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Skeletal muscle regeneration is impaired after myonecrosis induced by viperid snake venoms, but the mechanisms behind such poor regenerative outcome are not fully understood. This study compared the changes in basement membrane (BM) components in mouse skeletal muscle in two different scenarios of muscle injury: (a) injection of Bothrops asper venom, as a model of poor regeneration, and (b) injection of a myotoxic fraction (Mtx) isolated from this venom, as a model of successful regeneration. The degradation and reposition of laminin, type IV collagen and fibronectin were assessed over time by a combination of immunohistochemistry, Western blot, and real time polymerase chain reaction. Both treatments induced degradation of laminin and type IV collagen in areas of muscle necrosis since day one, however, there were differences in the pattern of degradation and reposition of these proteins along time. Overall, Mtx induced a higher synthesis of fibronectin and higher degradation of laminin at intermediate time points, together with higher levels of transcripts for the chains of the three proteins. Instead, venom induced a higher degradation of laminin and type IV collagen at early time intervals, followed by a reduced recovery of type IV collagen by 15 days. These differences in extracellular matrix degradation and remodeling between the two models could be associated to the poor muscle regeneration after myonecrosis induced by B. asper venom.

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.

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.

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 A2 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.

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 LD50s of venom by the intravenous route. Lower values of IC50 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.

Skin blistering as a result of snakebite envenomation is characteristic of some bites, however little is known regarding the mechanism of blister formation or the composition of the blister fluid. In order to investigate if blister fluid proteomes from humans suffering snakebite envenomation could provide insights on the pathophysiology of these skin alterations, blister fluid was collected from six patients upon presentation at a clinic in India bitten by three species of snakes, Daboia russelii (3), Hypnale hypnale (2), or Naja naja (1). Standard clinical data were recorded throughout the treatment. Approximately 805 proteins were identified in blister fluids using proteomic analyses. Informatics analyses of the proteomes identified the top biological response categories as: platelet degranulation, innate immune response, receptor-mediated endocytosis, complement activation, and blood coagulation. Hierarchical clustering did not show a clear segregation of patients&rsquo, proteomes being associated with the species of snake involved, suggesting that either the proteomic profiles described reflect a general response to venom-induced tissue damage or more patient data sets will be required to observe significant differences. Finally, it is of interest that venom proteins were also identified in the blister fluids suggesting that this fluid may serve as a reservoir of venom biologically active proteins/toxins, and as such, may indicate the clinical value of removing blister fluid to attenuate further tissue damage.

Abrogation of the hemorrhagic activity of BaP1, a PI Snake Venom Metalloproteinase (SVMP) from the venom of Bothrops asper, was achieved by the substitution of residues in the first part of the Ω loop surrounding the active site by the corresponding residues of a structurally-similar non-hemorrhagic PI SVMP from a related venom. Previous studies by molecular dynamic simulation showed higher flexibility in the first part of the loop in hemorrhagic SVMPs, as compared to non-hemorrhagic SVMPs. It has been suggested that the Ω loop is critical for protein-protein interface and may be involved in the interaction with extracellular matrix proteins, hence influencing the ability of the toxin to bind and hydrolyze basement membrane components. The SVMP with the site mutation completely lost hemorrhagic activity, and only had a partial reduction of proteolytic activity, indicating that this region in the loop plays a key role in the ability to induce hemorrhage. Our findings demonstrate a key structural determinant of the hemorrhagic capacity of PI SVMPs.

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 A2 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.

Renealmia alpinia (Rottb.) MAAS, obtained by micropropagation (in vitro) and wild forms have previously been shown to inhibit some toxic activities of Bothrops asper snake venom if preincubated before injection. In this study, assays were performed in a murine model in which extracts were administered for three days before venom injection. R. alpinia extracts inhibited lethal activity of B. asper venom injected by intraperitoneal route. Median Effective Dose (ED50) values were 36.6 ± 3.2 mg/kg and 31.7 ± 5.4 mg/kg (p &gt, 0.05) for R. alpinia wild and in vitro extracts, respectively. At a dose of 75 mg/kg, both extracts totally inhibited the lethal activity of the venom. Moreover, this dose prolonged survival time of mice receiving a lethal dose of venom by the intravenous route. At 75 mg/kg, both extracts of R. alpinia reduced the extent of venom-induced pulmonary hemorrhage by 48.0% (in vitro extract) and 34.7% (wild extract), in agreement with histological observations of lung tissue. R. alpinia extracts also inhibited hemorrhage in heart and kidneys, as evidenced by a decrease in mg of hemoglobin/g of organ. These results suggest the possibility of using R. alpinia as a prophylactic agent in snakebite, a hypothesis that needs to be further explored.

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. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP) UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicina

Bothrops ayerbei, a pitviper inhabiting the Patia River's basin (Valle Alto del Rio Patia) 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.

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. Mexican National Council for Science and Technology/[SEP-CONACYT-2008-82 833]/CONACYT/México UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Artículo electrónico, 18 páginas, 6 figuras, 1 tabla, Snake venom metalloproteinases (SVMPs) play key biological roles in prey immobilization and digestion. The majority of these activities depend on the hydrolysis of relevant protein substrates in the tissues. Hereby, we describe several isoforms and a cDNA clone sequence, corresponding to PII SVMP homologues from the venom of the Central American pit viper Bothriechis lateralis, which have modifications in the residues of the canonical sequence of the zinc-binding motif HEXXHXXGXXH. As a consequence, the proteolytic activity of the isolated proteins was undetectable when tested on azocasein and gelatin. These PII isoforms comprise metalloproteinase and disintegrin domains in the mature protein, thus belonging to the subclass PIIb of SVMPs. PII SVMP homologues were devoid of hemorrhagic and in vitro coagulant activities, effects attributed to the enzymatic activity of SVMPs, but induced a mild edema. One of the isoforms presents the characteristic RGD sequence in the disintegrin domain and inhibits ADP- and collagen-induced platelet aggregation. Catalytically-inactive SVMP homologues may have been hitherto missed in the characterization of snake venoms. The presence of such enzymatically-inactive homologues in snake venoms and their possible toxic and adaptive roles deserve further investigation., This study was supported by Vicerrectoría de Investigación, Universidad de Costa Rica (Projects 741-B0-528 and 741-B2-517), the International Foundation for Science (Grant F/4096-2) and Ministerio de Economía y Competitividad, Madrid, Spain (Grant BFU2013-42833-P). This work was performed in partial fulfillment of the requirements for the PhD degree of Erika Camacho at Universidad de Costa Rica. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A2, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM) and other extracellular matrix (ECM) proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs) or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms., Author Summary The local pathology induced by viperid snakes is characterized by a complex of alterations as consequence of direct and indirect effects of the toxins present in the venom, as well as the host response to tissue damage, and constitutes a dynamic process of degenerative and reparative events. The pathogenesis of local effects induced by Bothrops asper venom has been studied by traditional methodologies. Recently, proteomic analysis of wound exudates collected in the vicinity of affected tissue has become a powerful tool to study the pathogenesis of local envenoming from a more integrative perspective. Thus, in the present study we analyzed the dynamics of the local effects induced by B. asper venom in the gastrocnemius muscle of mice through a proteomic and immunochemistry approach in order to identify biomarkers of tissue damage and repair during the course of envenoming. Our results showed an early presence of cytosolic and mitochondrial proteins in exudates as compared to cytoskeletal proteins, which reflect the rapid cytotoxic effect of venom, followed by the action of endogenous proteinases in the cytoskeleton of damaged muscle fibers later on in the course of envenoming. On the other hand, the early presence of extracellular matrix components and the increment of some of them in exudates, reflect the rapid microvascular damage and hemorrhage induced by the venom, followed by the action of endogenous matrix metalloproteinases (MMPs) during tissue remodeling as part of the inflammatory response. Overall our study allowed the identification of key biomarkers of tissue damage and repair as part of the pathological effects induced by B. asper venom in skeletal muscle, which offer relevant insights for a better understanding of the complex dynamics of local pathology induced by viperid snakebite envenoming.

The historical development of discoveries and conceptual frames for understanding the hemorrhagic activity induced by viperid snake venoms and by hemorrhagic metalloproteinases (SVMPs) present in these venoms is reviewed. Histological and ultrastructural tools allowed the identification of the capillary network as the main site of action of SVMPs. After years of debate, biochemical developments demonstrated that all hemorrhagic toxins in viperid venoms are zinc-dependent metalloproteinases. Hemorrhagic SVMPs act by initially hydrolyzing key substrates at the basement membrane (BM) of capillaries. This degradation results in the weakening of the mechanical stability of the capillary wall, which becomes distended owing of the action of the hemodynamic biophysical forces operating in the circulation. As a consequence, the capillary wall is disrupted and extravasation occurs. SVMPs do not induce rapid toxicity to endothelial cells, and the pathological effects described in these cells in vivo result from the mechanical action of these hemodynamic forces. Experimental evidence suggests that degradation of type IV collagen, and perhaps also perlecan, is the key event in the onset of microvessel damage. It is necessary to study this phenomenon from a holistic, systemic perspective in which the action of other venom components is also taken into consideration. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

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.

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. Sweden-Central America NeTropica Universidad de Costa Rica International Foundation for Science (IFS) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

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 nonimmunologic 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 A2 and hemorrhagic metalloproteinases. Universidad de Costa Rica//UCR/Costa Rica Network for Research and Training in Tropical Diseases in Central America//NeTropica/ Universidad de Antioquia///Colombia Departamento Administrativo de Ciencia, Tecnología e Innovación//COLCIENCIAS/Colombia Wellcome Trust///Reino Unido International Foundation for Science//IFS/Suecia UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP) UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicina

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:16:52Z No. of bitstreams: 1 WOS000185425500016.pdf: 436693 bytes, checksum: 6e53e418baab576ea898d4d9383e0c12 (MD5) Made available in DSpace on 2014-02-26T17:16:52Z (GMT). No. of bitstreams: 1 WOS000185425500016.pdf: 436693 bytes, checksum: 6e53e418baab576ea898d4d9383e0c12 (MD5) Previous issue date: 2003-10-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:02:23Z No. of bitstreams: 1 WOS000185425500016.pdf: 436693 bytes, checksum: 6e53e418baab576ea898d4d9383e0c12 (MD5) Made available in DSpace on 2014-05-20T14:02:24Z (GMT). No. of bitstreams: 1 WOS000185425500016.pdf: 436693 bytes, checksum: 6e53e418baab576ea898d4d9383e0c12 (MD5) Previous issue date: 2003-10-01 BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated front the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C164I165M166, which characterize the metzincin superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four a-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single a-helix and several loops. The catalytic zinc ion is coordinated by the N-epsilon2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix. Univ Costa Rica, Fac Microbiol, Inst Clodomiro Picado, San Jose, Costa Rica Univ Costa Rica, Escuela Med, Dept Bioquim, San Jose, Costa Rica Univ Liverpool, Royal Liverpool Univ Hosp, Dept Haematol, Liverpool, Merseyside, England Univ Liverpool, Liverpool Sch Trop Med, Venom Res Unit, Liverpool L3 5QA, Merseyside, England Fiocruz MS, Dept Fisiol & Farmacodinam, BR-21045900 Rio de Janeiro, Brazil Univ Virginia, Hlth Sci Ctr, Dept Microbiol, Charlottesville, VA 22908 USA UNESP, IBILCE, Dept Phys, BR-15054000 Sao Jose do Rio Preto, Brazil UNESP, IBILCE, Dept Phys, BR-15054000 Sao Jose do Rio Preto, Brazil

A prothrombin activator, named ‘basparin A,’ was isolated from the venom of the crotaline snake Bothrops asper, the species responsible for the majority of snakebite cases in Central America. It is an acidic (pI 5.4), 70 kDa, single chain P-III metalloproteinase comprising, in addition to the metalloproteinase domain, disintegrin-like, and high-cysteine domains. Basparin A is a glycoprotein displaying immunological cross-reactivity with BaH1, a P-III hemorrhagic metalloproteinase isolated from the same venom. It activates prothrombin through the formation of meizothrombin, without requiring additional cofactors; it is, therefore, a class A snake venom prothrombin activator. In contrast with most venom metalloproteinases, it does not degrade components of the extracellular matrix. Apart from its clotting activity, basparin A inhibits collagen-dependent platelet aggregation in vitro, an effect that does not depend on proteolytic activity. Clotting activity on human plasma is not abrogated by the plasma proteinase inhibitors α2 macroglobulin and murinoglobulin, whereas activity is completely inhibited by Costa Rican polyvalent (Crotalinae) anti-venom. Basparin A does not induce local tissue alterations, such as hemorrhage, myonecrosis, and edema, in mice. Moreover, it does not induce systemic hemorrhage, thrombocytopenia nor prolongation of the bleeding time following intravenous administration. At low doses, the only observed effect induced by basparin A, when injected intravenously or intramuscularly into mice, is defibrin(ogen)ation. At higher doses, intravenous administration resulted in sudden death due to numerous occluding thrombi in pulmonary vessels. Basparin A is likely to play an important role in the coagulopathy associated with B. asper envenoming. Universidad de Costa Rica/[741-A2-036]/UCR/Costa Rica Universidad de Costa Rica/741-A1-504]/UCR/Costa Rica Wellcome Trust/[062043]//Inglaterra International Foundation for Science/[2707-2]/IFS/Suecia UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy using antibodies raised in horses. In order to obtain more specific and batch-to-batch consistent antivenons, recombinant antibodies are considered a good option compared to animal immunization. We constructed a recombinant single chain variable fragment (scFv) from a monoclonal antibody against BaP1 (MABaP1) formerly secreted by a hybridoma clone. This recombinant antibody was cloned into pMST3 vector in fusion with SUMO protein and contains VH and VL domains linked by a flexible (G4S)3 polypeptide (scFvBaP1). The aim of this work was to produce scFvBaP1 and to evaluate its potential concerning the neutralization of biologically important activities of BaP1. The cytoplasmic expression of this construct was successfully achieved in C43 (DE3) bacteria. Our results showed that scFvBaP1-SUMO fusion protein presented an electrophoretic band of around 43 kDa from which SUMO alone corresponded to 13.6 kDa, and only the scFv was able to recognize BaP1 as well as the whole venom by ELISA. In contrast, neither an irrelevant scFv anti-LDL nor its MoAb partner recognized it. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO, in a concentration-dependent manner. In addition, scFvBaP1, as well as MaBaP1, completely neutralized in vivo hemorrhage, muscle necrosis, and inflammation induced by the toxin. Docking analyses revealed possible modes of interaction of the recombinant antibody with BaP1. Our data showed that scFv recognized BaP1 and whole B. asper venom, and neutralized biological effects of this SVMP. This scFv antibody can be used for understanding the molecular mechanisms of neutralization of SVMPs, and for exploring the potential of recombinant antibody fragments for improving the neutralization of local tissue damage in snakebite envenoming. Fundação de Amparo à Pesquisa do Estado de São Paulo/[2012/01028-3]/FAPESP/Brasil Fundação de Amparo à Pesquisa do Estado de São Paulo/[PIPE/FAPESP 2004/08297-3]/FAPESP/Brasil Conselho Nacional de Desenvolvimento Científico e Tecnológico//CNPq/Brasil Universidad de Costa Rica//UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Stings by Thalassophryne nattereri are responsible for envenomation of fishermen in north-eastern Brazil. Its venom induces prominent local tissue damage, characterized by pain, oedema and necrosis. The pathogenesis of acute muscle damage induced by T. nattereri venom was studied in mice. Intramuscular injection induced myonecrosis within the first hours. Some muscle cells presented a hypercontracted morphology, but most necrotic fibres were not hypercontracted, being instead characterized by a disorganization of myofibrils, with Z line loss, mitochondrial swelling and sarcolemmal disruption. In addition, thrombosis was observed histologically in venules and veins, together with vascular congestion and stasis, evidenced by intravital microscopy. Venom induced a rapid increment in serum creatine kinase (CK) levels, concomitant with a reduction in gastrocnemius muscle CK activity, whereas no increments in muscle lactic acid were detected. A rapid cytolytic effect was induced by the venom on C2C12 murine myoblasts in culture. The inflammatory reaction in affected muscle was characterized by oedema and scarce cellular infiltrate of polymorphonuclear leucocytes and macrophages, with a consequent delay in the removal of necrotic material. Skeletal muscle regeneration was partially impaired, as evidenced by the presence of regenerating fibres of variable size and by the increase of fibrotic tissue in endomysium and perimysium. It is suggested that T. nattereri venom affects muscle fibres by a direct cytotoxic effect, and that the vascular alterations described preclude a successful regenerative process.

Anti-bothropic complex (ABC) was isolated from the serum of the South American opossum ( Didelphis albiventris ) by single-step affinity chromatography using a Sepharose-immobilized metalloprotease (BaP1) from Bothrops asper as the binding protein. Biochemical characterization of ABC showed the presence of two glycosylated subunits of 43 and 45 kDa, respectively, with an isoelectric point B. asper venom. In addition to the anti-hemorrhagic and anti-proteolytic activities, ABC also showed anti-myotoxic, anti-lethal, and anti-edematogenic effects against myotoxic phospholipases A 2 isolated from the same venom. Moreover, it had inhibitory effects on the phospholipase A 2 activity of the crude venom as well as the isolated venom phospholipases A 2 .

Aqueous extract from Casearia sylvestris leaves, a typical plant from Brazilian open pastures, was able to neutralize the hemorrhagic activity caused by Bothrops asper, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pirajai venoms. It also neutralized two hemorrhagic metalloproteinases from Bothrops asper venom. Proteolytic activity on casein induced by bothropic venoms and by isolated proteases, including Bn2 metalloproteinase from B. neuwiedi venom, was also inhibited by the C. sylvestris extract in different levels. The α-fibrinogen chain was partially protected against degradation caused by B. jararacussu venom, when this venom was incubated with C. sylvestris extract. We also observed that this extract partially increased the time of plasma coagulation caused by B. jararacussu, B. moojeni and B. neuwiedi venoms. C. sylvestris extract did not induce proteolysis in any substrate assayed.

SummaryThrombocytopenia occurs in a number of patients bitten by Bothrops asper, a species responsible for the majority of snakebites in Central America and southern Mexico. In this work we describe the isolation of a new platelet-aggregating protein, named aspercetin, from the venom of B. asper, which induces thrombocytopenia in mice. Isolation was carried out by a combination of ion-exchange chromatography on DEAE-Sepharose and affinity chromatography on Affi-Gel Blue. Aspercetin is a disulfide-linked heterodimer, with a pI of 4.5 and a molecular mass of 29,759 Da, detemined by MALDI-ESI mass spectrometry. N-terminal sequence shows homology with a number of venom proteins which belong to the C-type lectin family. Aspercetin has functional similarities with botrocetin, from B. jararaca venom, since it induces platelet aggregation only in the presence of plasma or purified von Willebrand factor. Aspercetin-mediated platelet aggregation results from the interaction of von Willebrand factor with platelet receptor GPIb. Aspercetin lacks anticoagulant effect and does not agglutinate erythrocytes, in contrast with other representatives of the C-type lectin family isolated from snake venoms. Moreover, aspercetin is not lethal, nor does it induce myonecrosis, hemorrhage and edema. When injected intravenously or intramuscularly in mice it induces a rapid, dose-dependent drop in platelet counts and prolongs the bleeding time, suggesting that it may play a role in the thrombocytopenia that develops in a number of B. asper envenomations. Moreover, mice injected intravenously with aspercetin and then receiving an intradermal injection of B. asper hemorrhagic metalloproteinase BaP1 develop a larger hemorrhagic lesion than mice receiving only BaP1. This suggests that aspercetin, by reducing platelet numbers, may contribute to the hemorrhagic effect characteristic of B. asper envenomations.

Batimastat (BB-94), a synthetic hydroxamate peptidomimetic matrix metalloproteinase inhibitor, was tested for its ability to inhibit proteolytic and toxic effects induced by BaP1, a 24-kDa hemorrhagic metalloproteinase isolated from the venom of Bothrops asper, the medically most important snake species in Central America and southern Mexico. Batimastat inhibited proteolytic activity on biotinylated casein, with anic50 of 80 nM. In addition, batimastat was effective in inhibiting hemorrhagic, dermonecrotic, and edema-forming activities of this metalloproteinase if incubated with the enzyme prior to the assays. When the inhibitor was administered i.m. at the site of the toxin injection without preincubation, rapidly after metalloproteinase administration, it totally abrogated the hemorrhagic and dermonecrotic effects of BaP1. Inhibition was less effective as the time lapse between toxin and batimastat injection increased, due to the extremely rapid development of BaP1-induced local tissue damage in this experimental model. On the other hand, batimastat was ineffective if administered by the i.p. route immediately after toxin injection. It is concluded that batimastat, and probably other synthetic metalloproteinase inhibitors, may become useful therapeutic tools aimed at the in situ inhibition of venom metalloproteinases, when injected at the site of the bite rapidly after envenomation. Universidad de Costa Rica/[741–98-202]/UCR/Costa Rica International Foundation for Science/[F/2707–1]/IFS/Suecia Consejo Nacional para Investigaciones Científicas y Tecnológicas/[98-012-F0]/CONICIT/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

A hemorrhagic metalloproteinase, named BaH4, was isolated from the venom of the snake Bothrops asper by a combination of ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sephacryl S-200. BaH4 is a 69 kDa protein with a pI of 5.3. It was recognized by antibodies raised against hemorrhagic metalloproteinase BaH1 isolated from B. asper venom, with a reaction of partial immunologic identity. BaH4 shows proteolytic activity on biotinylated casein, hide powder azure and fibrin, although having lower activity than crude B. asper venom and metalloproteinase BaP1 isolated from the same venom. BaH4 hydrolyzed fibronectin, laminin and type IV collagen in vitro, albeit at a relatively high enzyme:substrate ratio. Proteolytic activity was inhibited by chelating agents and 2-mercaptoethanol, but not by soybean trypsin inhibitor. Prominent hemorrhage developed in gastrocnemius and cremaster muscles after administration of BaH4. Moreover, it induced lethality in mice after intravenous injection, with an LD50 of 0.37 μg/g. Histological observations showed conspicuous pulmonary hemorrhage when the enzyme was injected intravenously. BaH4 is a hemorrhagic metalloproteinase which may play a relevant role in local and systemic bleeding characteristic of B. asper envenomations. Universidad de Costa Rica/[741-98-202]/UCR/Costa Rica International Foundation for Science/[F/2707-1]/IFS/Suecia Deutscher Akademischer Austauschdienst//DAAD/Alemania UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

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. Universidad de Costa Rica/[741-B0-528]/UCR/Costa Rica Universidad de Costa Rica/[741-B2-517]/UCR/Costa Rica International Foundation for Science/[F/4096-2]/IFS/Suecia Network for Research and Training in Tropical Diseases in Central America/[01N-2010]/NeTropica/ Ministerio de Educación y Ciencia/[BFU2010-17373]//España UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

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 A, 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.

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 (CaNa2EDTA). 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. Universidad de Costa Rica/[741-B0-606]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Introduction: Viperid snakebite envenomings are characterized by muscle necrosis and a deficient regenerative response. Methods: Homogenates from gastrocnemius muscles of mice injected with the venom of the snake Bothrops asper or with 2 tissue-damaging toxins were added to cultures of C2C12 myogenic cells. Myoblasts proliferation and fusion were assessed. Venom was detected by immunoassay in mouse muscle during the first week after injection. Results: Homogenates from venom-injected muscle induced a drop in the number of proliferating myoblasts and a complete elimination of myotube formation. The inhibitory effect induced by homogenates from venom-injected mice was abrogated by preincubation of the homogenate with antivenom antibodies but not with control antibodies. This finding provides evidence that the effect is due to the action of venom in the tissue. Conclusions: Our observations suggest that traces of venom in muscle tissue might inhibit myotube formation and preclude a successful regenerative response. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

No immunological cross-reactivity was observed between BaH1 and BaP1, two hemorrhagic metalloproteinases isolated from B. asper venom, by gel immunodiffusion, Western blotting and neutralization studies. Cross-reactivity was detected with antisera against these toxins in several crotaline and viperine snake venoms by ELISA, whereas no reactivity was observed with either antiserum against the venoms of Bothrops nummifer, Crotalus durissus terrificus, Vipera russelli and several elapid venoms. Antiserum against native BaH1 neutralized hemorrhagic activity of the venoms of B. asper, B. atrox, B. jararaca, Crotalus atrox, C. durissus durissus, Echis carinatus and Trimeresurus flavoviridis, being ineffective against the venoms of Agkistrodon bilineatus and Lachesis muta.

The majority of snakebite envenomations in Central America are caused by the viperid species Bothrops asper, whose venom contains a high proportion of zinc-dependent metalloproteinases that play a relevant role in the pathogenesis of hemorrhage characteristic of these envenomations. Broad metalloproteinase inhibitors, such as the peptidomimetic hydroxamate Batimastat, have been shown to inhibit snake venom metalloproteinases (SVMP). However, the difficulty in having open public access to Batimastat and similar molecules highlights the need to design new inhibitors of SVMPs that could be applied in the treatment of snakebite envenomations. We have chosen the SVMP BaP1 as a model to search for new inhibitors using different strategies, that is, screening of the Prestwick Chemical Library and rational peptide design. Results from these approaches provide clues on the structural requirements for efficient BaP1 inhibition and pave the way for the design of new inhibitors of SVMP. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP) UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Química

artículo (arbitrado) -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2011 Background: Viperid snakebite envenoming is characterized by prominent local tissue damage, including muscle necrosis. A frequent outcome of such local pathology is deficient skeletal muscle regeneration, which causes muscle dysfunction, muscle loss and fibrosis, thus provoking permanent sequelae that greatly affect the quality of life of patients. The causes of such poor regenerative outcome of skeletal muscle after viperid snakebites are not fully understood. Methodology/Principal Findings: A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America. Gastrocnemius muscle was injected with either B. asper venom, a myotoxic phospholipase A2 (Mtx), a hemorrhagic metalloproteinase (SVMP), or saline solution. At various time intervals, during one month, tissue samples were collected and analyzed by histology, and by immunocytochemical and immunohistochemical techniques aimed at detecting muscle fibers, collagen, endothelial cells, myoblasts, myotubes, macrophages, TUNEL-positive nuclei, and axons. A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature. In contrast, poor regeneration, with fibrosis and atrophic fibers, occurred when muscle was injected with venom or SVMP, both of which provoke necrosis, microvascular damage leading to hemorrhage, and poor axonal regeneration. Conclusions/Significance: The deficient skeletal muscle regeneration after injection of B. asper venom is likely to depend on the widespread damage to the microvasculature, which affects the removal of necrotic debris by phagocytes, and the provision of nutrients and oxygen required for regeneration. In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model. This study was supported by NeTropica (grant 2-N-2008), by Vicerrectoría de Investigación, Universidad de Costa Rica (project 741-A7-604). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (Kd), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The Kds of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure–function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation. Universidad de Costa Rica//UCR/Costa Rica Conselho Nacional de Desenvolvimento Científico e Tecnológico//CNPq/Brasil Fundação de Amparo a Pesquisa do Estado de São Paulo//FAPESP/Brasil NeTropica/// UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Snake venom metalloproteinases (SVMPs) play a prominent role in the local and systemic manifestations of viperid snakebite envenomations. Thus, the possibility of using metalloproteinase inhibitors in the treatment of these envenomations is a promising therapeutic alternative. This study assessed the ability of two metalloproteinase inhibitors, the biphosphonate clodronate and the tetracycline doxycycline, to inhibit proteolytic, hemorrhagic, coagulant and defibrinogenating effects of Bothrops asper venom. Both compounds were able to inhibit these activities, at concentrations in the mM range, when incubated with venom prior to testing. However, when inhibition of hemorrhage was assessed in assays involving independent injection of venom and drug, inhibition was poor, even when these compounds were injected immediately after envenomation. These findings support the concept that the effectiveness of compounds, such as clodronate and doxycycline, whose inhibitory action on SVMPs is based on zinc chelation alone, is limited, and stress the view that more specific molecules are required for an effective inhibition of SVMPs in vivo. Universidad de Costa Rica, Vicerrectoría de Investigación, projects 741-A4-061 and 741-A7-604 International Foundation for Science, project F/4096-1 NeTropica, project 2-N-2008 UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

The local and systemic pathophysiological alterations induced by BjussuSP-I, a thrombin-like serine proteinase from the venom of the snake Bothrops jararacussu, were assessed in mice. BjussuSP-I induced a mild edema but no local myonecrosis or hemorrhage. It did not induce any microvascular alteration in the cremaster muscle. Intramuscular injection of BjussuSP-I promoted an increase in the expression of proMMP-9, but it did not induce the activation of proMMP-2 or proMMP-9 synthesized in muscle tissue injected with a myotoxic phospholipase A2 homolog. BjussuSP-I induced defibrin(ogen)ation upon intravenous and intramuscular injections, with reduction in plasma fibrinogen concentration and increments in the levels of fibrin degradation products and D-dimer. When compared with animals having normal coagulation, mice defibrin(ogen)ated by BjussuSP-I developed a slightly larger hemorrhagic lesion in the skin when injected with metalloproteinase BaP1. Intravenous injection of sublethal doses of BjussuSP-I promoted a series of behavioral and motor changes similar to those previously described for ‘gyroxin’, i.e. opisthotonus and a circular body movement along the longitudinal axis. /[grant 2-N-2005]/NeTropica/Estados Unidos Vicerrectoría de Investigación/[741-A6-505]/UCR/Costa Rica Fundação de Amparo à Pesquisa do Estado de São Paulo//FAPESP/Brasil Conselho Nacional de De-senvolvimento Cientı́fico e Tecnológico//CNPq/Brasil UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Thrombocytopenia and platelet dysfunction occur in patients bitten by Bothrops sp snakes in Latin America. An experimental model was developed in mice to study the effects of B. asper venom in platelet numbers and function. Intravenous administration of this venom induces rapid and prominent thrombocytopenia and ex vivo platelet hypoaggregation.The drop in platelet numbers was primarily due to aspercetin, a protein of the C-type lectin family which induces von Willebrand factor-mediated platelet aggregation/agglutination.In addition,the effect of class P-III hemorrhagic metalloproteinases on the microvessel wall also contributes to thrombocytopenia since jararhagin, a P-III metalloproteinase, reduced platelet counts. Hypoaggregation was associated with the action of procoagulant and defibrin( ogen)ating proteinases jararacussin-I (a thrombin-like ser ine proteinase) and basparin A (a prothrombin activating metalloproteinase). At the doses which induced hypoaggregation, these enzymes caused defibrin(ogen)ation, increments in fibrin( ogen) degradation products and D-dimer and prolongation of the bleeding time. Incubation of B. asper venom with batimastat and a 2 -macroglobulin abrogated the hypoaggregating activity, confirming the role of venom proteinases in this effect. Neither aspercetin nor the defibrin(ogen)ating and hypoaggregating components induced hemorrhage upon intravenous injection. However, aspercetin, but not the thrombin-like or the prothrombin-activating proteinases, potentiated the hemorrhagic activity of two hemorrhagic metalloproteinases in the lungs. Universidad de Costa Rica//UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

The peptidomimetic hydroxamate metalloproteinase inhibitor batimastat (BB-94) was assessed for its ability to neutralize the systemic effects (lethality, hemorrhage and coagulopathy) induced by the venom of Bothrops asper, the most important snake from a medical standpoint in Central America. Batimastat inhibited lethality when a venom challenge dose of two LD50s was used by intraperitoneal and intravenous routes, with ED50s of 250 and 22 μM, respectively. With a challenge dose of three LD50s, lethality was not abrogated, but a conspicuous and dose-dependent delay in the time of death was observed in mice injected with mixtures of venom plus batimastat. Upon incubation with 500 μM batimastat, venom LD50 increased 2.86-fold (intraperitoneal route) and 2.37-fold (intravenous route), when compared with LD50 of venom alone. Batimastat also inhibited the hemorrhagic effect induced by venom in the lungs after intravenous injection. Moreover, batimastat exerted a significant inhibition of in vitro coagulant and in vivo defibrinogenating effects of venom, evidencing that metalloproteinases play a key role in the coagulopathy characteristic of B. asper envenomation. The remaining uninhibited coagulant effect is due to serine proteinases, i.e. thrombin-like enzymes, since this effect was completely abrogated by the combination of batimastat and PMSF. Our results stress the view that metalloproteinases play a relevant role in the systemic pathophysiology of B. asper envenomation and that metalloproteinase inhibitors may become a therapeutic alternative in this pathology. Universidad de Costa Rica/[741-A1-504]/UCR/Costa Rica Universidad de Costa Rica/[741-A2-036]/UCR/Costa Rica International Foundation for Science/[F-2707-3]/IFS/Suecia United Nations Educational, Scientific and Cultural Organization/[883.701-3]/UNESCO/ UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:13:39Z No. of bitstreams: 1 WOS000176271200016.pdf: 139681 bytes, checksum: f2320da6506df316df21bdaf2e6ca128 (MD5) Made available in DSpace on 2014-02-26T17:13:39Z (GMT). No. of bitstreams: 1 WOS000176271200016.pdf: 139681 bytes, checksum: f2320da6506df316df21bdaf2e6ca128 (MD5) Previous issue date: 2002-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:02:17Z No. of bitstreams: 1 WOS000176271200016.pdf: 139681 bytes, checksum: f2320da6506df316df21bdaf2e6ca128 (MD5) Made available in DSpace on 2014-05-20T14:02:17Z (GMT). No. of bitstreams: 1 WOS000176271200016.pdf: 139681 bytes, checksum: f2320da6506df316df21bdaf2e6ca128 (MD5) Previous issue date: 2002-06-01 BaP1 is a metalloproteinase isolated from the venom of the Central American snake Bothrops asper (terciopelo). It is a 24 kDa protein consisting of a single chain which includes the metalloproteinase domain only, therefore being classified as a class P-I snake-venom metalloproteinase. BaP1 induces prominent local tissue damage, such as haemorrhage, myonecrosis, blistering, dermonecrosis and oedema. In order to elucidate its structure, BaP1 was crystallized by the hanging-drop vapour-diffusion technique in 0.1 M bicine pH 9.0, 10% PEG 20 000 and 2%(v/v) dioxane. Diffraction data were observed to a resolution of 2.7 Angstrom. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.22, b = 60.17, c = 86.09 Angstrom. UNESP, IBILCE, Dept Phys, BR-15054000 Sao Jose do Rio Preto, Brazil Univ Costa Rica, Fac Microbiol, Inst Clodomiro Picado, San Jose, Costa Rica Univ Liverpool, Dept Haematol, Liverpool L69 3BX, Merseyside, England Univ Liverpool, Liverpool Sch Trop Med, Venom Res Unit, Liverpool L3 5QA, Merseyside, England UNESP, IBILCE, Dept Phys, BR-15054000 Sao Jose do Rio Preto, Brazil

The pathological alterations induced by neuwiedase, a 22 kDa class P-I metalloproteinase from the venom of the South American pit viper Bothrops neuwiedi, were studied in mice. Neuwiedase was devoid of hemorrhagic activity when tested in the skin up to a dose of 200 μg, and also after intramuscular injection in the gastrocnemius. However, it induced bleeding when applied onto the mouse cremaster muscle in intravital microscopy experiments, and caused pulmonary hemorrhage when injected intravenously at doses higher than 5 μg/g. Median lethal dose (LD50) by the intravenous route was 5 μg/g, whereas LD50 of crude venom was 0.47 μg/g. After intramuscular injection, neuwiedase induced a mild myotoxic effect, evidenced histologically and by the increment in plasma creatine kinase activity, but it was devoid of hemorrhagic and thrombotic effects. In contrast, crude B. neuwiedi venom induced prominent hemorrhage and myonecrosis in gastrocnemius muscle. Both venom and neuwiedase induced an inflammatory reaction in muscle tissue characterized by abundant polymorphonuclear leukocytes. Moreover, a conspicuous edema developed in the foot pad after subcutaneous injection of neuwiedase. Anti-neuwiedase antibodies produced in rabbits were effective in the neutralization of hemorrhagic activity of crude venom, evidencing immunological cross-reactivity between neuwiedase and other hemorrhagic metalloproteinases present in the venom, and suggesting that metalloproteinases devoid of, or having low, hemorrhagic activity could be good immunogens to generate antibodies effective against high molecular mass metalloproteinasas having potent hemorrhagic activity. It is concluded that neuwiedase, despite its lack of hemorrhagic effect when injected in the gastrocnemius muscle, contributes to local tissue damage by inducing edema, inflammatory infiltrate and mild myotoxicity, and by degrading extracellular matrix components. In addition, large doses of neuwiedase may contribute to pulmonary bleeding Conselho Nacional de Desenvolvimento Científico e Tecnológico//CNPq/Brasil Fundação de Amparo à Pesquisa do Estado de São Paulo//FAPESP/Brasil International Foundation for Science/[F/2707]/IFS/Suecia Universidad de Costa Rica/[741-98-202]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

The biochemical characteristics of hemorrhagic metalloproteinases isolated from snake venoms are reviewed, together with their role in the pathogenesis of the local tissue damage characteristic of crotaline and viperine snake envenomations. Venom metalloproteinases differ in their domain structure. Some enzymes comprise only the metalloproteinase domain, others have disintegrin-like and high cysteine domains and others present, besides these domains, an additional lectin-like subunit. All of them are zinc-dependent enzymes with highly similar zinc binding environments. Some metalloproteinases induce hemorrhage by directly affecting mostly capillary blood vessels. It is suggested that hemorrhagic enzymes cleave, in a highly selective fashion, key peptide bonds of basement membrane components, thereby affecting the interaction between basement membrane and endothelial cells. As a consequence, these cells undergo a series of morphological and functional alterations in vivo, probably associated with biophysical hemodynamic factors such as tangential fluid shear stress. Eventually, gaps are formed in endothelial cells through which extravasation occurs. In addition to hemorrhage, venom metalloproteinases induce skeletal muscle damage, myonecrosis, which seems to be secondary to the ischemia that ensues in muscle tissue as a consequence of bleeding and reduced perfusion. Microvessel disruption by metalloproteinases also impairs skeletal muscle regeneration, being therefore responsible of fibrosis and permanent tissue loss after snakebites. Moreover, venom metalloproteinases participate in the degradation of extracellular matrix components and play a relevant role in the prominent local inflammatory response that characterizes snakebite envenomations, since they induce edema, activate endogenous matrix metalloproteinases (MMPs) and are capable of releasing TNF-α from its membrane-bound precursor. Owing to their protagonic role in the pathogenesis of local tissue damage, snake venom metalloproteinases constitute relevant targets for natural and synthetic inhibitors which may complement antivenoms in the neutralization of these effects. Universidad de Costa Rica/[741-98-202]/UCR/Costa Rica International Foundation for Science/[F/2707-1]/IFS/Suecia Consejo Nacional para Investigaciones Científicas y Tecnológicas/[98-012-FO]/CONICIT/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

The venom of the snake Bothrops asper, the most important poisonous snake in Central America, evokes an inflammatory response, the mechanisms of which are not well characterized. The objectives of this study were to investigate whether B. asper venom and its purified toxins--phospholipases and metalloproteinase--activate the complement system and the contribution of the effect on leucocyte recruitment. In vitro chemotaxis assays were performed using Boyden's chamber model to investigate the ability of serum incubated with venom and its purified toxins to induce neutrophil migration. The complement consumption by the venom was evaluated using an in vitro haemolytic assay. The importance of complement activation by the venom on neutrophil migration was investigated in vivo by injecting the venom into the peritoneal cavity of C5-deficient mice. Data obtained demonstrated that serum incubated with crude venom and its purified metalloproteinase BaP-1 are able to induce rat neutrophil chemotaxis, probably mediated by agent(s) derived from the complement system. This hypothesis was corroborated by the capacity of the venom to activate this system in vitro. The involvement of C5a in neutrophil chemotaxis induced by venom-activated serum was demonstrated by abolishing migration when neutrophils were pre-incubated with antirat C5a receptor antibody. The relevance of the complement system in in vivo leucocyte mobilization was further demonstrated by the drastic decrease of this response in C5-deficient mice. Pre-incubation of serum with the soluble human recombinant complement receptor type 1 (sCR 1) did not prevent the response induced by the venom, but abolished the migration evoked by metalloproteinase-activated serum. These data show the role of the complement system in bothropic envenomation and the participation of metalloproteinase in the effect. Also, they suggest that the venom may contain other component(s) which can cause direct activation of C5a. Universidad de Costa Rica/[741-98-202]/UCR/Costa Rica Fundação de Amparo à Pesquisa do Estado de São Paulo//FAPESP/Brasil International Foundation for Science/[F/2707-1]/IFS/Suecia UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Local tissue damage represents a serious consequence of Bothrops asper envenomations. It encompasses a complex series of alterations, including myonecrosis, dermonecrosis, hemorrhage and edema. Due to its rapid development it is difficult to neutralize by antivenoms, especially if there is a delay in serotherapy. Experimental studies with this venom and the polyvalent (Crotalinae) antivenom produced in Costa Rica indicate that antivenom is effective in neutralizing these toxic activities when incubated with the venom prior to injection. However, if venom and antivenom are injected independently in mice, neutralization of these effects is only partial. Moreover, neutralization is not complete even if homologous or heterologous antibodies are present in the circulation before venom is injected. Despite differences in their pharmacokinetic profiles, equine whole IgG and F(ab')2 antivenoms show similar efficacy in the neutralization of edema, hemorrhage and myonecrosis induced by B. asper venom, suggesting that the use of antivenoms made of antibody fragments may not improve neutralization of these effects. This is due, at least in part, to the fact that microvessel disruption by venom components favors a similar antibody concentration in the affected tissues. Recent advances in the development of neutralizing substances of rapid diffusion, that could be injected locally in the field, may contribute to the neutralization of metalloproteinases and phospholipases A2. In addition, the rapid administration of antivenoms with high antibody titers against locally-acting toxins is very important in the treatment of these effects. Universidad de Costa Rica/[741-89-057]/UCR/Costa Rica International Foundation for Science/[F/0883-4]/IFS/Suecia International Foundation for Science/[F/1388-3F]/IFS/Suecia UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

A new myotoxic phospholipase A2was isolated from the venom of the arboreal snakeBothriechis schlegelii(formerlyBothrops schlegelii) from Costa Rica, by ion-exchange chromatography on CM-Sephadex.B. schlegeliimyotoxin I is a basic protein (pI> 9.3) with a subunit molecular weight of 15 kDa, which migrates as a dimer in sodium dodecyl sulfate–polyacrylamide gel electrophoresis under nonreducing conditions. This myotoxin is recognized by antibodies generated againstBothrops aspermyotoxin II (a lysine-49 phospholipase A2), by both enzyme-immunoassay and gel immunodiffusion, in the latter case with a pattern of partial identity. The toxin induces rapid myonecrosis upon intramuscular injection in mice, as evidenced by the early increase in plasma creatine kinase activity and by direct intravital microscopic observation.B. schlegeliimyotoxin I also induces edema in the mouse footpad assay and exerts lethal activity (LD50∼2.5 μg/g) upon intravenous injection. The toxin has a low phospholipase A2activity (4.2 μEq·mg−1·min−1) using egg yolk phospholipids as substrate. It also shows a weak anticoagulant effectin vitro.Its N-terminal sequence, SMYELGKMILLETGKNAATSYIAYG, shows 93% homology with bothBothrops aspermyotoxin II andB. jararacussubothropstoxin I, suggesting thatB. schlegeliimyotoxin I may be a new lysine-49 variant of this family of myotoxic phospholipases A2. Universidad de Costa Rica Proy. 741-95-264 y 741-96-265 International Foundation for Science F/1388-3F UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

The ability of pre-existing antibodies to neutralize locally-acting toxins of Bothrops asper snake venom was investigated. Hemorrhage, myonecrosis, and edema were markedly reduced in actively immunized mice, although none of these effects was completely abolished. In mice passively immunized with equine antivenom, hemorrhage was prevented completely, while myonecrosis and edema were partially reduced. Pre-existing antibodies did not modify the early stage ( < 3 hr) of venom-induced edema, but significantly accelerated the normalization of this effect within 24 hr. Passive administration of antivenom either 5 or 120 min before venom injection gave similar results, suggesting that the presence of antibodies in the intravascular compartment may fully neutralize locally acting toxins, in this experimental animal model. Overall, the homologous or heterologous origin of antibodies was not a significant factor influencing their in uit,o neutralizing efficiency against local venom effects. Antibody titrations by enzyme-immunoassay using purified toxins and whole venom indicated that serum from actively-immunized mice had a higher proportion of anti-myotoxin antibodies than equine antivenom. Universidad de Costa Rica/[VI-741-95-264]/UCR/Costa Rica International Foundation for Science/[F/1388-3F]/IFS/Suecia UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)