Assessment of the potential toxicological hazard of the Green Parrot Snake (Leptophis ahaetulla marginatus): Characterization of its venom and venom-delivery system.

Snakes are the major group of venomous vertebrates, and the rear-fanged snakes represent the vast majority of species and occur worldwide; however, relatively few studies have characterized their venoms and evaluated their potential hazards for humans. Herein we explore the protein composition and properties of the venom of the rear-fanged Green Parrot Snake, Leptophis ahaetulla marginatus, the most common snake found in the Iguazu National Park (Argentina), as well as the main features of its venom delivery system. This species has venom reminiscent of elapid venoms, composed mainly of components such as 3FTxs, CRiSPs and AChE, but it shows low toxicity toward mammals (LD50 > 20 µg/g mouse). The histology of its Duvernoy's venom gland is similar to that of other colubrids, with serous secretory cells arranged in densely packed secretory tubules. The posterior end of its maxilla exhibits 1-3 blade-shaped and slightly recurved fangs but without grooves. This study provides an initial analysis of the biological role of venom in Leptophis, with implications for potential symptoms that might be anticipated from bites by this species.

A proteomic analysis of Pakistan Daboia russelii russelii venom and assessment of potency of Indian polyvalent and monovalent antivenom.

UNLABELLED: To address the dearth of knowledge on the biochemical composition of Pakistan Russell's Viper (Daboia russelii russelii) venom (RVV), the venom proteome has been analyzed and several biochemical and pharmacological properties of the venom were investigated. SDS-PAGE (reduced) analysis indicated that proteins/peptides in the molecular mass range of ~56.0-105.0kDa, 31.6-51.0kDa, 15.6-30.0kDa, 9.0-14.2kDa and 5.6-7.2kDa contribute approximately 9.8%, 12.1%, 13.4%, 34.1% and 30.5%, respectively of Pakistan RVV. Proteomics analysis of gel-filtration peaks of RVV resulted in identification of 75 proteins/peptides which belong to 14 distinct snake venom protein families. Phospholipases A2 (32.8%), Kunitz type serine protease inhibitors (28.4%), and snake venom metalloproteases (21.8%) comprised the majority of Pakistan RVV proteins, while 11 additional families accounted for 6.5-0.2%. Occurrence of aminotransferase, endo-ß-glycosidase, and disintegrins is reported for the first time in RVV. Several of RVV proteins/peptides share significant sequence homology across Viperidae subfamilies. Pakistan RVV was well recognized by both the polyvalent (PAV) and monovalent (MAV) antivenom manufactured in India; nonetheless, immunological cross-reactivity determined by ELISA and neutralization of pro-coagulant/anticoagulant activity of RVV and its fractions by MAV surpassed that of PAV. BIOLOGICAL SIGNIFICANCE: The study establishes the proteome profile of the Pakistan RVV, thereby indicating the presence of diverse proteins and peptides that play a significant role in the pathophysiology of RVV bite. Further, the proteomic findings will contribute to understand the variation in venom composition owing to different geographical location and identification of pharmacologically important proteins in Pakistan RVV.

Comparative venomics of the Prairie Rattlesnake (Crotalus viridis viridis) from Colorado: Identification of a novel pattern of ontogenetic changes in venom composition and assessment of the immunoreactivity of the commercial antivenom CroFab®.

Here we describe and compare the venomic and antivenomic characteristics of both neonate and adult Prairie Rattlesnake (Crotalus viridis viridis) venoms. Although both neonate and adult venoms contain unique components, similarities among protein family content were seen. Both neonate and adult venoms consisted of myotoxin, bradykinin-potentiating peptide (BPP), phospholipase A2 (PLA2), Zn(2+)-dependent metalloproteinase (SVMP), serine proteinase, L-amino acid oxidase (LAAO), cysteine-rich secretory protein (CRISP) and disintegrin families. Quantitative differences, however, were observed, with venoms of adults containing significantly higher concentrations of the non-enzymatic toxic compounds and venoms of neonates containing higher concentrations of pre-digestive enzymatic proteins such as SVMPs. To assess the relevance of this venom variation in the context of snakebite and snakebite treatment, we tested the efficacy of the common antivenom CroFab® for recognition of both adult and neonate venoms in vitro. This comparison revealed that many of the major protein families (SVMPs, CRISP, PLA2, serine proteases, and LAAO) in both neonate and adult venoms were immunodepleted by the antivenom, whereas myotoxins, one of the major toxic components of C. v. viridis venom, in addition to many of the small peptides, were not efficiently depleted by CroFab®. These results therefore provide a comprehensive catalog of the venom compounds present in C. v. viridis venom and new molecular insight into the potential efficacy of CroFab® against human envenomations by one of the most widely distributed rattlesnake species in North America. BIOLOGICAL SIGNIFICANCE: Comparative proteomic analysis of venoms of neonate and adult Prairie Rattlesnake (Crotalus viridis viridis) from a discrete population in Colorado revealed a novel pattern of ontogenetic shifts in toxin composition for viperid snakes. The observed stage-dependent decrease of the relative content of disintegrins, catalytically active D49-PLA2s, L-amino acid oxidase, and SVMPs, and the concomitant increase of the relative abundance of paralytic small basic myotoxins and ohanin-like toxin, and hemostasis-disrupting serine proteinases, may represent an age-dependent strategy for securing prey and avoiding injury as the snake switches from small ectothermic prey and newborn rodents to larger endothermic prey. Such age-dependent shifts in venom composition may be relevant for antivenom efficacy and treatment of snakebite. However, applying a second-generation antivenomics approach, we show that CroFab®, developed against venom of three Crotalus and one Agkistrodon species, efficiently immunodepleted many, but not all, of the major compounds present in neonate and adult C. v. viridis venoms.

Biochemical and pharmacological properties of a new thrombin-like serine protease (Russelobin) from the venom of Russell's Viper (Daboia russelii russelii) and assessment of its therapeutic potential.

BACKGROUND: Snake venoms are rich sources of bioactive molecules, and several venom-derived proteins have entered clinical trials for use in ischemic disorders; however, late-stage failure of a recent drug candidate due to low in vivo efficacy demonstrated the need for new sources of fibrinogenolytic drug candidates. METHODS: A 51.3kDa thrombin-like serine protease (Russelobin) purified from the venom of Russell's Viper (Daboia russelii russelii) was subjected to extensive biochemical characterization, including N-terminal sequencing, substrate specificity, kinetic and inhibitor assays, glycosylation analysis and stability assays. Toxicity and pathology analyses were conducted in NSA mice. RESULTS: Russelobin has extensive N-terminus identity with a beta-fibrinogenase-like serine proteinase precursor from Daboia russelii siamensis venom, a mass of 51.3kDa and contains extensive N-linked oligosaccharides. Serine protease inhibitors and heparin significantly decreased activity, with much lower inhibition by DTT, antithrombin-III and α2-macroglobulin. Russelobin preferentially released FPA and slowly released FPB from human fibrinogen, forming a labile fibrin clot readily hydrolyzed by plasmin. The partially deglycosylated enzyme showed significantly lower activity toward fibrinogen and less resistance against neutralization by plasma α2MG and antithrombin-III. Russelobin was non-cytotoxic, non-lethal and produced no histopathologies in mice, and it demonstrated in vivo dose-dependent defibrinogenating activity. CONCLUSIONS: Russelobin is an A/B fibrinogenase with high specificity toward fibrinogen, both in vitro and in vivo. Extensive glycosylation appears to protect the molecule against endogenous protease inhibitors, prolonging its in vivo efficacy. GENERAL SIGNIFICANCE: Due to its low toxicity, stability and activity as a defibrinogenating agent, Russelobin shows high potential for cardiovascular drug development.