Hump-Nosed Pit Viper (Hypnale hypnale) Venom-Induced Irreversible Red Blood Cell Aggregation, Inhibition by Monovalent Anti-Venom and N-Acetylcysteine.

Envenomation by the Hypnale hypnale in the Western Ghats of India (particularly in the Malabar region of Kerala) and the subcontinent island nation of Sri Lanka is known to inflict devastating mortality and morbidity. Currently, H. hypnale bites in India are devoid of anti-venom regimens. A detailed characterization of the venom is essential to stress the need for therapeutic anti-venom. Notably, the deleterious effects of this venom on human blood cells have largely remained less explored. Therefore, in continuation of our previous study, in the present study, we envisioned investigating the effect of venom on the morphological and physiological properties of red blood cells (RBCs). The venom readily induced deleterious morphological changes and, finally, the aggregation of washed RBCs. The aggregation process was independent of the ROS and the intracellular Ca2+ ion concentration. Confocal and scanning electron microscopy (SEM) images revealed the loss of biconcave morphology and massive cytoskeletal disarray. Crenation or serrated plasma membrane projections were evenly distributed on the surface of the RBCs. The venom did not cause the formation of methemoglobin in washed RBCs but was significantly induced in whole blood. Venom did not affect glucose uptake and Na+/K+ -ATPase activity but inhibited glucose 6 phosphate dehydrogenase activity and decreased the fluidity of the plasma membrane. Venom-induced RBC aggregates exhibited pro-coagulant activity but without affecting platelet aggregation. In pre-incubation or co-treatment studies, none of the bioactive compounds, such as melatonin, curcumin, fisetin, berberine, and quercetin, sugars such as mannose and galactose, and therapeutic polyvalent anti-venoms (Bharat and VINS) were inhibited, whereas only N-acetylcysteine and H. hypnale monovalent anti-venom could inhibit venom-induced deleterious morphological changes and aggregation of RBCs. In post-treatment studies, paradoxically, none of the bioactives and anti-venoms, including N-acetylcysteine and H. hypnale monovalent anti-venom, reversed the venom-induced RBC aggregates.

Oxidative Stress-Induced Platelet Apoptosis/Activation: Alleviation by Purified Curcumin via ASK1-JNK/p-38 Pathway.

Platelets are known for their indispensable role in hemostasis and thrombosis. However, alteration in platelet function due to oxidative stress is known to mediate various health complications, including cardiovascular diseases and other health complications. To date, several synthetic molecules have displayed antiplatelet activity; however, their uses are associated with bleeding and other adverse effects. The commercially available curcumin is generally a mixture of three curcuminoids: curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Although crude curcumin is known to inhibit platelet aggregation, the effect of purified curcumin on platelet apoptosis, activation, and aggregation remains unclear. Therefore, in this study, curcumin was purified from a crude curcumin mixture and the effects of this preparation on the oxidative stress-induced platelet apoptosis and activation was evaluated. 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) compound was used as an inducer of oxidative stress. Purified curcumin restored AAPH-induced platelet apoptotic markers like reactive oxygen species, intracellular calcium level, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release from mitochondria to the cytosol, and phosphatidyl serine externalization. Further, it inhibited the agonist-induced platelet activation and aggregation, demonstrating its antiplatelet activity. Western blot analysis confirms protective effect of the purified curcumin against oxidative stress-induced platelet apoptosis and activation via downregulation of MAPKs protein activation, including ASK1, JNK, and p-38. Together, these results suggest that the purified curcumin could be a potential therapeutic bioactive molecule to treat the oxidative stress-induced platelet activation, apoptosis, and associated complications.

A comparative cross-reactivity and paraspecific neutralization study on Hypnale hypnale, Echis carinatus, and Daboia russelii monovalent and therapeutic polyvalent anti-venoms.

Envenoming by the hump-nosed pit viper (Hypnale hypnale) raises concern as it inflicts significant debilitation and death in the Western Ghats of India and in the adjacent island nation of Sri Lanka. In India, its medical significance was realized only during 2007 due to its misidentification as Echis carinatus and sometimes as Daboia russelii. Of late, several case reports have underlined the ineptness of the existing polyvalent anti-venom therapy against H. hypnale envenoming. Currently, H. hypnale bite has remained dreadful in India due to the lack of neutralizing anti-venom therapy. Hence, this study was undertaken to establish a systematic comparative, biochemical, pathological, and immunological properties of Sri Lankan H. hypnale venom alongside Indian E. carinatus, and D. russelii venoms. All three venoms differed markedly in the extent of biochemical activities including proteolytic, deoxyribonuclease, L-amino acid oxidase, 5'-nucleotidase, hyaluronidase, and indirect hemolytic activities. The venoms also differed markedly in their pathological properties such as edema, hemorrhage, myotoxic, cardiotoxic, and coagulant activities. The venoms showed stark differences in their protein banding pattern. Strikingly, the affinity-purified rabbit monovalent anti-venoms prepared against H. hypnale, E. carinatus, and D. russelii venoms readily reacted and neutralized the biochemical and pathological properties of their respective venoms, but they insignificantly cross-reacted with, and thus failed to show paraspecific neutralization of any of the effects of the other two venoms, demonstrating the large degree of variations between these venoms. Further, the Indian therapeutic polyvalent anti-venoms from VINS Bioproducts, and Bharath Serums and Vaccines failed to protect H. hypnale venom-induced lethal effects in mice.

Bisphenol AF elevates procoagulant platelets by inducing necroptosis via RIPK1-inflammasome axis.

Bisphenol AF, an analogue of Bisphenol A, is an important raw material used in the production of plastic and rubber substances like plastic bottles and containers, toys, and medical supplies. Increased contamination of air, water, dust, and food with BPA/BPAF, poses an enormous threat to humans, globally. BPAF/BPA are endocrine-disrupting chemicals that mimic estrogen hormone, thus increasing the risks of various metabolic and chronic disorders. Exposure of human blood cells to BPA/BPAF induces oxidative stress and genotoxicity. However, its effects on platelets, which play central roles in hemostasis and thrombosis, are not well-documented. In this study, we demonstrate that BPAF induces RIPK1-inflammasome axis-mediated necroptosis in platelets, increasing procoagulant platelet levels in vivo and in vitro. We also show that BPAF-induced rise in procoagulant platelets worsens pulmonary thromboembolism in vivo. The elevated procoagulant platelets are shown to increase platelet-neutrophil/monocyte aggregates that mediate pathogenesis of CVD, thrombosis, and chronic inflammatory diseases. Our results demonstrate the toxic effects of BPAF on platelets and how it propagates the clinical complications by elevating procoagulant platelet numbers. Altogether, our study sends a cautionary message against extensive use of BPAF in the plastic and rubber industries, resulting in frequent human exposure to it, thus endangering platelet functions.