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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. Author summary: Envenomings by the viperid species Bothrops lanceolatus, endemic of the Caribbean Island of Martinique, are characterized by a thrombotic effect responsible for infarcts in various organs. Until now, no experimental in vivo models of this effect have been described. In this study, we developed a mouse model of thrombosis by using the intraperitoneal route of venom injection. The venom of juvenile specimens of B. lanceolatus induced the formation of abundant thrombi in the lungs, whereas the effect was much less pronounced with the venom of adult specimens. This difference in the ability of juvenile and adult venoms occurs despite both venoms having highly similar proteomic profiles. Both adult and juvenile venoms showed a weak in vitro procoagulant effect on plasma and fibrinogen, underscoring a thrombin-like (pseudo-procoagulant) activity. In vivo, the venoms did not affect the classical clotting tests (prothrombin time and activated partial thromboplastin time) but induced a partial drop in fibrinogen concentration and limited alterations in rotational thromboelastometry parameters when injected by the i.v. route. In contrast, few alterations of these parameters were observed after i.p. injection of venoms, in conditions in which thrombosis occurred, hence evidencing the lack of a consumption coagulopathy. After i.p. injection both venoms induced a pronounced thrombocytopenia. This experimental model reproduces some of the main clinical manifestations of envenoming by this species. This model can be used to identify the toxins responsible for the thrombotic effect, to study the mechanism(s) of thrombosis and to assess the preclinical efficacy of antivenoms and novel therapies. [ABSTRACT FROM AUTHOR]

Viperid snake venoms induce severe tissue damage, characterized by the direct toxic action of venom components, i.e., phospholipases A2 (PLA2s) 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. [ABSTRACT FROM AUTHOR]

Pathological and inflammatory events in muscle after the injection of snake venoms vary in different regions of the affected tissue and at different time intervals. In order to study such heterogeneity in the immune cell microenvironment, a murine model of muscle necrosis based on the injection of the venom of Daboia russelii was used. Histological and immunohistochemical methods were utilized to identify areas in muscle tissue with a different extent of muscle cell damage, based on the presence of hypercontracted muscle cells, a landmark of necrosis, and on the immunostaining for desmin. A gradient of inflammatory cells (neutrophils and macrophages) was observed from heavily necrotic areas to less damaged and non-necrotic areas. GeoMx® Digital Spatial Profiler (NanoString, Seattle, WA, USA) was used for assessing the presence of markers of various immune cells by comparing high-desmin (nondamaged) and low-desmin (damaged) regions of muscle. Markers of monocytes, macrophages, M2 macrophages, dendritic cells, neutrophils, leukocyte adhesion and migration markers, and hematopoietic precursor cells showed higher levels in low-desmin regions, especially in samples collected 24 hr after venom injection, whereas several markers of lymphocytes did not. Moreover, apoptosis (BAD) and extracellular matrix (fibronectin) markers were also increased in low-desmin regions. Our findings reveal a hitherto-unknown picture of immune cell microheterogeneity in venom-injected muscle which greatly depends on the extent of muscle cell damage and the time lapse after venom injection. [ABSTRACT FROM AUTHOR]

Longitudinal metabolomics and lipidomics analyses were carried out on the blood plasma of mice injected intramuscularly with venoms of the viperid species Bothrops asper or Daboia russelii. Blood samples were collected 1, 3, 6, and 24 h after venom injection, and a control group of non-envenomed mice was included. Significant perturbations in metabolomics and lipidomics were observed at 1, 3, and 6 h, while values returned close to those of control mice by 24 h, hence reflecting a transient pattern of metabolic disturbance. Both venoms induced significant changes in amino acids, as well as in several purines and pyrimidines, and in some metabolites of the tricarboxylic acid cycle. KEGG analysis of metabolic pathways that showed those with the greatest change included aminoacyl tRNA synthesis and amino acid biosynthesis and metabolism pathways. With regard to lipid metabolism, there was an increase in triglycerides and some acyl carnitines and a concomitant drop in the levels of some phospholipids. In addition, envenomed mice had higher levels of cortisol, heme, and some oxidative stress markers. The overall pattern of metabolic changes in envenomed mice bears similarities with the patterns described in several traumatic injuries, thus underscoring a metabolic response/adaptation to the injurious action of the venoms. [ABSTRACT FROM AUTHOR]

Opossums in the tribe Didelphini are resistant to pit viper venoms and are hypothesized to be coevolving with venomous snakes. Specifically, a protein involved in blood clotting (von Willebrand factor [vWF] which is targeted by snake venom C-type lectins [CTLs]) has been found to undergo rapid adaptive evolution in Didelphini. Several unique amino acid changes in vWF could explain their resistance; however, experimental evidence that these changes disrupt binding to venom CTLs was lacking. Furthermore, without explicit testing of ancestral phenotypes to reveal the mode of evolution, the assertion that this system represents an example of coevolution rather than noncoevolutionary adaptation remains unsupported. Using expressed vWF proteins and purified venom CTLs, we quantified binding affinity for vWF proteins from all resistant taxa, their venom-sensitive relatives, and their ancestors. We show that CTL-resistant vWF is present in opossums outside clade Didelphini and likely across a wider swath of opossums (family Didelphidae) than previously thought. Ancestral reconstruction and in vitro testing of vWF phenotypes in a clade of rapidly evolving opossums reveal a pattern consistent with trench warfare coevolution between opossums and their venomous snake prey. [ABSTRACT FROM AUTHOR]