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Platelets play key roles in hemostasis, inflammation, immune response, and tissue repair. Although it is known that viperid snake venoms induce thrombocytopenia and platelet hypoaggregation, the roles of these effects in the overall outcome of envenoming are poorly known. This study aimed to assess the effect of platelet depletion on several toxic activities induced by the venom of the Central American viperid snake Bothrops asper in a mouse model. A profound thrombocytopenia was induced in mice by the administration of aspercetin, a C-type lectin-like protein that induces platelet agglutination and drop in platelet counts, while a control group was treated with saline solution instead. Upon envenoming, animals rendered thrombocytopenic developed a higher extent of local and systemic hemorrhage and local myonecrosis, as compared to control envenomed mice. In addition, the median lethal dose (LD

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.

A global strategy, under the coordination of the World Health Organization, is being unfolded to reduce the impact of snakebite envenoming. One of the pillars of this strategy is to ensure safe and effective treatments. The mainstay in the therapy of snakebite envenoming is the administration of animal-derived antivenoms. In addition, new therapeutic options are being explored, including recombinant antibodies and natural and synthetic toxin inhibitors. In this review, snake venom toxins are classified in terms of their abundance and toxicity, and priority actions are being proposed in the search for snake venom metalloproteinase (SVMP), phospholipase A2 (PLA2), three-finger toxin (3FTx), and serine proteinase (SVSP) inhibitors. Natural inhibitors include compounds isolated from plants, animal sera, and mast cells, whereas synthetic inhibitors comprise a wide range of molecules of a variable chemical nature. Some of the most promising inhibitors, especially SVMP and PLA2 inhibitors, have been developed for other diseases and are being repurposed for snakebite envenoming. In addition, the search for drugs aimed at controlling endogenous processes generated in the course of envenoming is being pursued. The present review summarizes some of the most promising developments in this field and discusses issues that need to be considered for the effective translation of this knowledge to improve therapies for tackling snakebite envenoming.

Binding of two P-III snake venom metalloproteinase (SVMPs), one procoagulant and one hemorrhagic, to microvessels was compared in an ex vivo model. The procoagulant SVMP did not bind to the microvasculature, in contrast to the clear localization on microvessels of the hemorrhagic SVMP. Deglycosylation of the procoagulant enzyme did not enable this toxin to bind to microvessels, suggesting that glycosylation is not interfering with binding. These observations suggest that procoagulant SVMPs lack exosites for interaction with microvessels components.

Snakebite envenoming is a neglected tropical disease affecting millions of people every year, especially in vulnerable rural populations in the developing world. Viperid snakes cause envenomings characterized by a complex pathophysiology which includes local and systemic hemorrhage due to the action of snake venom metalloproteinases (SVMPs). The pathogenesis of SVMP-induced systemic hemorrhage has not been investigated in detail. This study explored the pulmonary hemorrhage induced in a murine model by a P-III SVMP from the venom of Crotalus simus. Histological analysis revealed extravasation in the lungs as early as 15 min after intravenous injection of the toxin, and hemorrhage increased at 360 min. Western blot analysis demonstrated the cleavage of basement membrane (BM) proteins in lung homogenates and in bronchoalveolar lavage fluid, implying an enzymatic disruption of this extracellular matrix structure at the capillary-alveolar barrier. Likewise, alveolar edema was observed, with an increment in protein concentration in the bronchoalveolar lavage fluid, and a neutrophil-rich inflammatory infiltrate was present in the parenchyma of the lungs as part of the inflammatory reaction. Pretreatment of mice with indomethacin, pentoxifylline and an anti-neutrophil antibody resulted in a significant decrease in pulmonary hemorrhage at 360 min. These findings suggest that this P-III SVMP induces acute lung injury through the direct action of this enzyme in the capillary-alveolar barrier integrity, as revealed by BM degradation, and as a consequence of the inflammatory reaction that develops in lung tissue. Our findings provide novel clues to understand the mechanism of action of hemorrhagic SVMPs in the lungs.

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.

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.

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

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.

Snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) are among the most abundant enzymes in many snake venoms, particularly among viperids. These proteinases are responsible for some of the clinical manifestations classically seen in viperid envenomings, including hemorrhage, necrosis, and coagulopathies. The objective of this study was to investigate the enzymatic activities of these proteins using a high-throughput peptide library to screen for the proteinase targets of the venoms of five viperid (Echis carinatus, Bothrops asper, Daboia russelii, Bitis arietans, Bitis gabonica) and one elapid (Naja nigricollis) species of high medical importance. The proteinase activities of these venoms were each tested against 360 peptide substrates, yielding 2160 activity profiles. A nonlinear regression model that accurately described the observed enzymatic activities was fitted to the experimental data, allowing for the comparison of cleavage rates across species. In this study, previously unknown protein targets of snake venom proteinases were identified, potentially implicating novel human and animal proteins that may be involved in the pathophysiology of viper envenomings. The functional relevance of these targets was further evaluated and discussed. These new findings may contribute to our understanding of the clinical manifestations and underlying biochemical mechanisms of snakebite envenoming by viperid species.

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.

The variable domain of New Antigen Receptors (vNAR) from sharks, present special characteristics in comparison to the conventional antibody molecules such as: small size (12-15 kDa), thermal and chemical stability and great tissue penetration, that makes them a good alternative source as therapeutic or diagnostic agents. Therefore, it is essential to improve techniques used for the development and selection of vNAR antibodies that recognize distinct antigens. The development of synthetic antibody libraries offers a fast option for the generation of antibodies with the desired characteristics. In this work three synthetic antibody libraries were constructed; without cysteines (Cys), with one Cys and with two Cys residues within its CDR3, with the objective of determining whether the presence or absence of Cys in the CDR3 favors the isolation of vNAR clones from a synthetic library. The libraries were validated selecting against six mammalian proteins. At least one vNAR was found for each of the antigens, and a clone coming from the library without Cys in the CDR3 was selected with all the antigens. In vitro angiogenesis assay with the isolated anti-VEGF antibodies, suggest that these vNARs are capable of inhibiting in vitro angiogenesis. In silico analysis of anti-VEGF antibodies showed that vNARs from synthetic libraries could rival antibodies with affinity maturation by in silico modeling.

Viperid snakebite envenomation is characterized by inflammatory events including increase in vascular permeability. A copious exudate is generated in tissue injected with venom, whose proteomics analysis has provided insights into the mechanisms of venom-induced tissue damage. Hereby it is reported that wound exudate itself has the ability to induce increase in vascular permeability in the skin of mice. Proteomics analysis of exudate revealed the presence of cytokines and chemokines, together with abundant damage associated molecular pattern molecules (DAMPs) resulting from both proteolysis of extracellular matrix and cellular lysis. Moreover, significant differences in the amounts of cytokines/chemokines and DAMPs were detected between exudates collected 1 h and 24 h after envenomation, thus highlighting a complex temporal dynamic in the composition of exudate. Pretreatment of mice with Eritoran, an antagonist of Toll-like receptor 4 (TLR4), significantly reduced the exudate-induced increase in vascular permeability, thus suggesting that DAMPs might be acting through this receptor. It is hypothesized that an “Envenomation-induced DAMPs cycle of tissue damage” may be operating in viperid snakebite envenomation through which venom-induced tissue damage generates a variety of DAMPs which may further expand tissue alterations.

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).

Snake venom metalloproteinases (SVMPs) affect the extracellular matrix (ECM) in multiple and complex ways. Previously, the combination of various methodological platforms, including electron microscopy, histochemistry, immunohistochemistry, and Western blot, has allowed a partial understanding of such complex pathology. In recent years, the proteomics analysis of exudates collected in the vicinity of tissues affected by SVMPs has provided novel and exciting information on SVMP-induced ECM alterations. The presence of fragments of an array of ECM proteins, including those of the basement membrane, has revealed a complex pathological scenario caused by the direct action of SVMPs. In addition, the time-course analysis of these changes has underscored that degradation of some fibrillar collagens is likely to depend on the action of endogenous proteinases, such as matrix metalloproteinases (MMPs), synthesized as a consequence of the inflammatory process. The action of SVMPs on the ECM also results in the release of ECM-derived biologically-active peptides that exert diverse actions in the tissue, some of which might be associated with reparative events or with further tissue damage. The study of the effects of SVMP on the ECM is an open field of research which may bring a renewed understanding of snake venom-induced pathology.

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)

The precise mechanisms by which Snake Venom Metalloproteinases (SVMPs) disrupt the microvasculature and cause haemorrhage have not been completely elucidated, and novel in vivo models are needed. In the present study, we compared the effects induced by BaP1, a PI SVMP isolated from Bothrops asper venom, and CsH1, a PIII SVMP from Crotalus simus venom, on cremaster muscle microvasculature by topical application of the toxins on isolated tissue (i.e., ex vivo model), and by intra-scrotal administration of the toxins (i.e., in vivo model). The whole tissue was fixed and immunostained to visualize the three components of blood vessels by confocal microscopy. In the ex vivo model, BaP1 was able to degrade type IV collagen and laminin from the BM of microvessels. Moreover, both SVMPs degraded type IV collagen from the BM in capillaries to a higher extent than in PCV and arterioles. CsH1 had a stronger effect on type IV collagen than BaP1. In the in vivo model, the effect of BaP1 on type IV collagen was widespread to the BM of arterioles and PCV. On the other hand, BaP1 was able to disrupt the endothelial barrier in PCV and to increase vascular permeability. Moreover, this toxin increased the size of gaps between pericytes in PCV and created new gaps between smooth muscle cells in arterioles in ex vivo conditions. These effects were not observed in the case of CsH1. In conclusion, our findings demonstrate that both SVMPs degrade type IV collagen from the BM in capillaries in vivo. Moreover, while the action of CsH1 is more directed to the BM of microvessels, the effects of BaP1 are widespread to other microvascular components. This study provides new insights in the mechanism of haemorrhage and other pathological effects induced by these toxins.

2082-01 Embargo por política editorial 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(ab0)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(ab0)2 antivenom when administered after envenomation, probably as a consequence of differences in their pharmacokinetic profiles. Universidad de Costa Rica/[741-A9-003]/UCR/Costa Rica Universidad de Costa Rica/[741-B0-528]/UCR/Costa Rica Network for Research and Training in Tropical Diseases in Central America /[01-N-2010]/NeTropica/ UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Tissue damage analysis by traditional laboratory techniques is problematic. Proteomic analysis of exudates collected from affected tissue constitutes a powerful approach to assess tissue alterations, since biomarkers associated with pathologies can be identified in very low concentrations. In this study we proteomically explore the pathological effects induced by the venom of the viperid snake Bothrops asper in the gastrocnemius muscle of mice. Predominant proteins identified in the exudates included intracellular proteins, plasma proteins, extracellular matrix proteins and cell membrane-associated proteins. The presence of such proteins indicates cytotoxicity, plasma exudation, extracellular matrix degradation and shedding of membrane proteins. Some of these proteins may represent useful biomarkers for myonecrosis and microvascular damage. The effect of fucoidan, an inhibitor of myotoxic phospholipases A2, and batimastat, an inhibitor of metalloproteinases, on the pathological effects induced by B. asper venom were also investigated. Fucoidan reduced the presence of intracellular proteins in exudates, whereas batimastat reduced the amount of relevant extracellular matrix proteins. The combination of these inhibitors resulted in the abrogation of the most relevant pathological effects of this venom. Thus, proteomic analysis of exudates represents a valuable approach to assess the characteristics of tissue damage in pathological models and the success of therapeutic interventions. Universidad de Costa Rica/[741-A7-604]/UCR/Costa Rica Universidad de Costa Rica/[741-A7-502]/UCR/Costa Rica Universidad de Costa Rica/[741-B0-606]/UCR/Costa Rica International Foundation for Science//IFS/Suecia Organization for the Prohibition of Chemical Weapons/[F/4096-1]/OPCW/Países Bajos Network for Research and Training in Tropical Diseases in Central America/[2-N-2008]/NeTropica/Suecia Network for Research and Training in Tropical Diseases in Central America/[01-N-2010]/NeTropica/Suecia University of Virginia///Estados Unidos UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Skeletal muscle regeneration after myonecrosis involves the activation, proliferation and fusion of myogenic cells, and a coordinated inflammatory response encompassing phagocytosis of necrotic cell debris, and the concerted synthesis of cytokines and growth factors. Myonecrosis often occurs in snakebite envenomings. In the case of venoms that cause myotoxicity without affecting the vasculature, such as those of many elapid snakes, regeneration proceeds successfully. In contrast, in envenomings by most viperid snakes, which affect the vasculature and extracellular matrix in addition to muscle fibers, regeneration is largely impaired and, therefore, the muscle mass is reduced and replaced by fibro-adipose tissue. This review discusses possible causes for such poor regenerative outcome including: (a) damage to muscle microvasculature, which causes tissue hypoxia and affects the inflammatory response and the timely removal of necrotic tissue; (b) damage to intramuscular nerves, which results in atrophy of regenerating fibers; (c) degradation of muscle cell basement membrane, compromising the spatial niche for proliferating myoblasts; (d) widespread degradation of the extracellular matrix; and (e) persistence of venom components in the damaged tissue, which may affect myogenic cells at critical points in the regenerative process. Understanding the causes of poor muscle regeneration may pave the way for the development of novel therapeutic interventions aimed at fostering the regenerative process in envenomed patients.

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)

20 páginas, 4 figuras, 3 tablas y 7 tablas en material suplementario., Snake venom hemorrhagic metalloproteinases (SVMPs) of the PI, PII and PIII classes were compared in terms of tissue localization and their ability to hydrolyze basement membrane components in vivo, as well as by a proteomics analysis of exudates collected in tissue injected with these enzymes. Immunohistochemical analyses of co-localization of these SVMPs with type IV collagen revealed that PII and PIII enzymes co-localized with type IV collagen in capillaries, arterioles and post-capillary venules to a higher extent than PI SVMP, which showed a more widespread distribution in the tissue. The patterns of hydrolysis by these three SVMPs of laminin, type VI collagen and nidogen in vivo greatly differ, whereas the three enzymes showed a similar pattern of degradation of type IV collagen, supporting the concept that hydrolysis of this component is critical for the destabilization of microvessel structure leading to hemorrhage. Proteomic analysis of wound exudate revealed similarities and differences between the action of the three SVMPs. Higher extent of proteolysis was observed for the PI enzyme regarding several extracellular matrix components and fibrinogen, whereas exudates from mice injected with PII and PIII SVMPs had higher amounts of some intracellular proteins. Our results provide novel clues for understanding the mechanisms by which SVMPs induce damage to the microvasculature and generate hemorrhage., This work was performed in partial fulfillment of the requirements for the PhD degree for Cristina Herrera at Universidad de Costa Rica.

The hemorrhagic activity characteristic of viperid snake envenomations is due to the action of venom metalloproteinases (SVMPs) on the capillary vessel basement membrane (BM). This study compared the action of two SVMPs on BM in vitro (degradation of Matrigel) and in vivo (immunohistochemical assessment of BM markers in mouse gastrocnemius muscle). SVMPs BaP1 (belonging to the P-I class) and jararhagin (of the P-III class) had a similar proteolytic activity on azocasein and degraded Matrigel with a slightly different cleavage pattern, since BaP1 exerted a limited proteolysis of both laminin and nidogen, whereas jararhagin predominantly degraded nidogen. In contrast with this pattern of limited proteolysis of BM proteins observed in vitro, immunohistochemical analysis of laminin, nidogen and type IV collagen, as well as of the endothelial cell marker VEGFR-2, in the hemorrhagic areas in the muscle, revealed a pronounced reduction in the immunostaining of these three BM components, associated with a loss of the endothelial cell marker. BM of muscle fibers was affected to a lesser extent. In conclusion, in vitro results demonstrated that SVMPs induce a pattern of limited proteolysis on BM components. The drastic loss of these antigens in affected capillaries in vivo is likely to depend on the combination of limited proteolysis of BM and the action of hemodynamic biophysical forces, previously shown to play a role in SVMP-induced capillary damage, which may cause a mechanical disruption of BM structure. Universidad de Costa Rica/[741-A4-061]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Envenomations by the snake Bothrops asper are characterized by prominent local tissue damage (i.e. myonecrosis), blistering, hemorrhage and edema. Various phospholipases A2 and metalloproteinases that induce local pathological alterations have been purified from this venom. Since these toxins induce a conspicuous inflammatory response, it has been hypothesized that inflammatory mediators may contribute to the local pathological alterations described. This study evaluated the local production of cytokines and matrix metalloproteinases (MMPs) as a consequence of intramuscular injections of an Asp-49 myotoxic phospholipase A2 (myotoxin III (MT-III)) and a P-I type hemorrhagic metalloproteinase (BaP1) isolated from B. asper venom. Both enzymes induced prominent tissue alterations and conspicuous increments in interleukin (IL)-1beta, IL-6 and a number of MMPs, especially gelatinase MMP-9, rapidly after injection. In contrast, no increments in tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma were detected. In agreement, MT-III and BaP1 did not induce the synthesis of TNF-alpha by resident peritoneal macrophages in vitro. Despite the conspicuous expression of latent forms of MMPs in muscle, evidenced by zymography, there were no increments in activated MMP-2 and only a small increase in activated MMP-9, as detected by a functional enzymatic assay. This suggests that MMP activity was regulated by a highly controlled activation of latent forms and, probably, by a concomitant synthesis of MMP inhibitors. Since no hemorrhage nor dermonecrosis were observed after injection of MT-III, despite a prominent increase in MMP expression, and since inflammatory exudate did not enhance hemorrhage induced by BaP1, it is suggested that endogenous MMPs released in the tissue are not responsible for the dermonecrosis and hemorrhage characteristic of B. asper envenomation. Moreover, pretreatment of mice with the peptidomimetic MMP inhibitor batimastat did not reduce myotoxic nor edema-forming activities of MT-III, suggesting that MMPs do not play a prominent role in the pathogenesis of these effects in this experimental model. It is concluded that MT-III and BaP1 induce a local inflammatory response associated with the synthesis of IL-1beta, IL-6 and MMPs. MMPs do not seem to play a prominent role in the acute local pathological alterations induced by these toxins in this experimental model. International Foundation for Science/[F/2707-2]/IFS/Suecia Network for Research and Training in Tropical Diseases in Central America//NeTropica/ Universidad de costa Roca/[741-A1-529]/UCR/Costa Rica Universidad de costa Roca/[741-A2-036]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

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 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)

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)

artículo (arbitrado) -- Universidad de Costa Rica, Instituto de investigaciones Clodomiro Picado. 2011 Hemorrhage is a clinically important manifestation of viperid snakebite envenomings, and is induced by snake venom metalloproteinases (SVMPs). Hemorrhagic and non-hemorrhagic SVMPs hydrolyze some basement membrane (BM) and associated extracellular matrix (ECM) proteins. Nevertheless, only hemorrhagic SVMPs are able to disrupt microvessels; the mechanisms behind this functional difference remain largely unknown. We compared the proteolytic activity of the hemorrhagic P-I SVMP BaP1, from the venom of Bothrops asper, and the non-hemorrhagic P-I SVMP leucurolysin-a (leuc-a), from the venom of Bothrops leucurus, on several substrates in vitro and in vivo, focusing on BM proteins. When incubated with Matrigel, a soluble extract of BM, both enzymes hydrolyzed laminin, nidogen and perlecan, albeit BaP1 did it at a faster rate. Type IV collagen was readily digested by BaP1 while leuc-a only induced a slight hydrolysis. Degradation of BM proteins in vivo was studied in mouse gastrocnemius muscle. Western blot analysis of muscle tissue homogenates showed a similar degradation of laminin chains by both enzymes, whereas nidogen was cleaved to a higher extent by BaP1, and perlecan and type IV collagen were readily digested by BaP1 but not by leuc-a. Immunohistochemistry of muscle tissue samples showed a decrease in the immunostaining of type IV collagen after injection of BaP1, but not by leuc-a. Proteomic analysis by LC/MS/MS of exudates collected from injected muscle revealed higher amounts of perlecan, and types VI and XV collagens, in exudates from BaP1-injected tissue. The differences in the hemorrhagic activity of these SVMPs could be explained by their variable ability to degrade key BM and associated ECM substrates in vivo, particularly perlecan and several non-fibrillar collagens, which play a mechanical stabilizing role in microvessel structure. These results underscore the key role played by these ECM components in the mechanical stability of microvessels. Universidad de Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Both serine and metalloproteinases have been shown to play the role of toxins in the venoms of many snakes. Determination of the natural protein substrates of these toxins is an important feature in the toxinological characterization of these proteinases. Furthermore, characterization of their peptide bond specificity is of value for understanding active site preference of the proteinase associated with effective proteolysis as well as of use in the design of peptide substrates and inhibitor lead compounds. Typically the determination of peptide bond cleavage specificity of snake venom serine proteinases (SVSPs) and snake venom metalloproteinases (SVMPs) has been performed using limited sets of peptides or small oligopeptides as experimental substrates. Although this approach has yielded valuable data it is generally limited in scope due to the relatively small sets of substrates used to generate the consensus specificity sequences for these proteinases. In this study we use a large, plasma based, proteome-derived peptide library as substrates along with mass spectrometry to explore the peptide bond specificity of three PI SVMPs and one PIII SVMP to determine their individual peptide cleavage consensus sequences. All of the proteinases assayed displayed a clear preference for a leucine residue in the P1′ site. Careful analysis of the specificity profiles of the SVMPs examined showed interesting differences in the preferences at the other P and P′ sites suggesting functional differences between these proteinases. The PI SVMPs, leucurolysin-a, atrolysin C, and BaP1, showed preferences across the full P4 to P4′ range whereas the PIII SVMP bothropasin showed a narrower range of preferences across the sites. In silico docking experiments with the experimentally derived consensus sequences as well as with comparison of the results to those in the literature regarding peptide bond specificity based on both peptide and protein substrates give rise to a fresh understanding of the specificity of these SVMPS and may serve as a foundation for future experiments to better elucidate their mechanism of action in the complex pathophysiology of snakebite envenomation. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

The study of snake venom-induced local tissue pathology has been predominantly based on experimental designs and concepts that view tissue alterations from a dichotomous, homogeneous and reductionist perspective. Experimental observations, however, indicate that cells and extracellular matrix are affected by venoms and toxins in a complex and heterogeneous fashion, and that the effect of whole venoms in whole organisms cannot be properly assessed by studying only the action of isolated toxins in isolated systems. It is proposed that a novel conceptual and methodological frame is necessary to better understand the complexity of these pathological alterations from a holistic, systems biology perspective. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

In light of the complexity of wound tissue, proteomic analysis may not clearly reveal the nature of the wound or the processes involved in healing. However, exudate associated with wounds may provide a “window” on cellular events leading to the development of the wound and/or its healing. In this investigation we performed proteomic analysis on wound exudates from muscular wounds in mice caused by two very different types of snake venom toxins: BaP1, a snake venom metalloproteinase and Mtx-I, a snake venom phospholipase A2. Proteomic analysis of the exudates associated with these wounds clearly differentiated them and offered new perspectives on functional mechanisms by which these toxins cause tissue damage. In the case of wounds caused by the metalloproteinase, there was evidence of degradation of nonfibrillar collagens whereas the phospholipase wound exudate was noted by the presence of fibrillar collagen type I, apolipoproteins A-I, A-IV, and E, and fibronectin. These results suggest that the hemorrhage caused by snake venom metalloproteinases may be associated with the degradation of specific extracellular matrix proteins which play a role in matrix/capillary stabilization and that release of apolipoproteins from their complexes may be involved with the dysfunctional hemostatsis observed following snake envenoming. Universidad de Costa Rica/[741-A7-502]/UCR/Costa Rica Universidad de Costa Rica/[741-A7-604]/UCR/Costa Rica International Foundation for Science/[F/4096-1]/IFS/Suecia Network for Research and Training in Tropical Diseases in Central America/[2-N-2008]/NeTropica/Suecia University of Virginia School of Medicine///Estados Unidos UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

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 A2 (PLA2s). SVMPs induce hemorrhage, blistering, dermonecrosis and general extracellular matrix degradation, whereas PLA2s 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 PLA2s 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. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

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

To assess the indirect effects of snake venom metalloproteinases (SVMP) on host tissue local necrosis, we investigated the effect of the SVMP jararhagin on the gene expression profiles of human fibroblasts in vitro and mouse tissue in vivo. Two functional classes of up-regulated proteins, cell death and inflammatory disease were identified as being significantly populated. The changes in gene expression observed by qRT-PCR on laser microdissected mouse muscle tissue treated with jararhagin were similar with significant up-regulation of proinflammatory transcripts such as IL-1β, IL-6, CXCL1, CXCL2, IL-8, and apoptosis, inflammation responsive transcripts such as TNF-α induced protein 6. Proteolytically inactive jararhagin had no effect on the gene expression profile of fibroblasts, indicating proteolysis as the primary mechanism affecting gene expression of cells and tissues resulting in a proinflammatory, pro-apoptotic host response which likely exacerbates the local necrosis frequently observed at the site of envenoming. University of Cologne/[10/2004]//Alemania University of Cologne/[SFB 589]//Alemania UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

The effects of blood flow interruption on the ultrastructural alterations induced by a snake venom hemorrhagic metalloproteinase on skeletal muscle capillary endothelial cells were studied. Saline solution or the metalloproteinase BaP1, from the venom of Bothrops asper, was injected into the gastrocnemius muscles of mice with normal blood flow perfusion or with blood supply abrogated by two different protocols. Tissue was collected 5 min after injection, and both histological and ultrastructural analyses of the muscle capillary vessels were performed. Muscle with normal perfusion injected with saline solution had the typical morphology of normal capillaries, whereas injection of metalloproteinase to muscle with normal blood flow induced prominent degenerative changes in endothelial cells, such as reduction in cell thickness, decrease in the amount of pinocytotic vesicles, prominent distention and rupture leading to extravasation. In contrast, endothelial cells of capillaries from tissue devoid of blood flow and injected with the metalloproteinase did not show degenerative changes. The only alterations observed were a reduction in the capillary lumen and the presence of cytoplasmic projections, or 'pseudopods', both of which were also present in capillaries from tissue devoid of blood flow and injected with saline solution, thus suggesting that such changes are due to the drop in transmural pressure as a consequence of blood flow interruption. Our observations support the hypothesis that biophysical forces operating in the microvasculature, i.e., transmural pressure-dependent wall tension and shear stress, play a significant role in the pathogenesis of endothelial cell damage and hemorrhage induced by snake venom metalloproteinases. International Foundation for Science/[F-2707-3]/IFS/Suecia Universidad de Costa Rica/[741-A2-036]/UCR/Costa Rica 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)

Zinc-dependent metalloproteinases are responsible for the hemorrhagic activity characteristic of viperid snake venoms. Snake venom metalloproteinases (SVMPs) are classified in various groups (P-I–IV), according to their domain composition. P-III SVMPs, comprising metalloproteinase, disintegrin-like and cysteine-rich domains, exert more potent hemorrhagic activity than P-I SVMPs, which present only the metalloproteinase domain. SVMPs degrade various components of the basement membrane and are also able to hydrolyze endothelial cell membrane proteins, such as integrins and cadherins, involved in cell–matrix and cell–cell adhesion. In addition, disintegrin-like and cysteine-rich domains interact with endothelial cell integrins, interfering with their adhesion to extracellular matrix. Hemorrhage induced by SVMPs is an extremely rapid event in vivo, with capillary endothelial cells showing drastic structural alterations within few minutes. In contrast, observations in cell culture conditions do not evidence such rapid endothelial cell damage. Instead, the main effect is detachment and rounding of these cells; it is only after several hours of incubation that cells show evidence of apoptotic damage. This apparent discrepancy between in vivo and in vitro observations can be explained if biophysical forces operating on microvessels in vivo are taken into consideration. It is proposed that SVMP-induced hemorrhage occurs in vivo by a ‘two-step’ mechanism. Initially, SVMPs degrade basement membrane and adhesion proteins, thus weakening the capillary wall and perturbing the interactions between endothelial cells and the basement membrane. Then, transmural pressure acting on the weakened capillary wall causes distention. As a consequence, endothelial cells become very thin, until the integrity of the capillary wall is lost at some points, where extravasation occurs. In addition, endothelial cells become more susceptible to blood flow-dependent shear stress, which further contributes to capillary wall disruption. Universidad de Costa Rica//UCR/Cota Rica Consejo Nacional para Investigaciones Científicas y Tecnológicas//CONICIT/Costa Rica International Foundation for Science//IFS/Suecia Wellcome Trust///Inglaterra //AID/ UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

The ability of the P-I metalloproteinase BaP1, isolated from the venom of the snake Bothrops asper, to induce systemic bleeding, thrombocytopenia and defibrinogenation was assessed in an experimental mouse model. Intravenous administration of BaP1 caused neither systemic bleeding nor any evidence of pathology in lungs, kidneys, liver, heart and brain. Moreover, there were no alterations in the whole blood clotting time or in platelet numbers. In addition, BaP1 did not inhibit collagen-induced platelet aggregation in vitro. Proteolytic and hemorrhagic activities of BaP1 were readily inhibited by the plasma proteinase inhibitor, α2-macroglobulin, and normal mouse serum also inhibited hemorrhage. Such inhibition may explain why BaP1 induces multiple local tissue-damaging effects, but is largely devoid of systemic toxicity. Wellcome Trust([062043]/Inglaterra UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Jararhagin is the most important hemorrhagic component in the venom of the snake Bothrops jararaca, a species of medical importance in South America. It is a P-III zinc-dependent metalloproteinase comprising catalytic, disintegrin-like, and cysteine-rich domains. Jararhagin injected intravenously into mice induced rapid and prominent bleeding in the lungs, whereas other organs were devoid of overt hemorrhagic manifestations. This action depends on the proteolytic activity of jararhagin, since it was abrogated by the synthetic inhibitor batimastat. There were conspicuous ultrastructural alterations in cells at the alveolo-capillary unit, i.e., capillary endothelial cells and type I pneumocytes, with a characteristic pattern of “regional alveolar damage” associated with extravasation. These pathological effects were observed under conditions in which the whole blood clotting time, bleeding time, and fibrinogen levels were not affected. 125I-labeled jararhagin is concentrated mainly in liver and kidneys after iv injection, with little radioactivity observed in the lungs, thereby indicating that the predominance of pulmonary microvascular damage is not due to a preferential concentration of this enzyme in the lungs. Despite the fact that jararhagin is complexed by plasma proteins after iv injection, its hemorrhagic activity was not inhibited by the plasma proteinase inhibitor α2-macroglobulin, and was only partially reduced by normal mouse serum, suggesting that resistance to inhibition may contribute to its ability to cause pulmonary hemorrhage. Wellcome Trust/[062043]//Inglaterra International Foundation for Science/[F-2707-2]/IFS/Suecia Universidad de Costa Rica/[741-A1-504]/UCR/Costa Rica Universidad de Costa Rica/[741-A2-036]/UCR/Costa Rica Fundação de Amparo à Pesquisa do Estado de São Paulo//FAPESP/Brasil 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)

The effectiveness of the chelating agent CaNa2EDTA and the peptidomimetic matrix metalloproteinase inhibitor batimastat (BB-94) to inhibit local tissue damage induced by Bothrops asper snake venom was studied in mice. Both compounds totally inhibited proteolytic, hemorrhagic, and dermonecrotic effects, and partially reduced edema-forming activity, when incubated with venom prior to injection. Much lower concentrations of batimastat than of CaNa2EDTA were required to inhibit these effects. In addition, batimastat, but not CaNa2EDTA, partially reduced myotoxic activity of the venom. When the inhibitors were administered at various time intervals after envenomation at the same site of venom injection, both compounds were effective in neutralizing local hemorrhage and dermonecrosis if administered rapidly after venom. Inhibition was not as effective as the time lapse between venom and inhibitor injections increased. Owing to the relevance of metalloproteinases in the pathogenesis of local tissue damage induced by B. asper and other pit viper venoms, it is suggested that administration of peptidomimetic metalloproteinase inhibitors or CaNa2EDTA at the site of venom injection may represent a useful alternative to complement antivenoms in the neutralization of venom-induced local tissue damage. Universidad de Costa Rica/[741-98-202]/UCR/Costa Rica Universidad de Costa Rica/[741-99-220]/UCR/Costa Rica International Foundation for Science/[F/2707-1]/IFS/Suecia UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)