Search

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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Envenomations by the snake Bothrops asper are characterized by prominent local tissue damage (i.e. myonecrosis), blistering, hemorrhage and edema. Various phospholipases A[sub 2] 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 A[sub 2] (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)-1β, IL-6 and a number of MMPs, especially gelatinase MMP-9, rapidly after injection. In contrast, no increments in tumor necrosis factor-α (TNF-α) and interferon-γ were detected. In agreement, MT-III and BaP1 did not induce the synthesis of TNF-α 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-1β, 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. [ABSTRACT FROM AUTHOR]

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. aspervenom and its purified toxins - phospholipases and metalloproteinase - activate the complement system and the contribution of the effect on leucocyte recruitment. In vitrochemotaxis 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 vitrohaemolytic assay. The importance of complement activation by the venom on neutrophil migration was investigated in vivoby 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 vivoleucocyte 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. [ABSTRACT FROM AUTHOR]

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' 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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • A P-III hemorrhagic metalloproteinase from Crotalus simus venom induces hemorrhage of rapid onset in the lungs in mice. • There is enzymatic degradation of pulmonary basement membrane components as soon as 15 min after toxin injection. • An inflammatory reaction develops in the lungs as a consequence of the action of CsH1. • The administration of indomethacin, pentoxifylline, and anti-neutrophil antibodies reduced the extent of hemorrhage. • Hemorrhage results from a direct action of CsH1 in the pulmonary vasculature and from the effect of inflammation. [ABSTRACT FROM AUTHOR]

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. Image 1 • Snake venom and myotoxin induced muscle fiber necrosis and basement membrane degradation. • Myotoxin induced a faster and higher upregulation of basement membrane proteins. • Venom induced a higher degradation and incomplete recovery of type IV collagen. • Deficient regeneration is associated to a higher degradation and lower synthesis of basement membrane proteins. [ABSTRACT FROM AUTHOR]

Inflammatory joint conditions are characterized by synovial inflammation, which involves activation of fibroblast-like synoviocytes (FLSs) and production of inflammatory mediators and matrix metalloproteases (MMPs) in joints. This study showed that the snake venom metalloprotease (SVMP) BaP1 activates FLSs to produce PGE2 by a mechanism dependent on COX-2, mPGES-1 and iPLA2s. BaP1 also induces IL-1β release, which up-regulates the production of PGE2 at a late stage of the stimulation. Expression of COX-2 and mPGES-1 are induced by BaP1 via activation of NF-κB pathway. While NF-κB p50 and p65 subunits are involved in up-regulation of COX-2 expression, only p65 is involved in BaP1-induced mPGES-1 expression. In addition, BaP1 up-regulates EP4 receptor expression. Engagement of this receptor by PGE2 triggers a positive feedback loop for its production by up-regulating expression of key components of the PGE2 biosynthetic cascade (COX-2, mPGES-1 and the EP4 receptor), thus contributing to amplification of BaP1-induced effects in FLSs. These data highlight the importance of FLS as a target for metalloproteases in joint inflammation and provide new insights into the roles of MMPs in inflammatory joint diseases. Moreover, our results may give insights into the importance of the catalytic domain, of MMPs for the inflammatory activity of these enzymes. [ABSTRACT FROM AUTHOR]

Venom-induced consumption coagulopathy and thrombocytopenia are common and potentially severe manifestations of viperid snakebite envenoming since they contribute to local and systemic hemorrhage. Therefore, the assessment of the efficacy of antivenoms to neutralize coagulopathic and thrombocytopenic toxins should be part of the preclinical evaluation of these drugs. To evaluate the efficacy of the polyvalent (Crotalinae) antivenom produced in Costa Rica, in this study we have used a mouse model of coagulopathy and thrombocytopenia induced by the venom of Bothrops asper , based on the bolus intravenous (i.v.) injection of venom. When venom and antivenom were incubated before injection, or when antivenom was administered i.v. immediately after venom injection, venom-induced hemostatic alterations were largely abrogated. We also studied the recovery rate of clotting parameters in conditions where antivenom was administered when mice were coagulopathic. Some parameters recovered more rapidly in antivenom-treated mice than in control envenomed animals, but others showed a spontaneous recovery without antivenom. This is due to a rapid clearance of plasma venom levels in these experimental conditions. This implies that models based on the bolus i.v. injection of venom have limitations for assessing the effect of antivenom in the recovery of clotting alterations once coagulopathy has developed. It is suggested that alternative models should be developed based on a slower systemic absorption of venom. Overall, our findings provide a protocol for the preclinical evaluation of antivenoms and demonstrate that the polyvalent antivenom is effective in neutralizing the toxins of B. asper venom responsible for coagulopathy and thrombocytopenia. [Display omitted] • The efficacy of a polyvalent antivenom to neutralize Bothrops asper venom coagulopathic effects was studied. • Venom affected classical coagulation tests, rotational thromboelastometry, and platelet counts. • When preincubated with the venom, the antivenom neutralized venom-induced coagulopathy and thrombocytopenia. • When administered i.v. immediately after venom injection, antivenom abrogated coagulopathy and thrombocytopenia. • Some clotting parameters recovered more rapidly in mice receiving antivenom than in control envenomed mice. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

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 50), determined by the intraperitoneal route, was significantly lower in thrombocytopenic mice, underscoring a higher toxicity of venom in these conditions. No difference in the value of LD 50 between the two groups was observed when using the intravenous route of injection, and no difference was observed in the magnitude and time-course of footpad edema. Skeletal muscle regeneration was assessed 14 days after venom injection in muscle. Both experimental groups showed a similarly poor regeneration, suggesting that platelets do not play a key role in the regenerative process in these experimental conditions. Results indicate that depletion of platelets increases hemorrhagic and myotoxic effects, as well as overall toxicity, of B. asper venom, implying that platelets play a protective hemostatic role in this model of envenoming. [Display omitted] • The impact of thrombocytopenia in Bothrops asper envenoming was studied in mice. • Thrombocytopenic mice developed stronger hemorrhagic and myotoxic effects than envenomed controls. • Thrombocytopenia increased the lethality of venom by the i.p. route but not by the i.v. route. • Thrombocytopenia did not modify the magnitude and time-course of venom-induced edema. • Thrombocytopenia did not modify the impaired muscle regeneration after venom-induced myonecrosis. [ABSTRACT FROM AUTHOR]

Background: The venom of Bothrops lanceolatus, a viperid species endemic to the Lesser Antillean Island of Martinique, induces thrombosis in a number of patients. Previous clinical observations indicate that thrombotic events are more common in patients bitten by juvenile specimens. There is a need to develop an experimental model of this effect in order to study the mechanisms involved., Methodology/principal Findings: The venoms of juvenile and adult specimens of B. lanceolatus were compared by (a) describing their proteome, (b) assessing their ability to induce thrombosis in a mouse model, and (c) evaluating their in vitro procoagulant activity and in vivo hemostasis alterations. Venom proteomes of juvenile and adult specimens were highly similar, albeit showing some differences. When injected by the intraperitoneal (i.p.) route, the venom of juvenile specimens induced the formation of abundant thrombi in the pulmonary vasculature, whereas this effect was less frequent in the case of adult venom. Thrombosis was not abrogated by the metalloproteinase inhibitor Batimastat. Both venoms showed a weak in vitro procoagulant effect on citrated mouse plasma and bovine fibrinogen. When administered intravenously (i.v.) venoms did not affect classical clotting tests (prothrombin time and activated partial thromboplastin time) but caused a partial drop in fibrinogen concentration. The venom of juvenile specimens induced partial alterations in some rotational thromboelastometry parameters after i.v. injection. When venoms were administered i.p., only minor alterations in classical clotting tests were observed with juvenile venom, and no changes occurred for either venom in rotational thromboelastometry parameters. Both juvenile and adult venoms induced a marked thrombocytopenia after i.p. injection., Conclusions/significance: An experimental model of the thrombotic effect induced by B. lanceolatus venom was developed. This effect is more pronounced in the case of venom of juvenile specimens, despite the observation that juvenile and adult venom proteomes are similar. Adult and juvenile venoms do not induce a consumption coagulopathy characteristic of other Bothrops sp venoms. Both venoms induce a conspicuous thrombocytopenia. This experimental model reproduces the main clinical findings described in these envenomings and should be useful to understand the mechanisms of the thrombotic effect., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rucavado et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Viperid snake venoms induce severe tissue damage, characterized by the direct toxic action of venom components, i.e., phospholipases A 2 (PLA 2 s) and metalloproteinases (SVMPs), concomitantly with the onset of endogenous inflammatory processes, in an intricate scenario of tissue alterations. Understanding the expression of relevant genes in muscle tissue will provide valuable insights into the undergoing pathological and inflammatory processes. In this study, we have used the Nanostring technology to evaluate the patterns of gene expression in mouse skeletal muscle 1 h, 6 h, and 24 h after injection of the venoms of Bothrops asper and Daboia russelii , two medically relevant species in Latin America and Asia, respectively, with somewhat different clinical manifestations. The dose of venoms injected (30 µg) induced local pathological effects and inflammation in muscle tissue. We focused our analysis on genes related to extracellular matrix (ECM) metabolism, immune system, programmed cell death, and autophagy. The results revealed a complex pattern of expression of genes. Regarding ECM metabolism and regulation, up-regulated genes included proteinase inhibitor Serpine 1, thrombospondin 1, collagens 1A1 and 4A1 (at 1 h in the case of B. asper ), TIMP1, MMP-3 (at 24 h), and lysil oxidase (LOX). In contrast, collagen chains 5A3 and 5A1 were down-regulated, especially at 6 h. Transforming growth factor β (TGF-β) and several genes related to myofibroblast regulation were also up-regulated, which might be related to the development of fibrosis. Several genes related to cytokine and chemokine synthesis and regulation and NFκB signaling were also up-regulated. Our observations show a variable expression of genes associated with programmed cell death and autophagy, thus revealing a hitherto unknown role of autophagy in tissue affected by snake venoms. These results provide clues to understanding the complex pattern of gene expression in tissue affected by viperid snake venoms, which likely impacts the final pathophysiology of damaged tissue in envenomings.

Envenomings by Russell's viper ( Daboia russelii ), a species of high medical importance in India and other Asian countries, commonly result in hemorrhage, coagulopathies, necrosis, and acute kidney injury. Although bleeding complications are frequently reported following viper envenomings, thrombotic events occur rarely (reported only in coronary and carotid arteries) with serious consequences. For the first time, we report three serious cases of peripheral arterial thrombosis following Russell's viper bites and their diagnostic, clinical management, and mechanistic insights. These patients developed occlusive thrombi in their peripheral arteries and symptoms despite antivenom treatment. In addition to clinical features, computed tomography angiography was used to diagnose arterial thrombosis and ascertain its precise locations. They were treated using thrombectomy or amputation in one case that presented with gangrenous digits. Mechanistic insights into the pathology through investigations revealed the procoagulant actions of Russell's viper venom in standard clotting tests as well as in rotational thromboelastometry analysis. Notably, Russell's viper venom inhibited agonist-induced platelet activation. The procoagulant effects of Russell's viper venom were inhibited by a matrix metalloprotease inhibitor, marimastat, although a phospholipase A 2 inhibitor (varespladib) did not show any inhibitory effects. Russell's viper venom induced pulmonary thrombosis when injected intravenously in mice and thrombi in the microvasculature and affected skeletal muscle when administered locally. These data emphasize the significance of peripheral arterial thrombosis in snakebite victims and provide awareness, mechanisms, and robust strategies for clinicians to tackle this issue in patients., Competing Interests: Conflict of Interest None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. ( https://creativecommons.org/licenses/by/4.0/ ).)

Viperid snakebite envenoming is often characterized by a venom-induced consumption coagulopathy due to the procoagulant effect of venom components, resulting in the alteration of clotting laboratory tests. There is a growing trend to use rotational thromboelastometry in the assessment of clotting disturbances in a variety of pathologies, although its use in experimental models of envenoming has been limited. An in vivo murine model was implemented to assess the coagulopathy induced by three Central American viperid venoms which have different mechanisms of action on clotting factors, i.e., Bothrops asper, Crotalus simus and Bothriechis lateralis. Venom was injected by the intravenous route and blood samples were collected at 1, 3, 5 and 24 h after envenoming. Coagulopathy was assessed by standard clotting tests and by routine rotational thromboelastometric parameters. In addition, the changes in platelet number were followed. B. asper and C. simus venoms induced coagulopathy and thrombocytopenia 1 h after injection, followed by a slow recovery at 3, 5 and 24 h, although the majority of clotting parameters were still significantly affected by 3 and 5 h, and were corrected by 24 h. In general, a similar time-course of alterations was observed for standard clotting tests and most rotational thromboelastomeric assays. However, some thromboelastometric parameters, especially those related to Fibtem, showed more drastic alterations than standard tests and remained altered even at 24 h in some cases. This is likely related to the low fibrinogen concentration observed at most time intervals. B. lateralis venom did not induce a consumption coagulopathy, although it caused a marked thrombocytopenia., (Copyright © 2022 Elsevier Ltd. All rights reserved.)