No abstract

Echis carinatus (EC) is known as saw-scaled viper and it is endemic to the Indian subcontinent. Envenoming by EC represents a major cause of snakebite mortality and morbidity in the Indian subcontinent. Zinc (Zn++) dependent snake venom metalloproteases (SVMPs) present in Echis carinatus venom is well known to cause systemic hemorrhage and coagulopathy in experimental animals. An earlier report has shown that Echis carinatus venom (ECV) activates neutrophils and releases neutrophil extracellular traps (NETs) that blocks blood vessels leading to severe tissue necrosis. However, the direct involvement of SVMPs in the release of NETs is not clear. Here, we investigated the direct involvement of EC SVMPs in observed pathological symptoms in a preclinical setup using specific Zn++ metal chelator, Tetraethyl thiuram disulfide (TTD)/disulfiram. TTD potently antagonizes the activity of SVMPs-mediated ECM protein degradation in vitro and skin hemorrhage in mice. In addition, TTD protected mice from ECV-induced footpad tissue necrosis by reduced expression of citrullinated H3 (citH3) and myeloperoxidase (MPO) in footpad tissue. TTD also neutralized ECV-induced systemic hemorrhage and conferred protection against lethality in mice. Moreover, TTD inhibited ECV-induced NETosis in human neutrophils and decreased the expression of peptidyl arginine deiminase (PAD) 4, citH3, MPO, and p-ERK. Further, we demonstrated that ECV-induced NETosis and tissue necrosis are mediated via PAR-1-ERK axis. Overall, our results provide an insight into SVMPs-induced toxicities and the promising protective efficacy of TTD can be extrapolated to treat severe tissue necrosis complementing anti-snake venom (ASV).

No abstract

Hemostasis is a tightly regulated process which maintains a fluid state of blood within the vasculature and provides thrombotic response upon tissue injury. Various scientific studies have implicated the role of plant latex proteases in hemostasis using in vitro experiments. However, in vivo models substantiate their role in hemostasis. Therefore, in the present study, the effect of plant latex thrombin-like proteases (PTLPs) on hemostasis was investigated systematically using mice tail bleeding as a preclinical model. In this direction, latex protease fractions (LPFs), which showed potent thrombin-like activity, were selected as they act directly on fibrinogen to form clot and quickly stop bleeding.Thrombin-like activity was exhibited mainly by cysteine proteases. Calotropis gigantea, Carica papaya, Jatropha curcas, Oxystelma esculentum, Tabernaemontana divaricata, and Vallaris solanacea LPFs and papain from C. papaya latex significantly reduced bleeding on a topical application in normal and aspirin administered mice. In addition, PTLPs accelerated the clotting of factor VIII deficient plasma, while, papain brought back the clotting time to normal levels acting like a bypassing agent. Further, papain failed to show activity in the presence of specific cysteine protease inhibitor iodoacetic acid; confirming protease role in all the activities exhibited. At the tested dose, PTLPs except C. gigantea did not show toxicity. Further, structural and sequence comparison between PTLPs and human thrombin revealed structural and sequence dissimilarity indicating their unique nature. The findings of the present study may open up a new avenue for considering PTLPs including papain in the treatment of bleeding wounds. K E Y W O R D S bypassing agent, hemophilia, hemostasis, intrinsic pathway, papain, procoagulant protease

Context and objective: Viperid venom-induced chronic local-toxicity continues even after anti-snake venom treatment. Therefore, traditional antidote Albizia lebbeck L. (Fabaceae) seed extract was tested against Echis carinatus S. (Viperidae) venom (ECV)-induced local toxicity to evaluate its complementary remedy. Materials and methods: Soxhlet extraction of A. lebbeck seeds was performed with the increasing polarity of solvents (n-hexane to water); the extract was screened for phytochemicals (alkaloids, anthraquinones, flavonoids, glycosides, phenolics, saponins, steroids and tannins). In preliminary in vitro analysis, A. lebbeck methanolic extract (ALME) demonstrated significant inhibition of ECV proteases, the major enzyme-toxin responsible for local-toxicity. Therefore, in vitro neutralizing potential of ALME was further evaluated against hyaluronidases and phospholipase A 2 (1:1-1:100 w/w). In addition, alleviation of ECV induced characteristic local-toxicity [haemorrhage (i.d.) and myotoxicity (i.m.)] was determined in mice. Results: ALME contained high concentrations of phenolics and flavonoids and demonstrated significant in vitro inhibition of ECV protease (IC 50 ¼ 36.32 lg, p < 0.0001) and hyaluronidase (IC 50 ¼ 91.95 lg, p < 0.0001) at 1:100 w/w. ALME significantly neutralized ECV induced haemorrhage (ED 50 ¼ 26.37 lg, p < 0.0001) and myotoxicity by significantly reducing serum creatinine kinase (ED 50 ¼ 37.5 lg, p < 0.0001) and lactate dehydrogenase (ED 50 ¼ 31.44 lg, p ¼ 0

No abstract

Hemostasis is a proteolytically regulated process that requires activation of platelets and the blood coagulation cascade upon vascular injury. Activated platelets create a thrombogenic environment and amplify the coagulation process. Plant latex proteases (PLPs) have been used as therapeutic components to treat various ailments by folk healers. One of the main applications of plant latices is to stop bleeding from minor injuries and to enhance wound healing activity. Although many studies have reported the pro‐coagulant activities of PLPs, an in‐depth investigation is required to understand the mechanism of action of PLPs on platelets. Here, the effect of PLPs on platelet aggregation was studied systematically to validate the observed pharmacological effect by folk healers. Among 29 latices from the Ficus genus tested, Ficus drupacea exhibited potent pro‐coagulant and thrombin‐like activity. Drupin, a thrombin‐like cysteine protease responsible for platelet aggregation was purified from F. drupacea latex. Drupin exhibits pro‐coagulant activity and reduces the bleeding time in mice tail. It induces platelet aggregation by activating mitogen‐activated protein kinases and the nuclear factor‐κB and PI3K/Akt signalling cascade, which, in turn, phosphorylats, cytosolic phospholipase A2 leading to the release of thromboxane A2 from the granules to activate the nearby platelets to aggregate. Furthermore, we investigated the involvement of protease‐activated receptors in drupin‐induced platelet aggregation using specific protease activated receptor 1 (PAR1) and PAR4 receptor antagonists. The results confirmed that the drupin‐induced platelet aggregation was mediated by both PAR1 and PAR4, synergistically. Overall, drupin reduces the bleeding time by exerting pro‐coagulant activity and induces platelet aggregation by activating the intracellular signalling cascade.

Echis carinatus envenomation leads to severe tissue necrosis at the bitten site by releasing DNA from immune cells that blocks the blood flow. An earlier report has shown that exogenous DNase 1 offers protection against such severe local tissue necrosis. Tricosanthus tricuspidata is a medicinal plant and the paste prepared from its leaves has been used extensively for the treatment of snakebite‐induced tissue necrosis. Most studies including reports from our laboratory focused on plant secondary metabolite as therapeutic molecules against snakebite envenomation. However, the involvement of hydrolytic enzymes including DNase in treating snake venom‐induced tissue necrosis has not been addressed. Several folk medicinal plants used against snakebite treatment showed the presence of DNase activity and found to be rich in T. tricuspidata. Further, purified T. tricuspidata DNase showed a single sharp peak in reversed‐phase high‐performance liquid chromatography (RP‐HPLC) with an apparent molecular mass of 17 kDa. T. tricuspidata DNase exhibited potent DNA degrading activity performed using agarose gel electrophoresis, spectrophotometric assay, and DNA zymography. In addition, purified DNase from T. tricuspidata was able to neutralize E. carinatus venom‐induced mouse tail tissue necrosis and normalized elevated serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels 30 minutes post venom injection. T. tricuspidata DNase was also able to reverse E. carinatus venom‐induced histopathological changes and collagen depletion in mice tail tissue. All these observed pharmacological actions of T. tricuspidata DNase were inhibited by sodium fluoride (NaF). This study provides scientific validation of the traditional use of T. tricuspidata leaf paste in the healing of snakebite‐induced tissue necrosis and might be exploited to treat snake venom‐induced local toxicity.