Clinical implications of ontogenetic differences in the coagulotoxic activity of Bothrops jararacussu venoms.

Is snake venom activity influenced by size? This is a long-standing question that can have important consequences for the treatment of snake envenomation. Ontogenetic shifts in venom composition are a well-documented characteristic of numerous snake species. Although snake venoms can cause a range of pathophysiological disturbances, establishing the coagulotoxic profiles related to such shifts is a justified approach because coagulotoxicity can be deadly, and its neutralisation is a challenge for current antivenom therapy. Thus, we aimed to assess the coagulotoxicity patterns on plasma and fibrinogen produced by B othrops jararacussu venoms from individuals of different sizes and sex, and the neutralisation potential of SAB (anti bothropic serum produced by Butantan Institute). The use of a metalloproteinase inhibitor (Prinomastat) and a serine proteinase inhibitor (AEBSF) enabled us to determine the toxin class responsible for the observed coagulopathy: activity on plasma was found to be metalloprotease driven, while the activity on fibrinogen is serine protease driven. To further explore differences in venom activity, the activation of Factor X and prothrombin as a function of snake size was also evaluated. All the venoms exhibited a potent procoagulant effect upon plasma and were less potent in their pseudo-procoagulant clotting effect upon fibrinogen. On human plasma, the venoms from smaller snakes produced more rapid clotting than the larger ones. In contrast, the venom activity on fibrinogen had no relation with size or sex. The difference in procoagulant potency was correlated with the bigger snakes being proportionally better neutralized by antivenom due to the lower levels of procoagulant toxins, than the smaller. Thus, while the antivenom ultimately neutralized the venoms, proportionally more would be needed for an equal mass of venom from a small snake than a large one. Similarly, the neutralisation by SAB of the pseudo-procoagulant clotting effects was also correlated with relative potency, with the smaller and bigger snakes being neutralized proportional to potency, but with no correlation to size. Thromboelastography (TEG) tests on human and toad plasma revealed that small snakes' venoms acted quicker than large snakes' venom on both plasmas, with the action upon amphibian plasma consistent with smaller snakes taking a larger proportion of anuran prey than adults. Altogether, the ontogenetic differences regarding coagulotoxic potency and corresponding impact upon relative antivenom neutralisation of snakes with different sizes were shown, underscoring the medical importance of investigating ontogenetic changes in order to provide data crucial for evidence-based design of clinical management strategies.

Clinical implications of ontogenetic differences in the coagulotoxic activity of Bothrops jararacussu venoms

Is snake venom activity influenced by size? This is a long-standing question that can have important consequences for the treatment of snake envenomation. Ontogenetic shifts in venom composition are a well-documented characteristic of numerous snake species. Although snake venoms can cause a range of pathophysiological disturbances, establishing the coagulotoxic profiles related to such shifts is a justified approach because coagulotoxicity can be deadly, and its neutralisation is a challenge for current antivenom therapy. Thus, we aimed to assess the coagulotoxicity patterns on plasma and fibrinogen produced by B. jararacussu venoms from individuals of different sizes and sex, and the neutralisation potential of SAB (anti bothropic serum produced by Butantan Institute). The use of a metalloproteinase inhibitor (Prinomastat) and a serine proteinase inhibitor (AEBSF) enabled us to determine the toxin class responsible for the observed coagulopathy: activity on plasma was found to be metalloprotease driven, while the activity on fibrinogen is serine protease driven. To further explore differences in venom activity, the activation of Factor X and prothrombin by as a function of snake size was also evaluated. All the venoms exhibited a potent procoagulant effect upon plasma and were less potent in their pseudo-procoagulant clotting effect upon fibrinogen. On human plasma, the venoms from smaller snakes produced more rapid clotting than the larger ones. In contrast, the venom activity on fibrinogen had no relation with size or sex. The difference in procoagulant potency was correlated with the bigger snakes being proportionally better neutralized by antivenom due to the lower levels of procoagulant toxins, than the smaller. Thus, while the antivenom ultimately neutralized the venoms, proportionally more would be needed for an equal mass of venom from a small snake than a large one. Similarly, the neutralisation by SAB of the pseudo-procoagulant clotting effects was also correlated with relative potency, with the smaller and bigger snakes being neutralized proportional to potency, but with no correlation to size. Thromboelastography (TEG) tests on human and toad plasma revealed that small snakes’ venoms acted quicker than large snakes’ venom on both plasmas, with the action upon amphibian plasma consistent with smaller snakes taking a larger proportion of anuran prey than adults. Altogether, the ontogenetic differences regarding coagulotoxic potency and corresponding impact upon relative antivenom neutralisation of snakes with different sizes were shown, underscores the medical importance of investigating ontogenetic changes in order to provide data crucial for evidence-based design of clinical management strategies.

Photobiomodulation reduces cell death and cytokine production in C2C12 cells exposed to Bothrops venoms.

Snakebites caused by the genus Bothrops are often associated with severe and complex local manifestations such as edema, pain, hemorrhage, and myonecrosis. Conventional treatment minimizes the systemic effects of venom; however, their local action is not neutralized. The purpose of this study was to evaluate the effect of photobiomodulation (PBM) on C2C12 muscle cells exposed to B. jararaca, B. jararacussu, and B. moojeni venoms on events involved in cell death and the release of inflammatory mediators. Cells were exposed to venoms and immediately irradiated with low-level laser (LLL) application in continuous wave at the wavelength of 660 nm, energy density of 4.4 J/cm2, power of 10 mW, area of 0.045 cm2, and time of 20 s. Cell integrity was analyzed by phase contrast microscope and cell death was performed by flow cytometry. In addition, interleukin IL1-ß, IL-6, and IL-10 levels were measured in the supernatant. Our results showed that the application of PBM increases cell viability and decreases cell death by apoptosis and necrosis. Moreover, the release of pro-inflammatory interleukins was also reduced. The data reported here indicate that PBM resulted in cytoprotection on myoblast C2C12 cells after venom exposure. This protection involves the modulation of cell death mechanism and decreased pro-inflammatory cytokine release.

Molecular and phenotypic data reveal a new Amazonian species of pit vipers (Serpentes: Viperidae: Bothrops)

Recent genetic studies have found unclear species boundaries and evidence of undescribed diversity in the poorly studied jararacussu species group within Bothrops. In this contribution, we investigate phenotypic and genetic diversity in the Amazonian snake Bothrops brazili to test previous assertions of unrecognised species diversity within this taxon. Our phylogenetic results and inferences of independently evolving lineages based on molecular data recover two divergent clades within B. brazili, one restricted to areas north and another to areas south of the Amazon River. Phylogenetic relationships between these lineages and other species in the jararacussu species group reveal B. brazili to be paraphyletic, with the northern clade inferred as sister to a clade composed of Atlantic Forest taxa (B. jararacussu, B. muriciensis, B. pirajai). External morphology (number of ventral and subcaudal scales) and colouration patterns (lateral trapezoidal marks) consistently separate the two lineages of B. brazili. We therefore recognise and describe the northern lineage as a new species of Bothrops, improving our knowledge of species diversity within a medically important clade of venomous South American snakes.