated by snake venom metalloproteinases (SVMPs). Here, we describe the characterization of BJ-PI2, a P-I class SVMP from B. jararaca venom, and its local tissue actions.

Methods

BJ-PI2 was purified by a combination of gel filtration, anion-exchange chromatography and reverse phase HPLC, and identified by mass spectrometry. Clotting and fibrin(ogen)olytic activities were assayed using conventional methods. Hemorrhagic activity and changes in vascular permeability were examined in rat dorsal skin. Myonecrosis and inflammatory activity were examined in mouse gastrocnemius muscle.

Results

BJ-PI2 was a 23.08 kDa single-chain polypeptide. Tryptic fragments showed highest homology with SVMP insularinase A from Bothrops insularis, but also with B. jararaca SVMP bothrojaractivase; less similarity was observed with B. jararaca SVMPs BJ-PI and jararafibrases II and IV. BJ-PI2 did not clot fibrinogen or rat citrated plasma but had α- and β-fibrinogenolytic activity (inhibited by EDTA and 1,10-phenanthroline but not by PMSF) and attenuated coagulation after plasma recalcification. BJ-PI2 had fibrinolytic activity. BJ-PI2 increased the vascular permeability of rat dorsal skin (inhibited by 1,10-phenanthroline). BJ-PI2 was not hemorrhagic or myonecrotic but caused migration of inflammatory cells. In contrast, venom was strongly hemorrhagic and myonecrotic but caused less infiltration of inflammatory cells.

Conclusions

BJ-PI2 is a non-hemorrhagic, non-myonecrotic, non-coagulant P-I class SVMP that may enhance vascular permeability and inflammatory cell migration in vivo.

General significance

BJ-PI2 contributes to enhanced vascular permeability and inflammatory cell migration after envenoming, but not to venom-induced hemorrhage and necrosis.     

Viper and Cobra Venom Neutralization by Alginate Coated Multicomponent Polyvalent Antivenom Administered by the Oral Route

Background

Snake bite causes greater mortality than most of the other neglected tropical diseases. Snake antivenom, although effective in minimizing mortality in developed countries, is not equally so in developing countries due to its poor availability in remote snake infested areas as, and when, required. An alternative approach in this direction could be taken by making orally deliverable polyvalent antivenom formulation, preferably under a globally integrated strategy, for using it as a first aid during transit time from remote trauma sites to hospitals.

Methodology/Principal Findings

To address this problem, multiple components of polyvalent antivenom were entrapped in alginate. Structural analysis, scanning electron microscopy, entrapment efficiency, loading capacity, swelling study, in vitro pH sensitive release, acid digestion, mucoadhesive property and venom neutralization were studied in in vitro and in vivo models. Results showed that alginate retained its mucoadhesive, acid protective and pH sensitive swelling property after entrapping antivenom. After pH dependent release from alginate beads, antivenom (ASVS) significantly neutralized phospholipaseA₂ activity, hemolysis, lactate dehydrogenase activity and lethality of venom. In ex vivo mice intestinal preparation, ASVS was absorbed significantly through the intestine and it inhibited venom lethality which indicated that all the components of antivenom required for neutralization of venom lethality were retained despite absorption across the intestinal layer. Results from in vivo studies indicated that orally delivered ASVS can significantly neutralize venom effects, depicted by protection against lethality, decreased hemotoxicity and renal toxicity caused by russell viper venom.

Conclusions/Significance

Alginate was effective in entrapping all the structural components of ASVS, which on release and intestinal absorption effectively reconstituted the function of antivenom in neutralizing viper and cobra venom. Further research in this direction can strategize to counter such dilemma in snake bite management by promoting control release and oral antivenom rendered as a first aid.     

Detection of Venom after Antivenom Is Not Associated with Persistent Coagulopathy in a Prospective Cohort of Russell's Viper (Daboia russelii) Envenomings

Background

Venom recurrence or persistence in the circulation after antivenom treatment has been documented many times in viper envenoming. However, it has not been associated with clinical recurrence for many snakes, including Russell''s viper (Daboia spp.). We compare the recovery of coagulopathy to the recurrence or persistence of venom in patients with Russell''s viper envenoming.

Methodology/Principal Findings

The study included patients with Russell''s viper (D. russelii) envenoming presenting over a 30 month period who had Russell''s viper venom detected by enzyme immunoassay. Demographics, information on the snake bite, and clinical effects were collected for all patients. All patients had serum collected for venom specific enzyme immunoassay and citrate plasma to measure fibrinogen levels and prothrombin time (international normalised ratio; INR). Patients with venom recurrence/persistence were compared to those with no detectable recurrence of venom. There were 55 patients with confirmed Russell''s viper envenoming and coagulopathy with low fibrinogen concentrations: 31 with venom recurrence/persistence, and 24 with no venom detected post-antivenom. Fibrinogen concentrations increased and INR decreased after antivenom in both the recurrence and non-recurrence patients. Clinical features, laboratory parameters, antivenom dose and length of hospital were similar for both groups. Pre-antivenom venom concentrations were higher in patients with venom recurrence/persistence with a median venom concentration of 385 ng/mL (16–1521 ng/mL) compared to 128 ng/mL (14–1492 ng/mL; p = 0.008).

Conclusion

Recurrence of Russell''s viper venom was not associated with a recurrence of coagulopathy and length of hospital stay. Further work is required to determine if the detection of venom recurrence is due to the venom specific enzyme immunoassay detecting both venom-antivenom complexes as well as free venom.     

Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

Background

Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption.

Methodology/Principal Findings

In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues.

Conclusions/Significance

These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.     

Tissue Localization and Extracellular Matrix Degradation by PI,PII and PIII Snake Venom Metalloproteinases: Clues on the Mechanisms of Venom-Induced Hemorrhage

Snake venom hemorrhagic metalloproteinases (SVMPs) of the PI, PII and PIII classes were compared in terms of tissue localization and their ability to hydrolyze basement membrane components in vivo, as well as by a proteomics analysis of exudates collected in tissue injected with these enzymes. Immunohistochemical analyses of co-localization of these SVMPs with type IV collagen revealed that PII and PIII enzymes co-localized with type IV collagen in capillaries, arterioles and post-capillary venules to a higher extent than PI SVMP, which showed a more widespread distribution in the tissue. The patterns of hydrolysis by these three SVMPs of laminin, type VI collagen and nidogen in vivo greatly differ, whereas the three enzymes showed a similar pattern of degradation of type IV collagen, supporting the concept that hydrolysis of this component is critical for the destabilization of microvessel structure leading to hemorrhage. Proteomic analysis of wound exudate revealed similarities and differences between the action of the three SVMPs. Higher extent of proteolysis was observed for the PI enzyme regarding several extracellular matrix components and fibrinogen, whereas exudates from mice injected with PII and PIII SVMPs had higher amounts of some intracellular proteins. Our results provide novel clues for understanding the mechanisms by which SVMPs induce damage to the microvasculature and generate hemorrhage.     

Viper and cobra venom neutralization by β-sitosterol and stigmasterol isolated from the root extract of Pluchea indica Less. (Asteraceae)

We reported previously that the methanolic root extract of the Indian medicinal plant Pluchea indica Less. (Asteraceae) could neutralize viper venom-induced action [Alam, M.I., Auddy, B., Gomes, A., 1996. Viper venom neutralization by Indian medicinal plant (Hemidesmus indicus and P. indica) root extracts. Phytother. Res. 10, 58-61]. The present study reports the neutralization of viper and cobra venom by beta-sitosterol and stigmasterol isolated from the root extract of P. indica Less. (Asteraceae). The active fraction (containing the major compound beta-sitosterol and the minor compound stigmasterol) was isolated and purified by silica gel column chromatography and the structure was determined using spectroscopic analysis (EIMS, (1)H NMR, (13)C NMR). Anti-snake venom activity was studied in experimental animals. The active fraction was found to significantly neutralize viper venom-induced lethal, hemorrhagic, defibrinogenation, edema and PLA(2) activity. Cobra venom-induced lethality, cardiotoxicity, neurotoxicity, respiratory changes and PLA(2) activity were also antagonized by the active component. It potentiated commercial snake venom antiserum action against venom-induced lethality in male albino mice. The active fraction could antagonize venom-induced changes in lipid peroxidation and superoxide dismutase activity. This study suggests that beta-sitosterol and stigmasterol may play an important role, along with antiserum, in neutralizing snake venom-induced actions.     

Triacontyl p-coumarate: An inhibitor of snake venom metalloproteinases

Snake venom metalloproteinases (SVMPs) participate in a number of important biological, physiological and pathophysiological processes and are primarily responsible for the local tissue damage characteristic of viperid snake envenomations. The use of medicinal plant extracts as antidotes against animal venoms is an old practice, especially against snake envenomations. Such plants are sources of many pharmacologically active compounds and have been shown to antagonize the effects of some venoms and toxins. The present study explores the activity of triacontyl p-coumarate (PCT), an active compound isolated from root bark of Bombacopsis glabra vegetal extract (Bg), against harmful effects of Bothropoides pauloensis snake venom and isolated toxins (SVMPs or phospholipase A₂). Before inhibition assays, Bg or PCT was incubated with venom or toxins at ratios of 1:1 and 1:5 (w/w; venom or isolated toxins/PCT) for 30 min at 37 °C. Treatment conditions were also assayed to simulate snakebite with PCT inoculated at either the same venom or toxin site. PCT neutralized fibrinogenolytic activity and plasmatic fibrinogen depletion induced by B. pauloensis venom or isolated toxin. PCT also efficiently inhibited the hemorrhagic (3MDH – minimum hemorrhagic dose injected i.d into mice) and myotoxic activities induced by Jararhagin, a metalloproteinase from B. jararaca at 1:5 ratio (toxin: inhibitor, w/w) when it was previously incubated with PCT and injected into mice or when PCT was administered after toxin injection. Docking simulations using data on a metalloproteinase (Neuwiedase) structure suggest that the binding between the protein and the inhibitor occurs mainly in the active site region causing blockade of the enzymatic reaction by displacement of catalytic water. Steric hindrance may also play a role in the mechanism since the PCT hydrophobic tail was found to interact with the loop associated with substrate anchorage. Thus, PCT may provide a alternative to complement ophidian envenomation treatments.     

Identification of potent inhibitors against snake venom metalloproteinase (SVMP) using molecular docking and molecular dynamics studies

Snake venom metalloproteinase (SVMP) (Echis coloratus (Carpet viper) is a multifunctional enzyme that is involved in producing several symptoms that follow a snakebite, such as severe local hemorrhage, nervous system effects and tissue necrosis. Because the three-dimensional (3D) structure of SVMP is not known, models were constructed, and the best model was selected based on its stereo-chemical quality. The stability of the modeled protein was analyzed through molecular dynamics (MD) simulation studies. Structure-based virtual screening was performed, and 15 potential molecules with the highest binding energies were selected. Further analysis was carried out with induced fit docking, Prime/MM–GBSA (ΔG_Bind calculations), quantum-polarized ligand docking, and density functional theory calculations. Further, the stability of the lead molecules in the SVMP-active site was examined using MD simulation. The results showed that the selected lead molecules were highly stable in the active site of SVMP. Hence, these molecules could potentially be selective inhibitors of SVMP. These lead molecules can be experimentally validated, and their backbone structural scaffold could serve as building blocks in designing drug-like molecules for snake antivenom.     

Iron protects porcine plasma coagulation kinetics from degradation by <Emphasis Type="Italic">Crotalus atrox</Emphasis> venom

While the administration of antivenom to treat hemotoxic snake bite injury remains the gold standard of therapy, we have demonstrated that modifying human fibrinogen with iron and carbon monoxide renders it resistant to fibrinogenolytic snake venom enzymes. In order to translate these findings into a possible biometal-based therapy complementary to antivenom administration, a preclinical model that possesses fibrinogen that closely mimics the human molecule in response to iron and carbon monoxide needed to be identified. The goal of this investigation was to determine if a swine model could serve in this capacity by assessing the thrombelastographic response of porcine plasma to iron and carbon monoxide exposure, without or with further exposure to the fibrinogenolytic venom of the viper Crotalus atrox. Using plasma obtained from eight swine, it was determined that their plasma responded to iron and carbon monoxide in a manner similar to that of human plasma by displaying enhanced coagulation kinetics. However, in sharp contrast to the response seen with human plasma, only iron significantly protected porcine plasma coagulation kinetics from C. atrox venom degradation. Therefore the pig is an animal beyond humans that could derive benefit from the biometal-focused therapy of iron infusion to protect against venom mediated compromise of coagulation. Thus, future investigation to assess the effects of iron administration to attenuate the effects of fibrinogenolytic envenomation with a pig model is justified.     

Bites by foreign venomous snakes in Britain.

In 1970-7 17 people in Britain were the victims of 32 bites by foreign venomous snakes. Crotalus atrox caused eight of these bites, Bitis arietans five, and the remaining 19 bites were caused by 12 different species. All the victims were bitten while handling the snake, and 24 bites were incurred by private individuals in their own homes. Poisoning was negligible in 17 of the 32 bites but life-threatening in at least two cases. Thus in the early stages snake bite may be unpredictable as a clinical problem. All victims of snake bite should be observed for at least 12 hours to assess the severity of poisoning and to ensure rational treatment. Local necrosis developed in six cases and resulted in prolonged illness in five of these cases; local incision was carried out and many have been a casual factor. Comprehensive stocks of antivenoms for treating bites by foreign venomous snakes are held by the National Health Services in Liverpool and London. Antivenom is indicated (a) for potentially serious systemic poisoning, as evidenced by hypotension, electrocardiographic changes, neurtrophilia, and acidosis (after viper or elapid bites), abnormal bleeding or non-clotting blood after viper bites; and ptosis or glossopharyngeal palsy after elapid bites; and (b) for bites from snakes whose venom causes local necrosis, to prevent or minimise this unpleasant complication. For effective antivenom treatment intravenous infusion is mandatory.     

Priapism following a juvenile Russell’s viper bite: An unusual case report

Following a bite from a juvenile Russell’s viper (Daboia russelii), a priapism (painful erection) developed rapidly in a 16-year-old male and only subsided after administration of antivenom 3 hours later. Potential mechanisms for this snakebite-induced priapism are unclear but likely due to venom toxins causing nitric oxide (NO) release and subsequent vasodilation of endothelium in the corpus cavernosum, although the possible involvement of other mechanisms cannot be ruled out. We strongly believe that this unusual case report may lead to further scientific research in order to improve the clinical understanding of the pathophysiology of envenomation due to Russell’s viper bites. Although it is too early to speculate, further research may also discover the possibilities of developing venom-based candidate molecules to treat sexual dysfunction in males and females.     

Production of high titre antibody response against Russell's viper venom in mice immunized with ethanolic extract of fruits of Piper longum L. (Piperaceae) and piperine

Piper longum L. fruits have been traditionally used against snakebites in north-eastern and southern region of India. The aim of the study was to assess the production of antibody response against Russell's viper venom in mice after prophylactic immunization with ethanolic extract of fruits of Piper longum L. and piperine. The mice sera were tested for the presence of antibodies against Russell's viper venom by in vitro lethality neutralization assay and in vivo lethality neutralization assay. Polyvalent anti-snake venom serum (antivenom) manufactured by Haffkine Bio-Pharmaceutical Corporation Ltd. was used as standard. Further confirmation of presence of antibodies against the venom in sera of mice immunized with PLE and piperine was done using indirect enzyme-linked immunosorbent assay (ELISA) and double immunodiffusion test. Treatment with PLE-treated mice serum and piperine-treated mice serum was found to inhibit the lethal action of venom both in the in vitro lethality neutralization assay and in vivo lethality neutralization assay. ELISA testing indicated that there were significantly high (p < 0.01) levels of cross reactions between the PLE and piperine treated mice serum and the venom antigens. In double immunodiffusion test, a white band was observed between the two wells of antigen and antibodies for both the PLE-treated and piperine-treated mice serum. Thus it can be concluded that immunization with ethanolic extract of fruits of Piper longum and piperine produced a high titre antibody response against Russell's viper venom in mice. The antibodies against PLE and piperine could be useful in antivenom therapy of Russell's viper bites. PLE and piperine may also have a potential interest in view of the development of antivenom formulations used as antidote against snake bites.     

Role of hyaluronidase inhibitors in the neutralization of toxicity of Egyptian horned viper Cerastes cerastes venom

Hyaluronidase “venom spreading factor” is a common component of snake venoms and indirectly potentiates venom toxicity. It may cause permanent local tissue destruction at the bite site/systemic collapse of the envenomated victim. The present study was performed to assess the benefits of inhibiting the hyaluronidase activity of Egyptian horned viper, Cerastes cerastes (Cc). The aqueous extracts of some medicinal plants were screened for their inhibitory effect on hyaluronidase activity of Cc venom. The results revealed that the Rosmarinus officinalis (Ro) extract is the most potent hyaluronidase inhibitor among the tested extracts. The Ro extract is more potent inhibitory effect on the hyaluronidase activity than the prepared rabbit monoclonal antiserum of previously purified hyaluronidase enzyme from Cc venom (anti-CcHaseII). In addition, the Ro extract is efficiently inhibited the activity of hemorrhagic toxin previously purified from Cc venom, and it also neutralized the edema inducing activity of the Cc venom in vivo. Furthermore, the Ro extract markedly increased the survival time of experimental mice injected with lethal dose of Cc venom up to 7 h in compared to mice injected with venom alone or with venom/anti-CcHaseII (15 ± 5, 75 ± 4 min), respectively. Our findings imply the significance of plant-derived hyaluronidase inhibitor in the neutralization of local effects of Cc venom and retardation of death time. Therefore, it may use as a therapeutic value in complementary snakebite therapy.     

Protective Effect of the Sulfated Agaran Isolated from the Red Seaweed <Emphasis Type="Italic">Laurencia aldingensis</Emphasis> Against Toxic Effects of the Venom of the Snake, <Emphasis Type="Italic">Lachesis muta</Emphasis>

Snakebite is a serious occupational hazard affecting mainly rural populations of tropical and subtropical developing countries. Lachesis muta (Bushmaster) bites are extremely serious but are rarely reported in the literature. Bushmaster envenomings are characterized by intense local pain, edema, neurotoxicity, hypotension, local hemorrhage, and dramatic systemic alterations. Antivenom treatment has regularly been used for more than a century; however, it fails to neutralize local tissue damage and hemorrhage, leading to morbidity or disabilities in victims. Thus, the production and clinical use of antivenom must be improved. The present work characterizes, for the first time, a sulfated polysaccharide from the red seaweed, Laurencia aldingensis, including its neutralizing effect on some toxic activities of L. muta venom. Chemical and spectroscopic analyses showed that L. aldingensis produces sulfated agarans with the A-units partially C-2 sulfated or 6-O-methoxylated presetting the B-units in the cyclized (3,6-anhydro-α-L-galactose) or in the non-cyclized form (α-L-galactose). The latter is significantly substituted by sulfate groups on C-6. In vitro and in vivo assays showed that this sulfated agaran inhibited hemolysis, coagulation, proteolysis, edema, and hemorrhage of L. muta venom. Neutralization of hemorrhagic activity was also observed when the agaran was administered by different routes and after or before the venom injection. Furthermore, the agaran blocked the edema caused by a phospholipase A₂ isolated from the L. muta venom. Experimental evidence therefore indicates that the sulfated agaran of L. aldingensis has potential to aid antivenom therapy of accidents caused by L. muta venom and may help to develop more effective antivenom treatments of snake bites in general.     

Biochemical and enzymatic characterization of BpMP-I, a fibrinogenolytic metalloproteinase isolated from Bothropoides pauloensis snake venom

Snake Venom Metalloproteinases (SVMPs) are the most abundant components present in Viperidae venom. They are important in the induction of systemic alterations and local tissue damage after envenomation. In the present study, a metalloproteinase named BpMPI was isolated from Bothropoides pauloensis snake venom and its biochemical and enzymatic characteristics were determined. BpMPI was purified in two chromatography steps on ion exchange CM-Sepharose Fast flow and Sephacryl S-300. This protease was homogeneous on SDS-PAGE and showed a single chain polypeptide of 20 kDa under non reducing conditions. The partial amino acid sequence of the enzyme showed high similarity with other SVMPs enzymes from snake venoms. BpMPI showed proteolytic activity upon azocasein and bovine fibrinogen and was inhibited by EDTA, 1,10 phenanthroline and β-mercaptoethanol. Moreover, this enzyme showed stability at neutral and alkaline pH and it was inactivated at high temperatures. BpMPI was able to hydrolyze glandular and tissue kallikrein substrates, but was unable to act upon factor Xa and plasmin substrates. The enzyme did not induce local hemorrhage in the dorsal region of mice even at high doses. Taken together, our data showed that BpMP-I is in fact a fibrinogenolytic metalloproteinase and a non hemorrhagic enzyme.     

Coagulopathy after snake bite by Bothrops neuwiedi: case report and results of in vitro experiments

Coagulation studies were performed in a patient who had been bitten by a snake of the species Bothrops neuwiedi. The patient presented with hemorrhagic necrosis at the envenomization site and considerable bleeding from venous puncture sites. He developed a severe defibrination syndrome with a clottable fibrinogen level of approximately 0.1 g/l. Fibrinogen was not measurable by clotting time assay. Fibrin degradation products were greatly elevated. Treatment with antivenom caused an anaphylactic reaction within ten minutes and serum sickness after three days. In vitro experiments revealed that B. neuwiedi venom directly activates Factors II and X, but does not activate Factor XIII. In vivo consumption of Factor XIII after B. neuwiedi envenomization is ascribed to the action of Factor IIa. At low venom concentrations clotting is initiated by activation of prothrombin by the venom either directly or via Factor X activation. Treatment with heparin might be beneficial in coagulopathy secondary to snake bite by reducing circulating active thrombin. The venom contains thrombin-like proteases which cause slow clotting of fibrinogen, and plasmin-like components causing further proteolysis of fibrinogen and fibrin. Antivenom has no effect on the proteolytic action of the snake venom. The in vivo effects of antivenom are presumably caused by acceleration of the elimination of venom components from the circulation. Intravenous administration of antivenom caused normalization of blood coagulation parameters within 48 h.     

Snake venomics and antivenomics of the arboreal neotropical pitvipers Bothriechis lateralis and Bothriechis schlegelii

We report the comparative proteomic characterization of the venoms of two related neotropical arboreal pitvipers from Costa Rica of the genus Bothriechis, B. lateralis (side-striped palm pit viper) and B. schlegelii (eyelash pit viper). The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venom proteomes of B. lateralis and B. schlegelii comprise similar number of distinct proteins belonging, respectively, to 8 and 7 protein families. The two Bothriechis venoms contain bradykinin-potentiating peptides (BPPs), and proteins from the phospholipase A 2 (PLA 2), serine proteinase, l-amino acid oxidase (LAO), cysteine-rich secretory protein (CRISP), and Zn (2+)-dependent metalloproteinase (SVMP) families, albeit each species exhibit different relative abundances. Each venom also contains unique components, for example, snake venom vascular endothelial growth factor (svVEGF) and C-type lectin-like molecules in B. lateralis, and Kazal-type serine proteinase inhibitor-like proteins in B. schlegelii. Using a similarity coefficient, we estimate that the similarity of the venom proteins between the two Bothriechis taxa may be <10%, indicating a high divergence in their venom compositions, in spite of the fact that both species have evolved to adapt to arboreal habits. The major toxin families of B. lateralis and B. schlegelii are SVMP (55% of the total venom proteins) and PLA 2 (44%), respectively. Their different venom toxin compositions provide clues for rationalizing the distinct signs of envenomation caused by B. schlegelii and B. lateralis. An antivenomic study of the immunoreactivity of the Instituto Clodomiro Picado (ICP) polyvalent antivenom toward Bothriechis venoms revealed that l-amino acid oxidase and SVMPs represent the major antigenic protein species in both venoms. Our results provide a ground for rationalizing the reported protection of the ICP polyvalent antivenom against the hemorrhagic, coagulant, defibrinating, caseinolytic and fibrin(ogen)olytic activities of Bothriechis ( schlegelii, lateralis) venoms. However, these analyses also evidenced the limited recognition capability of the polyvalent antivenom toward a number of Bothriechis venom components, predominantly BPPs, svVEGF, Kazal-type inhibitors, some PLA 2 proteins, some serine proteinases, and CRISP molecules.