Within Neotropical pit-vipers, the Mexican/Central-American clade consisting of Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium is a wide-ranging, morphologically and ecologically diverse group of snakes. Despite their prevalence, little is known of the functional aspects of their venoms. This study aimed to fill the knowledge gap regarding coagulotoxic effects and to examine the potential of different therapeutic approaches. As a general trait, the venoms were shown to be anticoagulant but were underpinned by diverse biochemical actions. Pseudo-procoagulant activity (i.e., thrombin-like), characterized by the direct cleavage of fibrinogen to form weak fibrin clots, was evident for Atropoides picadoi, Cerrophidiontzotzilorum, Metlapilcoatlus mexicanus, M. nummifer, M. occiduus, M. olmec, and Porthidium porrasi. In contrast, other venoms cleaved fibrinogen in a destructive (non-clotting) manner, with C. godmani and C. wilsoni being the most potent. In addition to actions on fibrinogen, clotting enzymes were also inhibited. FXa was only weakly inhibited by most species, but Cerrophidion godmani and C. wilsoni were extremely strong in their inhibitory action. Other clotting enzymes were more widely inhibited by diverse species spanning the full taxonomical range, but in each case, there were species that had these traits notably amplified relatively to the others. C. godmani and C. wilsoni were the most potent amongst those that inhibited the formation of the prothrombinase complex and were also amongst the most potent inhibitors of Factor XIa. While most species displayed only low levels of thrombin inhibition, Porthidium dunni potently inhibited this clotting factor. The regional polyvalent antivenom produced by Instituto Picado Clodomiro was tested and was shown to be effective against the diverse anticoagulant pathophysiological effects. In contrast to the anticoagulant activities of the other species, Porthidium volcanicum was uniquely procoagulant through the activation of Factor VII and Factor XII. This viperid species is the first snake outside of the Oxyuranus/Pseudonaja elapid snake clade to be shown to activate FVII and the first snake venom of any kind to activate FXII. Interestingly, while small-molecule metalloprotease inhibitors prinomastat and marimastat demonstrated the ability to prevent the procoagulant toxicity of P. volcanicum, neither ICP antivenom nor inhibitor DMPS showed this effect. The extreme variation among the snakes here studied underscores how venom is a dynamic trait and how this can shape clinical outcomes and influence evolving treatment strategies.

The Eastern Long-Nosed Viper (Vipera ammodytes meridionalis) is considered one of the most venomous snakes in Europe. However, it is unknown whether ontogenetic variation in venom effects occurs in this subspecies and how this may impact antivenom efficacy. In this study, we compared the procoagulant activities of V. a. meridionalis venom on human plasma between neonate and adult venom phenotypes. We also examined the efficacy of three antivenoms—Viperfav, ViperaTAb, and Inoserp Europe—across our neonate and adult venom samples. While both neonate and adult V. a. meridionalis venoms produced procoagulant effects, the effects produced by neonate venom were more potent. Consistent with this, neonate venom was a stronger activator of blood-clotting zymogens, converting them into their active forms, with a rank order of Factor X >> Factor VII > Factor XII. Conversely, the less potent adult venom had a rank order of FXII marginally more activated than Factor VII, and both much more so than Factor X. This adds to the growing body of evidence that activation of factors besides FII (prothrombin) and FX are significant variables in reptile venom-induced coagulopathy. Although all three examined antivenoms displayed effective neutralization of both neonate and adult V. a. meridionalis venoms, they generally showed higher efficacy on adult venom than on neonate venom. The ranking of antivenom efficacy against neonate venom, from the most effective to the least effective, were Viperfav, Inoserp Europe, ViperaTAb; for adult venom, the ranking was Inoserp Europe, Viperfav, ViperaTAb. Our data reveal ontogenetic variation in V. a meridionalis, but this difference may not be of clinical concern as antivenom was effective at neutralizing both adult and neonate venom phenotypes. Regardless, our results highlight a previously undocumented ontogenetic shift, likely driven by the documented difference in prey preference observed for this species across age classes

The distribution and relative potency of post-synaptic neurotoxic activity within Crotalinae venoms has been the subject of less investigation in comparison with Elapidae snake venoms. No previous studies have investigated post-synaptic neurotoxic activity within the Atropoides, Metlapilcoatlus, Cerrophidion, and Porthidium clade. Given the specificity of neurotoxins to relevant prey types, we aimed to uncover any activity present within this clade of snakes that may have been overlooked due to lower potency upon humans and thus not appearing as a clinical feature. Using biolayer interferometry, we assessed the relative binding of crude venoms to amphibian, lizard, bird, rodent and human α-1 nAChR orthosteric sites. We report potent alpha-1 orthosteric site binding in venoms from Atropoides picadoi, Metlapilcoatlus occiduus, M. olmec, M. mexicanus, M. nummifer. Lower levels of binding, but still notable, were evident for Cerrophidion godmani, C. tzotzilorum and C. wilsoni venoms. No activity was observed for Porthidium venoms, which is consistent with significant alpha-1 orthosteric site neurotoxicity being a trait that was amplified in the last common ancestor of Atropoides/Cerrophidion/Metlapilcoatlus subsequent to the split by Porthidium. We also observed potent taxon-selective activity, with strong selection for non-mammalian targets (amphibian, lizard, and bird). As these are poorly studied snakes, much of what is known about them is from clinical reports. The lack of affinity towards mammalian targets may explain the knowledge gap in neurotoxic activity within these species, since symptoms would not appear in bite reports. This study reports novel venom activity, which was previously unreported, indicating toxins that bind to post-synaptic receptors may be more widespread in pit vipers than previously considered. While these effects appear to not be clinically significant due to lineage-specific effects, they are of significant evolutionary novelty and of biodiscovery interest. This work sets the stage for future research directions, such as the use of in vitro and in vivo models to determine whether the alpha-1 orthosteric site binding observed within this study confers neurotoxic venom activity.

Ontogenetic variation in venom activity and composition has been documented across several pit viper genera, but these toxicological variations are poorly understood in Cerrophidion, Metlapilcoatlus, Ophryacus, and Porthidum. This study investigated ontogenetic changes in coagulotoxic venom activity across adult and juvenile specimens of Cerrophidion petlalcalensis, Metlapilcoatlus olmec, M. nummifer, Ophryacus undulatus, Porthidium ophryomegas, and P. yucatanicum. Adult C. petlalcalensis, M. olmec, and M. nummifer displayed potent thrombin-like activity, resulting in a pseudo-procoagulant action whereby the venoms directly acted upon fibrinogen to produce weak, unstable fibrin clots that rapidly break down, thereby contributing to anticoagulant pathophysiological effects by depleting fibrinogen levels. In contrast, their juvenile counterparts showed either weaker or no such activity. Ophryacus undulatus displayed an ontogenetic shift in anticoagulant targets, where adults inhibited clotting factors and juveniles did not. The anticoagulant target for juvenile O. undulatus remains unknown, with this study ruling out fibrinogen degradation, factor inhibition and phospholipid degradation. Both adult and juvenile P. ophryomegas and P. yucatanicum exhibited potent anticoagulant effects through fibrinogen degradation and factor inhibition. These species exhibited previously undocumented factor VIIa inhibition, a mechanism not previously reported in viperid venoms. The regionally available PoliVal-ICP polyvalent antivenom effectively cross-neutralized the pseudo-procoagulant activities of Cerrophidion and Metlapilcoatlus, across both life stages tested. These findings reveal significant ontogenetic variation in venom activity and antivenom efficacy, with implications for both clinical management of snakebite and our understanding of venom evolution in these species.

Venom is an adaptative trait that allows predators to subjugate prey. Despite this, there remains a distinct lack of research regarding the coagulotoxic effects of snake venom upon a species preferred prey. Using thrombelastography, this study aimed to characterize coagulotoxic venom effects from the Central American pit viper clade of Atropoides, Cerrophidion, Metlpailcoatlus, and Porthidium against the plasma of model species: amphibian, avian, lizard and rodent. Venom from Atropoides and Metlapilcoaltus exhibited anticoagulant activity in rodent and lizard plasma by disrupting the formation of stable clots. While clotting time was reduced, the clots formed were weak and transient, indicating impaired coagulation. This activity did not correspond to avian and amphibian plasma, where no activity was observed. Porthidium volcanicum, Cerrophidion tzotilorum and Porthidium porrasi showed coagulotoxic activity against all plasmas tested. Consistent with the described procoagulant effect on human plasma, Porthidium volcanicum reduced clotting time and produced strong, stable clots in amphibian, lizard and rodent plasma. Intriguingly, this venom displayed an anticoagulant effect upon avian plasma, with clotting time significantly delayed. Cerrophidion tzotzilorum and Porthidium porrasi venoms produced an anticoagulant effect on avian plasma but had no effect against amphibian plasma. In addition, C. tzotzilorum and P. porrasi venoms displayed the fibrinogen-depleting pseudo-procoagulant/thrombin-like type of anticoagulant activity previously observed in human plasma against lizard plasma. However, they differed in their effects on rodent plasma. While C. tzotzilorum retained the pseudo-procoagulant/thrombin-like activity, P. porrasi delayed clotting time. The wide variation in activity observed within this study highlights the importance of studying venom activity upon relevant prey models and the limitations of extrapolating animal models to predict human related envenoming.

This study investigated the individual variation in the procoagulant toxicity of Vipera aspis venoms collected from multiple Swiss localities, in addition to the neutralisation capacity of three currently available antivenoms. Functional coagulation assays revealed quantifiable geographic differences in venom potency and clotting kinetics. Mechanistic analyses demonstrated that V. aspis venoms exert their procoagulant effects primarily through Factor Va-dependent action of the coagulation cascade, leading to accelerated thrombin generation and clot formation. These findings highlight the complexity of venom-host interactions, showing that Factor Va acts as a multifactorial cofactor that mediates both Factor X and Factor VII activation and varies between individual specimens. From a clinical perspective, this toxicological variability underscores the challenges or relying on a single antivenom for effective treatment across the vast range of a single species. Understanding the molecular basis of these pathophysiological variations through proteomic or transcriptomic profiling will be critical for developing antivenoms with improved neutralising efficacy. Beyond its clinical toxicology relevance, this work provides novel insight into the ecological and evolutionary forces shaping venom diversity in medically important species and identifies procoagulant enzymes from V. aspis venom as promising lead candidates for therapeutic development.