A Genus-Wide Bioactivity Analysis of Daboia (Viperinae: Viperidae) Viper Venoms Reveals Widespread Variation in Haemotoxic Properties

The snake genus Daboia (Viperidae: Viperinae; Oppel, 1811) contains five species: D. deserti, D. mauritanica, and D. palaestinae, found in Afro-Arabia, and the Russell’s vipers D. russelii and D. siamensis, found in Asia. Russell’s vipers are responsible for a major proportion of the medically important snakebites that occur in the regions they inhabit, and their venoms are notorious for their coagulopathic effects. While widely documented, the extent of venom variation within the Russell’s vipers is poorly characterised, as is the venom activity of other species within the genus. In this study we investigated variation in the haemotoxic activity of Daboia using twelve venoms from all five species, including multiple variants of D. russelii, D. siamensis, and D. palaestinae. We tested the venoms on human plasma using thromboelastography, dose-response coagulometry analyses, and calibrated automated thrombography, and on human fibrinogen by thromboelastography and fibrinogen gels. We assessed activation of blood factors X and prothrombin by the venoms using fluorometry. Variation in venom activity was evident in all experiments. The Asian species D. russelii and D. siamensis and the African species D. mauritanica possessed procoagulant venom, while D. deserti and D. palaestinae were net-anticoagulant. Of the Russell’s vipers, the venom of D. siamensis from Myanmar was most toxic and D. russelli of Sri Lanka the least. Activation of both factor X and prothrombin was evident by all venoms, though at differential levels. Fibrinogenolytic activity varied extensively throughout the genus and followed no phylogenetic trends. This venom variability underpins one of the many challenges facing treatment of Daboia snakebite envenoming. Comprehensive analyses of available antivenoms in neutralising these variable venom activities are therefore of utmost importance.

Pharmacological Characterisation of Pseudocerastes and Eristicophis Viper Venoms Reveal Anticancer (Melanoma) Properties and a Potentially Novel Mode of Fibrinogenolysis

Venoms are a rich source of potential lead compounds for drug discovery, and descriptive studies of venom form the first phase of the biodiscovery process. In this study, we investigated the pharmacological potential of crude Pseudocerastes and Eristicophis snake venoms in haematological disorders and cancer treatment. We assessed their antithrombotic potential using fibrinogen thromboelastography, fibrinogen gels with and without protease inhibitors, and colourimetric fibrinolysis assays. These assays indicated that the anticoagulant properties of the venoms are likely induced by the hydrolysis of phospholipids and by selective fibrinogenolysis. Furthermore, while most fibrinogenolysis occurred by the direct activity of snake venom metalloproteases and serine proteases, modest evidence indicated that fibrinogenolytic activity may also be mediated by selective venom phospholipases and an inhibitory venom-derived serine protease. We also found that the Pseudocerastes venoms significantly reduced the viability of human melanoma (MM96L) cells by more than 80%, while it had almost no effect on the healthy neonatal foreskin fibroblasts (NFF) as determined by viability assays. The bioactive properties of these venoms suggest that they contain a number of toxins suitable for downstream pharmacological development as candidates for antithrombotic or anticancer agents.

The hemostatic activity of Manilkara zapota (L.) P. Royen latex associated with fibrinogenolytic activity

Manilkara zapota (L.) P. Royen (Sapotaceae), is widely used in traditional medicine for various ailments like, diarrhea, pulmonary diseases, piles, ulcers and to treat wounds. The present study evaluates the role of M. zapota latex in hemostasis. The processed latex named as M. zapota natant latex (MzNL), has proteins at the concentration of 8 mg/ml and showed protein bands in Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteolytic activity of MzNL was evaluated using casein in comparison with trypsin. The phenylmethylsulfonyl fluoride (PMSF) inhibited the protease activity indicating the possible presence of serine protease. The effect of temperature, pH and metal ions on proteolytic activity was evaluated. MzNL exhibited fibrinogenolytic activity by hydrolysing A? and B? subunits of fibrinogen. However, ? subunit remained resistant for hydrolysis. MzNL hydrolyzed all the subunits of collagen type I and IV at the concentration of 8 µg and 25 µg in 20 µl each respectively. MzNL showed procoagulant activity and is devoid of hemolytic activity. Fibrinogenolytic activity and procoagulant nature of MzNL suggests its possible role in blood coagulation that in turn restores hemostasis.

A Web of Coagulotoxicity: Failure of Antivenom to Neutralize the Destructive (Non-Clotting) Fibrinogenolytic Activity of Loxosceles and Sicarius Spider Venoms

Envenomations are complex medical emergencies that can have a range of symptoms and sequelae. The only specific, scientifically-validated treatment for envenomation is antivenom administration, which is designed to alleviate venom effects. A paucity of efficacy testing exists for numerous antivenoms worldwide, and understanding venom effects and venom potency can help identify antivenom improvement options. Some spider venoms can produce debilitating injuries or even death, yet have been largely neglected in venom and antivenom studies because of the low venom yields. Coagulation disturbances have been particularly under studied due to difficulties in working with blood and the coagulation cascade. These circumstances have resulted in suboptimal spider bite treatment for medically significant spider genera such as Loxosceles and Sicarius. This study identifies and quantifies the anticoagulant effects produced by venoms of three Loxoscles species (L. reclusa, L. boneti, and L. laeta) and that of Sicarius terrosus. We showed that the venoms of all studied species are able to cleave the fibrinogen Aα-chain with varying degrees of potency, with L. reclusa and S. terrosus venom cleaving the Aα-chain most rapidly. Thromboelastography analysis revealed that only L. reclusa venom is able to reduce clot strength, thereby presumably causing anticoagulant effects in the patient. Using the same thromboelastography assays, antivenom efficacy tests revealed that the commonly used Loxoscles-specific SMase D recombinant based antivenom failed to neutralize the anticoagulant effects produced by Loxosceles venom. This study demonstrates the fibrinogenolytic activity of Loxosceles and Sicarius venom and the neutralization failure of Loxosceles antivenom, thus providing impetus for antivenom improvement.

Effects of Heme Modulation on Ovophis and Trimeresurus Venom Activity in Human Plasma

Geographic isolation and other factors result in evolution-driven diversity of the enzymatic composition of venom of pit vipers in the same genus. The present investigation sought to characterize venoms obtained from such genetically diverse Ovophis and Trimeresurus pit vipers utilizing thrombelastographic coagulation kinetic analyses. The coagulation kinetics of human plasma were assessed after exposure to venom obtained from two Ovophis and three Trimeresurus species. The potency of each venom was defined (µg/mL required to equivalently change coagulation); additionally, venoms were exposed to carbon monoxide (CO) or a metheme-inducing agent to modulate any enzyme-associated heme. All venoms had fibrinogenolytic activity, with four being CO-inhibitable. While Ovophis venoms had similar potency, one demonstrated the presence of a thrombin-like activity, whereas the other demonstrated a thrombin-generating activity. There was a 10-fold difference in potency and 10-fold different vulnerability to CO inhibition between the Trimeresurus species. Metheme formation enhanced fibrinogenolytic-like activity in both Ovophis species venoms, whereas the three Trimeresurus species venoms had fibrinogenolytic-like activity enhanced, inhibited, or not changed. This novel “venom kinetomic” approach has potential to identify clinically relevant enzymatic activity and assess efficacy of antivenoms between genetically and geographically diverse species.

Anti-snake venom potential of Clerodendrum serratum extracts on Bungarus caeruleus and Daboia russelii venom

<p class="Abstract">Anti-snake venom therapy is the only treatment for snake bite but leads to acute and chronic conditions which may be severe. The medicinal plants have gained importance over years to find an effective alternative to anti-snake venom. The present study focused on evaluating the potential of <em>Clerodendrum serratum</em> for the anti-snake venom activity. Phytochemicals were extracted from the <em>C. serratum</em> with different solvents. The ethyl acetate and methanolic extracts were found to neutralize the major enzyme toxins (phospholipase A₂, protease and hyaluronidase) of <em>Bungarus caeruleus</em> and <em>Daboia russelii</em> venom at a concentration of 100 µg/mL. The fibrinogenolytic activity of both the venoms were neutralized. The study proves that the plant <em>C. serratum</em> possesses certain compounds which inhibit the toxins present in the venom of <em>B. caeruleus</em> and <em>D. russelii</em>.</p><p class="Abstract"><strong>Video Clip of Methodology</strong>:</p><p class="Abstract">Hyaluronidase assay: 3 min 30 sec <a href="https://www.youtube.com/v/Xz5C6bbwgW0">Full Screen</a> <a href="https://www.youtube.com/watch?v=Xz5C6bbwgW0">Alternate</a></p>