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Clinical manifestations of envenomings by bites of the viperid snakes Bothrops asper and Daboia russelii show marked differences. Both venoms elicit the typical effects induced by viperid venoms (local tissue damage, bleeding, coagulopathies, shock). In addition, envenomings by D. russelii are characterized by a high incidence of acute kidney injury and by systemic capillary leak syndrome. The present investigation aimed to compare the local pathological and inflammatory events induced by the intramuscular injection of these venoms in a mouse model. B. asper venom induced stronger local hemorrhage, whereas D. russelii venom caused a higher extent of myonecrosis, and both venoms induced inflammation. Exudates collected from the site of tissue damage showed higher proteolytic activity in the case of samples from B. asper venom-treated mice. This activity was abrogated by antivenoms, indicating that it is the result of the action of venom proteinases. In addition, an increase in matrix metalloproteinases (MMPs) over time was detected in exudates induced by both venoms. Proteome analysis of exudates revealed higher abundance of extracellular matrix (ECM)-derived protein fragments in samples collected from B. asper venom-injected mice, whereas those from D. russelii venom-injected animals had higher amounts of intracellular proteins. Analysis of the subproteome of inflammatory mediators in exudates showed various patterns of change over time. Some mediators peaked at 180 min and decreased afterwards, whereas others increased and remained elevated during the 360 min observation period. Interestingly, various mediators (MIP-1α, MIP-1β, KC, MIP-2, GM-CSF, VEGF, and LIX) increased and then decreased in the case of B. asper venom, while they remained elevated at 360 min in the case of D. russelii venom. Our findings show that these venoms induce a different pattern of local tissue damage and suggest that the venom of D. russelii induces a more sustained inflammatory reaction, an observation that may have implications for the pathophysiology of envenomings., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Opossums in the clade Didelphini are well known to be resistant to snake venom due to endogenous circulating inhibitors which target metalloproteinases and phospholipases. However, the mechanisms through which these opossums cope with a variety of other damaging venom proteins are unknown. A protein involved in blood clotting (von Willebrand Factor) has been found to have undergone rapid adaptive evolution in venom-resistant opossums. This protein is a known target for a subset of snake venom C-type lectins (CTLs), which bind it and then induce it to bind platelets, causing hemostatic disruption. Several amino acid changes in vWF unique to these opossums could explain their resistance; however, experimental evidence that these changes disrupt venom CTL binding was lacking. We used platelet aggregation assays to quantify resistance to a venom-induced platelet response in two species of venom-resistant opossums (Didelphis virginiana, Didelphis aurita), and one venom-sensitive opossum (Monodelphis domestica). We found that all three species have lost nearly all their aggregation response to the venom CTLs tested. Using washed platelet assays we showed that this loss of aggregation response is not due to inhibitors in the plasma, but rather to the failure of either vWF or platelets (or both) to respond to venom CTLs. These results demonstrate the potential adaptive function of a trait previously shown to be evolving under positive selection. Surprisingly, these findings also expand the list of potentially venom tolerant species to include Monodelphis domestica and suggest that an ecological relationship between opossums and vipers may be a broader driver of adaptive evolution across South American marsupials than previously thought., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Inflammatory joint conditions are characterized by synovial inflammation, which involves activation of fibroblast-like synoviocytes (FLSs) and production of inflammatory mediators and matrix metalloproteases (MMPs) in joints. This study showed that the snake venom metalloprotease (SVMP) BaP1 activates FLSs to produce PGE 2 by a mechanism dependent on COX-2, mPGES-1 and iPLA 2 s. BaP1 also induces IL-1β release, which up-regulates the production of PGE 2 at a late stage of the stimulation. Expression of COX-2 and mPGES-1 are induced by BaP1 via activation of NF-κB pathway. While NF-κB p50 and p65 subunits are involved in up-regulation of COX-2 expression, only p65 is involved in BaP1-induced mPGES-1 expression. In addition, BaP1 up-regulates EP4 receptor expression. Engagement of this receptor by PGE 2 triggers a positive feedback loop for its production by up-regulating expression of key components of the PGE 2 biosynthetic cascade (COX-2, mPGES-1 and the EP4 receptor), thus contributing to amplification of BaP1-induced effects in FLSs. These data highlight the importance of FLS as a target for metalloproteases in joint inflammation and provide new insights into the roles of MMPs in inflammatory joint diseases. Moreover, our results may give insights into the importance of the catalytic domain, of MMPs for the inflammatory activity of these enzymes.

Envenomings by some populations of the Russell's viper (Daboia russelii) are characterized by a systemic capillary leak syndrome (CLS) which causes hemoconcentration, and is associated with the severity of envenoming. We adapted a model of CLS in mice by assessing hemoconcentration. The venom of D. russelii from Pakistan, but not that of another viperid, Bothrops asper, induced hemoconcentration and an increment in vascular permeability, being devoid of hemorrhagic activity at the doses tested. These findings reveal a dichotomous pattern of vasculotoxicity in viperid snake venoms. This difference might depend on variations in venom composition, especially regarding metalloproteinases (SVMPs), which are low in Pakistani D. russelii and high in B. asper. Inhibition of SVMPs and phospholipases A 2 in D. russelii venom did not abrogate hemoconcentration. An hemoconcentration-inducing fraction was obtained by chromatography, which contains vascular endothelial growth factor (VEGF), a known potent inducer of increment in vascular permeability. Exudates collected from tissue injected with venom also induced hemoconcentration, and the effect was inhibited by antivenom. However, the amount of venom in exudate required to induce the effect is low, as compared with venom dissolved in saline solution, hence suggesting that endogenous proteins present in the exudate, probably inflammatory mediators, potentiate the effect.

Bothrops lanceolatus is an endemic viperid species in the Lesser Caribbean island of Martinique. Envenomings by this species are characterized by local and systemic effects, among which the development of thrombosis in various organs is the most severe complication. An experimental toxicological characterization of this venom was performed using in vivo mouse tests and various in vitro assays. The venom induced lethal, local and systemic hemorrhagic, edema-forming, myotoxic, thrombocytopenic, proteinase and phospholipase A 2 activities. The preclinical efficacy of a batch of monospecific Bothrofav ® antivenom currently in use in Martinique was assessed. The antivenom was highly effective in the neutralization of all activities tested, in agreement with its described clinical efficacy. This batch of antivenom showed a higher preclinical efficacy as compared to a previous batch used in the past., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Snakebite envenoming by viperid species, and by some elapids, is characterized by a complex pattern of tissue damage at the anatomical site of venom injection. In severe cases, tissue destruction may be so extensive as to lead to permanent sequelae, with serious pathophysiological, social and psychological consequences. Significant advances have been performed in the study of venom-induced tissue damage, including identification and characterization of the toxins involved, insights into the mechanisms of action of venoms and toxins, and study of tissue responses to venom-induced injury. Nevertheless, much remains to be known and understood on the pathogenesis of these alterations. This review focuses on some of the pending issues in the topic of snake venom-induced local tissue damage. The traditional 'reductionist' approach, which has predominated in the study of snake venoms and their actions, needs to be complemented by more integrative and holistic perspectives aimed at capturing the complexity of these pathological alterations. Future advances in the study of these topics will certainly pave the way for innovative therapeutic interventions, with the goal of reducing the impact of this aspect of snakebite envenoming., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Pterois volitans/miles lionfish (adult and juvenile) dorsal spines and caudal fin extracts were compared in their general composition, enzymatic activities and hemolytic and cytotoxic effects on bovine aortic endothelial cells and murine myoblasts, to distinguish between the activities present in the venom and epidermal mucus. Intradermal and intramuscular injections were also administered in mice to determine in vivo effects. This work shows that crude venom of Caribbean species of lionfish, present in dorsal spines, induces several in vitro effects including hemolysis, weak cytotoxicity, proteolytic and hyaluronidase activities, whereas in vivo, it is not hemorrhagic nor myotoxic, but causes edema, plasma extravasation and a thrombotic-associated lesion on the skin. Some small differences were observed between adult and juvenile venomous secretions. Gelatinolytic activity of the epidermal mucus, the only activity found in caudal fin extracts, could contribute to the in vivo toxicity of the venom., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The ability of two peptidomimetic hydroxamate metalloproteinase inhibitors, Batimastat and Marimastat, to abrogate toxic and proteinase activities of the venom of Echis ocellatus from Cameroon and Ghana was assessed. Since this venom largely relies for its toxicity on the action of zinc-dependent metalloproteinases (SVMPs), the hypothesis was raised that toxicity could be largely eliminated by using SVMP inhibitors. Both hydroxamate molecules inhibited local and pulmonary hemorrhagic, in vitro coagulant, defibrinogenating, and proteinase activities of the venoms in conditions in which venom and inhibitors were incubated prior to the test. In addition, the inhibitors prolonged the time of death of mice receiving 4 LD 50 s of venom by the intravenous route. Lower values of IC 50 were observed for in vitro and local hemorrhagic activities than for systemic effects. When experiments were performed in conditions that simulated the actual circumstances of snakebite, i.e. by administering the inhibitor after envenoming, Batimastat completely abrogated local hemorrhage if injected immediately after venom. Moreover, it was also effective at inhibiting lethality and defibrinogenation when venom and inhibitor were injected by the intraperitoneal route. Results suggest that these, and possibly other, metalloproteinase inhibitors may become an effective adjunct therapy in envenomings by E. ocellatus when administered at the anatomic site of venom injection rapidly after the bite., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The potency of antivenoms is assessed by analyzing the neutralization of venom-induced lethality, and is expressed as the Median Effective Dose (ED50). The present study was designed to investigate the pathophysiological mechanisms responsible for lethality induced by the venom of Bothrops asper, in the experimental conditions used for the evaluation of the neutralizing potency of antivenoms. Mice injected with 4 LD50s of venom by the intraperitoneal route died within ∼25 min with drastic alterations in the abdominal organs, characterized by hemorrhage, increment in plasma extravasation, and hemoconcentration, thus leading to hypovolemia and cardiovascular collapse. Snake venom metalloproteinases (SVMPs) play a predominat role in lethality, as judged by partial inhibition by the chelating agent CaNa2EDTA. When venom was mixed with antivenom, there was a venom/antivenom ratio at which hemorrhage was significantly reduced, but mice died at later time intervals with evident hemoconcentration, indicating that other components in addition to SVMPs also contribute to plasma extravasation and lethality. Pretreatment with the analgesic tramadol did not affect the outcome of the neutralization test, thus suggesting that prophylactic (precautionary) analgesia can be introduced in this assay. Neutralization of lethality in mice correlated with neutralization of in vitro coagulant activity in human plasma., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Four disintegrins were isolated from the venoms of the Central American viperid snakes Atropoides mexicanus (atropoimin), Bothrops asper (bothrasperin), Cerrophidion sasai (sasaimin), and Crotalus simus (simusmin). Purifications were performed by reverse-phase HPLC. The four disintegrins have biochemical characteristics, i.e. molecular mass and location of Cys, which allow their classification within the group of medium-size disintegrins. All of them present the canonical RGD sequence, which determines their interaction with integrins in cell membranes. The disintegrins inhibited ADP and collagen-induced human platelet aggregation, with similar IC50s in the nM range. In addition, disintegrins inhibited the adhesion of an endothelial cell line and a melanoma cell line to the extracellular matrix proteins type I collagen, laminin, fibronectin, and vitronectin, albeit showing variable ability to exert this activity. This study expands the inventory of this family of viperid venom proteins, and reports, for the first time, disintegrins from the venoms of species of the genera Atropoides and Cerrophidion.

A new homodimeric PII metalloproteinase, named BlatH1, was purified from the venom of the Central American arboreal viperid snake Bothriechis lateralis by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. BlatH1 is a glycoprotein of 84 kDa. The mature protein contains a metalloproteinase domain, with the characteristic zinc-binding motif (HEXXHXXGXXH) followed by the sequence CIM at the Met-turn. In the disintegrin domain, the tripeptide sequence TDN substitutes the characteristic RGD motif found in many disintegrins. BlatH1 hydrolyzed azocasein, gelatin and fibrinogen, and exerts a potent local and systemic hemorrhagic activity in mice. The hemorrhagic activity of BlatH1 is not inhibited by the plasma proteinase inhibitor α2-macroglobulin, although the SVMP is able to cleave this plasma inhibitor, generating a 90 kDa product. BlatH1 inhibits ADP- and collagen-induced human platelet aggregation (IC50 = 0.3 μM and 0.7 μM for ADP and collagen, respectively). This activity is abrogated when the enzyme is preincubated with the metalloproteinase inhibitor Batimastat, implying that it depends on proteolysis. In agreement, a synthetic peptide containing the sequence TDN of the disintegrin domain is unable to inhibit platelet aggregation. BlatH1 is a valuable tool to understand the structural determinants of toxicity in PII SVMPs., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Bothrops ayerbei, a pitviper inhabiting the Patía River's basin (Valle Alto del Río Patía) in the Southwestern Department of Cauca, Colombia, was considered as a variant form of Bothrops asper prior to being proposed as a new species in 2010, on the basis of subtle morphological differences. This study reports the proteomic and functional profiling of B. ayerbei venom. Its most striking feature is an almost complete absence (0.7%) of phospholipases A2 (PLA2), which is in contrast to the high proportion of these enzymes (25.3%) in the venom of B. asper from Cauca, as well as in other species of Bothrops. The predominant proteins in B. ayerbei venom are metalloproteinases (53.7%), in agreement with its higher hemorrhagic and lethal activities compared to B. asper venom. Moreover, the negligible content of PLA2s in B. ayerbei venom correlates with its weaker myotoxic effect, in contrast to B. asper venom, here shown to contain abundant Asp49- and Lys49-type PLA2s responsible for its strong myotoxic activity. Other components identified in B. ayerbei venom include bradykinin-potentiating-like peptides and proteins belonging to the C-type lectin/lectin-like, serine proteinase, l-amino acid oxidase, disintegrin, cysteine-rich secretory protein, nerve growth factor, and phosphodiesterase families. The venom composition of B. ayerbei resembles that of neonate specimens of B. asper, which shows a predominance of metalloproteinases, with only low amounts of PLA2s. Therefore, the present findings suggest that the expression of venom proteins in B. ayerbei, in contrast to B. asper, might retain a marked 'paedomorphic' condition. Altogether, the proteomic and toxicological characterization of the venom of B. ayerbei here reported argues in favor of its taxonomical separation from B. asper in Cauca, Colombia., Biological Significance: B. ayerbei, a pitviper found in Cauca, Colombia, had been considered as a variant form of B. asper, but was recently described as a new species on the basis of subtle morphological differences. Our study provides the first detailed proteomic and functional analysis of the venom of B. ayerbei, revealing striking interspecific variation from B. asper, thus arguing in favor of their taxonomical separation. In addition, the observed composition of the venom of B. ayerbei correlates well with its functional and toxicological properties, helping to predict the main clinical manifestations in envenomings by this species, which inflicts a considerable number of snakebites in the Southwestern regions of Colombia., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The rock rattlesnakes Crotalus lepidus comprise a group (lepidus, klauberi, morulus and maculosus) of poorly known mountain cold-tolerant snakes in Mexico. In particular, Crotalus lepidus morulus is a snake endemic of the northeast of Mexico, whereas Crotalus lepidus klauberi and C. l. lepidus are distributed in some regions of the north and central Mexico and southern U. S. Until now very little data are available from C. lepidus subspecies from Mexico, as the terrain inhabited by these snakes is generally steep and rugged. In this work, we have determined some biochemical and biological properties of C. l. morulus, C. l. klauberi and C. l. lepidus crude venoms. Some minor differences in venoms were noted in SDS-PAGE, HPLC profile and MALDI-TOF mass spectrometry analysis. Partial sequences of metalloproteinases, phospholipases A₂ (PLA₂) and galactose-specific lectins were identified in the venoms. Venoms of C. l. klauberi and C. l. lepidus had significantly higher hemorrhagic and lethal activities than C. l. morulus venom. Proteolytic activity in azocasein was higher in C. l. morulus venom, whereas gelatin hydrolysis was higher in C. l. klauberi. Fibrinogenolytic and PLA₂ activities were very similar in all venoms tested. The histological observations in the gastrocnemius muscle damaged by venoms from all the subspecies confirmed myonecrotic and hemorrhagic activities (at 3 and 24 h), which resulted in a poor regenerative response after 14 days. However, C. l. lepidus and C. l. klauberi venom induced a higher increase in the plasma activity of creatine kinase (CK), evidencing higher myotoxicity, whereas paw edema-inducing activity was higher in C. l. lepidus venom. The results indicate that the venoms from the three subspecies have similar protein profiles in electrophoresis, HPLC and molecular weight determinations. However, differences were found in the biological activities in mice. Notably, the venoms of C. l. lepidus and C. l. klauberi present higher toxicity (lower LD₅₀) and hemorrhagic activity than C. l. morulus venom., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Glycolic acid (GA) (2-Hydroxyethanoic acid) is widely used as chemical peeling agent in Dermatology and, more recently, as a therapeutic and cosmetic compound in the field of skin care and disease treatment. In this work we tested the inhibitory ability of glycolic acid on the enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper venom, which induces a variety of toxic actions. Glycolic acid inhibited the proteolytic activity of BaP1 on azocasein, with an IC₅₀ of 1.67 mM. The compound was also effective at inhibiting the hemorrhagic activity of BaP1 in skin and muscle in experiments involving preincubation of enzyme and inhibitor prior to injection. When BaP1 was injected i.m. and then, at the same site, different concentrations of glycolic acid were administered at either 0 or 5 min, 7 mM solutions of the inhibitor partially abrogated hemorrhagic activity when administered at 0 min. Moreover, glycolic acid inhibited, in a concentration-dependent manner, edema-forming activity of BaP1 in the footpad. In order to have insights on the mode of action of glycolic acid, UV-vis and intrinsic fluorescence studies were performed. Results of these assays suggest that glycolic acid interacts directly with BaP1 and chelates the Zn²⁺ ion at the active site. These findings were supported by molecular docking results, which suggested that glycolic acid forms hydrogen bonds with residues Glu143, Arg110 and Ala111 of the enzyme. Additionally, molecular modeling results suggest that the inhibitor chelates Zn²⁺, with a distance of 3.58 Å, and may occupy part of substrate binding cleft of BaP1. Our results suggest that glycolic acid is a candidate for the development of inhibitors to be used in snakebite envenomation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bothrops pirajai snake venom was analyzed by a proteomic strategy. Proteins were separated by RP-HPLC, followed by SDS-PAGE, in-gel tryptic digestion, identification by MALDI-TOF/TOF mass spectrometry, and assignment to known protein families by similarity. Proteins belonging to six families were found in B. pirajai venom, including abundant PLA2s and metalloproteinases, with the remaining proteins distributed among l-amino acid oxidase, serine proteinase, disintegrin and lectin-like families. A P-I class metalloproteinase, named BpirMP, was isolated from this venom by three chromatographic steps. The enzyme has a molecular mass of 23.1kDa, as determined by mass spectrometry. Its proteolytic activity on azocasein was inhibited by chelating and reducing agents, with optimum activity at higher pH values and 37°C. BpirMP presented weak hemorrhagic activity, with an MHD of 50μg, and was able to hydrolyze basement membrane components in vivo and in vitro. The toxin cleaved both Aα and Bβ chains of fibrinogen and was also able to degrade fibrin and blood clots in vitro. The primary sequence analysis indicates that BpirMP contains a zinc ligand motif and a CVM motif that is associated with a Met-turn structure. These results demonstrate that BpirMP is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activities., Biological Significance: This manuscript describes the diversity of protein components present in the venom of Bothops pirajai, a threatened snake species from northeastern Brazil, as well as the isolation and biochemical properties of a PI-SVMP. The results showed distinct mechanisms of action that should contribute in the elucidation of the differences in the hemorrhagic potential of SVMPs, allowing a better understanding of this class of enzymes and of the biology of Bothrops pirajai species., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Snakebite envenoming by Bothrops caribbaeus, an endemic viperid from the Lesser Antillean island of Saint Lucia, is clinically characterized by local tissue damage and systemic thrombosis that can lead to cerebral, myocardial or pulmonary infarctions and venous thromboses. Systemic effects (lethality, pulmonary hemorrhage, thrombocytopenia and coagulopathy) induced by intravenous (i.v.) administration of B. caribbaeus venom were studied in mice. The role of snake venom metalloproteinases (SVMPs) in these systemic alterations was assessed by inhibition with the chelating agent calcium disodium ethylenediaminetetraacetic acid (CaNa(2)EDTA). A snake C-type lectin-like (snaclec) and a type P-III hemorrhagic SVMP were isolated and characterized from this venom, and the effect of venom and the isolated snaclec on human platelet aggregation was studied in vitro. Results indicate that SVMPs play an important role in the overall toxicity of B. caribbaeus venom, being responsible for systemic hemorrhage and lethality, but not thrombocytopenia, whereas the isolated snaclec is involved in the thrombocytopenic effect. Both venom and snaclec induce platelet aggregation/agglutination. Moreover, the snaclec binds directly to glycoprotein Ib (GPIb) and induces agglutination in washed fixed platelets. On the other hand, B. caribbaeus venom hydrolyzed fibrinogen in vitro and induced a partial drop of fibrinogen levels with an increase in fibrin/fibrinogen degradation products (FDP) levels in vivo. The negative result for D-dimer (DD) in plasma is consistent with the lack of microscopic evidence of pulmonary thrombosis and endothelial cell damage. Likewise, no increments in plasma sE-selectin levels were detected. The absence of thrombosis in this murine model suggests that this effect may be species-specific., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Two clotting serine proteinases, named Cdc SI and Cdc SII, were isolated and characterized for the first time from Colombian Crotalus durissus cumanensis snake venom. The enzymes were purified using two chromatographic steps: molecular exclusion on Sephacryl S-200 and RP-HPLC on C8 Column. The molecular masses of the proteins, determined by MALDI-TOF mass spectrometry, were 28,561.4 and 28,799.2 Da for Cdc SI and Cdc SII, respectively. The aim of the present study was to evaluate enzymatic, coagulant and toxic properties of the two enzymes. The serine proteinases hydrolyzed specific chromogenic substrate (BaPNA) and exhibited a Michaelis-Menten behavior. Cdc SI had V(max) of 0.038 ± 0.003 nmol/min and K(M) of 0.034 ± 0.017 mM, while Cdc SII displayed values of V(max) of 0.267 ± 0.011 nmol/min and K(M) of 0.145 ± 0.023 mM. N-terminal sequences were VIGGDEXNIN and VIGGDICNINEHNFLVALYE for Cdc SI and Cdc SII, respectively. Molecular masses, N-terminal sequences, inhibition assays, and enzymatic profile suggest that Cdc SI and Cdc SII belong to the family of snake venom thrombin-like enzymes. These serine proteinases differed in their clotting activity on human plasma, showing a minimum coagulant dose of 25 μg and 0.571 μg for Cdc SI and Cdc SII, respectively. Enzymes also showed coagulant activity on bovine fibrinogen and degraded chain α of this protein. Toxins lack hemorrhagic and myotoxic activities, but are capable to induce defibrin(ogen)ation, moderate edema, and an increase in vascular permeability. These serine proteinases may contribute indirectly to the local hemorrhage induced by metalloproteinases, by causing blood clotting disturbances, and might also contribute to cardiovascular alterations characteristic of patients envenomed by C. d. cumanensis in Colombia., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Severe arenaviral infections in humans are characterized by clinical findings common to other viral hemorrhagic fevers (VHFs), including thrombocytopenia, leukopenia, skin and internal organ hemorrhages, high viral replication, splenic necrosis, and death. Host responses, rather than direct damage by the arenaviral replication, account for most of the observed pathology, but it is not known what protective roles platelets may have in each of the manifestations. To address this issue in an animal model, we compared nondepleted (100%), partially depleted (15%), and profoundly (< 2.5%) platelet depleted mice infected with the mouse arenavirus lymphocytic choriomeningitis virus (LCMV). Here, we describe that systemic bleedings and death were seen only in those animals receiving the stronger depletion treatment. Furthermore, we showed that the nonhemorrhagic but partially platelet-depleted mice were unable to control the viral replication because of generalized splenic necrosis, affecting innate and adaptive immune cells.These data suggest that, by their supportive roles in hemostasis, platelets may be preventing the severe pathology observed in human arenaviral infections.

The aim of this study was the isolation of the LAAO from Lachesis muta venom (LmLAAO) and its biochemical, functional and structural characterization. Two different purification protocols were developed and both provided highly homogeneous and active LmLAAO. It is a homodimeric enzyme with molar mass around 120 kDa under non-reducing conditions, 60 kDa under reducing conditions in SDS-PAGE and 60852 Da by mass spectrometry. Forty amino acid residues were directly sequenced from LmLAAO and its complete cDNA was identified and characterized from an Expressed Sequence Tags data bank obtained from a venom gland. A model based on sequence homology was manually built in order to predict its three-dimensional structure. LmLAAO showed a catalytic preference for hydrophobic amino acids (K(m) of 0.97 mmol/L with Leu). A mild myonecrosis was observed histologically in mice after injection of 100 μg of LmLAAO and confirmed by a 15-fold increase in CK activity. LmLAAO induced cytotoxicity on AGS cell line (gastric adenocarcinoma, IC₅₀: 22.7 μg/mL) and on MCF-7 cell line (breast adenocarcinoma, IC₅₀:1.41 μg/mL). It presents antiparasitic activity on Leishmania brasiliensis (IC₅₀: 2.22 μg/mL), but Trypanosoma cruzi was resistant to LmLAAO. In conclusion, LmLAAO showed low systemic toxicity but important in vitro pharmacological actions., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Proteomic analysis of wound exudates represents a valuable tool to investigate tissue pathology and to assess the therapeutic success of various interventions. In this study, the ability of horse-derived IgG and F(ab')(2) antivenoms to neutralize local pathological effects induced by the venom of the snake Bothrops asper in mouse muscle was investigated by the proteomic analysis of exudates collected in the vicinity of affected tissue. In experiments involving the incubation of venom and antivenom prior to injection in mice, hemorrhagic activity was completely abolished and local muscle-damaging activity was significantly reduced by the antivenoms. In these conditions, the relative amounts of several intracellular and extracellular matrix proteins were reduced by the action of antivenoms, whereas the relative amounts of various plasma proteins were not modified. Because not all intracellular proteins were reduced, it is likely that there is a residual cytotoxicity not neutralized by antivenoms. In experiments designed to more closely reproduce the actual circumstances of envenoming, that is, when antivenom is administered after envenomation, the number of proteins whose amounts in exudates were reduced by antivenoms decreased, underscoring the difficulty in neutralizing local pathology due to the very rapid onset of venom-induced pathology. In these experiments, IgG antivenom was more efficient than F(ab')(2) antivenom when administered after envenomation, probably as a consequence of differences in their pharmacokinetic profiles.

Hemorrhage is one of the most significant effects in envenomings induced by viperid snakebites. Damage to the microvasculature, induced by snake venom metalloproteinases (SVMPs), is the main event responsible for this effect. The precise mechanism by which SVMPs disrupt the microvasculature has remained elusive, although recent developments provide valuable clues to deciphering the details of this pathological effect. The main targets of hemorrhagic SVMPs are components of basement membrane (BM) and surrounding extracellular matrix (ECM), which provide mechanical stability to capillaries. P-III SVMPs, comprising disintegrin-like and cysteine-rich domains in addition to the catalytic domain, are more potent hemorrhagic toxins than P-I SVMPs, constituted only by the metalloproteinase domain. This is likely due to the presence of exosites in the additional domains, which contribute to the binding of SVMPs to relevant targets in the microvasculature. Recent in vivo studies have shown that P-III SVMPs are preferentially located in microvessels. On the other hand, the structural determinants responsible for the different hemorrhagic potential of P-I SVMPs remain largely unknown, although backbone flexibility in a loop located near the active site is likely to play a role. Moreover, hemorrhagic and non-hemorrhagic SVMPs differ in their capacity to hydrolyze in vivo key BM proteins, such as type IV collagen and perlecan, as well as other ECM proteins, like types VI and XV collagens, which play a critical role by connecting BM components to perivascular fibrillar collagens. The evidence gathered support a two-step model for the pathogenesis of SVMP-induced hemorrhage: initially, hemorrhagic SVMPs bind to and hydrolyze components of the BM and associated extracellular matrix proteins that play a key role in the mechanical stability of BM. In conditions of normal blood flow in the tissues, such cleavage results in the weakening, distension and eventual disruption of capillary wall due to the action of biophysical forces operating in vivo., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A hemorrhagic metalloproteinase, named Batx-I, was isolated from the venom of Bothrops atrox specimens (from Southeastern Colombian region) by a combination of CM-Sephadex C25 ion-exchange and Affi-gel Blue affinity chromatographies. This enzyme accounts for about 45% of venom proteins, and it has an ESI-MS isotope-averaged molecular mass of 23296.2 Da and a blocked N-terminus. Two internal fragments sequenced by mass spectrometric analysis showed similarity to other SVMPs from Bothrops venoms. To investigate the possible participation of Batx-I in the envenomation pathophysiology, proteolytic, fibrinogenolytic, hemorrhagic, and other biological activities were evaluated. The minimal hemorrhagic dose obtained was 17 microg/20 g body weight. The enzyme showed proteolytic activity on azocasein, comparable with activity of BaP1. This activity was inhibited by EDTA and 1, 10 o-phenanthroline but not by aprotinin, pepstatin A or PMSF. Fibrinogenolytic activity was analyzed by SDS-PAGE, revealing a preference for degrading the A alpha- and B beta-chains, although partial degradation of the gamma-chain was also detected. The protein lacks coagulant and defibrinating activity. The CK levels obtained, clearly reflects a myotoxic activity induced by Batx-I. The hemorrhagic and fibrinogenolytic activities exhibited by the isolated PI-SVMP may play a role in the hemorrhagic and blood-clotting disorders observed in patients bitten by B. atrox in Colombia., ((c) 2010 Elsevier Ltd. All rights reserved.)

Interest in studies on the neutralization of snake venoms and toxins by diverse types of inhibitors is two-fold. From an applied perspective, results enclose the potential to be translated into useful therapeutic products or procedures, to benefit patients suffering from envenomings. From a basic point of view, on the other hand, neutralizing agents may be used as powerful dissecting tools to determine the relative role of toxins within the context of the overall pathology induced by a venom, or to increase our understanding on the molecular mechanisms by which toxins exert their harmful actions upon particular targets. The venom of the snake Bothrops asper has been the subject of a number of experimental studies addressing its neutralization by antibodies, as well as by non-immunologic inhibitors, including natural products derived from plants or animals, or synthetic drugs. As summarized in the present review, neutralization studies on this venom and some of its isolated toxins have contributed to a better understanding of envenomings by this species, and their treatment. In addition, such studies have provided valuable knowledge on the mechanisms of action and the relative functional importance of particular toxins of this venom, especially in the case of its myotoxic phospholipases A(2) and hemorrhagic metalloproteinases.

Envenomations by Bothrops asper are often associated with complex and severe local pathological manifestations, including edema, blistering, dermonecrosis, myonecrosis and hemorrhage. The pathogenesis of these alterations has been investigated at the experimental level. These effects are mostly the consequence of the direct action of zinc-dependent metalloproteinases (SVMPs) and myotoxic phospholipases A(2) (PLA(2)s). SVMPs induce hemorrhage, blistering, dermonecrosis and general extracellular matrix degradation, whereas PLA(2)s induce myonecrosis and also affect lymphatic vessels. In addition, the prominent vascular alterations leading to hemorrhage and edema may contribute to ischemia and further tissue necrosis. The mechanisms of action of SVMPs and PLA(2)s are discussed in detail in this review. Venom-induced tissue damage plays also a role in promoting bacterial infection. A prominent inflammatory reaction develops as a consequence of these local pathological alterations, with the synthesis and release of abundant mediators, resulting in edema and pain. However, whether inflammatory cells and mediators contribute to further tissue damage is not clear at present. Muscle tissue regeneration after venom-induced pathological effects is often impaired, thus resulting in permanent tissue loss and dysfunction. SVMP-induced microvessel damage is likely to be responsible of this poor regenerative outcome. Antivenoms are only partially effective in the neutralization of B. asper-induced local effects, and the search for novel toxin inhibitors represents a potential avenue for improving the treatment of this serious aspect of snakebite envenomation.

Moderate and severe envenomations by the snake Bothrops asper provoke systemic alterations, such as systemic bleeding, coagulopathy, hypovolemia, hemodynamic instability and shock, and acute renal failure. Systemic hemorrhage is a typical finding of these envenomations, and is primarily caused by the action of P-III snake venom metalloproteinases (SVMPs). This venom also contains a thrombin-like serine proteinase and a prothrombin-activating P-III SVMP, both of which cause defibrin(ogen)ation. Thrombocytopenia, predominantly induced by a C-type lectin-like protein, and platelet hypoaggregation, caused by the two defibrin(ogen)ating enzymes, also contribute to hemostatic disturbances, which potentiate the systemic bleeding induced by hemorrhagic SVMPs. Cardiovascular disturbances leading to shock are due to the combined effects of hemorrhagic toxins, other venom components that increase vascular permeability, the action of hypotensive agents in the venom and of endogenous mediators, and the potential cardiotoxic effect of some toxins. Renal alterations are likely to be caused by direct cytotoxicity of venom components in the kidney, and by renal ischemia resultant from hypovolemia and hypoperfusion. Lethality induced by B. asper venom is the consequence of several combined effects among which the action of P-III SVMPs is especially relevant.

Viperid snakebite envenomation induces blistering and dermonecrosis. The pathological alterations induced by a snake venom metalloproteinase in the skin were investigated in a mouse ear model. Metalloproteinase BaP1, from Bothrops asper, induced rapid edema, hemorrhage, and blistering; the latter two effects were abrogated by preincubation with the metalloproteinase inhibitor batimastat. Neutrophils did not play a role in the pathology, as depletion of these cells resulted in a similar histological picture. Blisters are likely to result from the direct proteolytic activity of BaP1 of proteins at the dermal-epidermal junction, probably at the lamina lucida, as revealed by immunostaining for type IV collagen and laminin. Widespread apoptosis of keratinocytes was detected by the TUNEL assay, whereas no apoptosis of capillary endothelial cells was observed. BaP1 induced a drastic reduction in the microvessel density, revealed by immunostaining for the endothelial marker vascular endothelial growth factor receptor-2. This was followed by a rapid angiogenic response, leading to a partial revascularization. Skin damage was followed by inflammation and granulation tissue formation. Then, a successful re-epithelization process occurred, and the skin of the ear regained its normal structure by 2 weeks. Venom metalloproteinase-induced skin damage reproduces the pathological changes described in snakebitten patients.