A hemorrhagin as a metalloprotease in the venom of Trimeresurus purpureomaculatus: purification and characterization.

A major hemorrhagin was purified from the venom of the Thai green pit viper (Trimeresurus purpureomaculatus) by gel filtration, ion-exchange and affinity chromatography. A 15-fold purification was achieved with an overall yield of 7% of hemorrhagic activity. The hemorrhagin was homogeneous according to disc- and SDS-PAGE as well as on immunodiffusion. The molecular weight determined by SDS-PAGE was 72kDa. The purified hemorrhagin expresses proteolytic activity with heat-denatured casein and hide powder azure, but it was free of AE-hydrolase and phospholipase activities. Both hemorrhagic and proteolytic activities were inhibited by EDTA, suggesting that the hemorrhagin is a metalloprotease. The hemorrhagin hydrolyzed all gelatin preparations derived from types I, II, III and IV collagen, whereas it hydrolyzed only type IV native collagen. The hemorrhagic activity was neutralized by Thai green pit viper antivenom raised to Trimeresurus albolabris venom.

Isolation of the major lethal toxin in the venom of Bungarus flaviceps.

The major lethal toxin in the venom of Bungarus flaviceps has been isolated by ion-exchange chromatography, absorption chromatography and RP-HPLC with a 14-fold purification and an overall yield of 16.5% of the lethal toxicity contained in crude venom. Its sublethal dose (LD(50)) determined in mice weighing 18-20 g was 0.25 (0.19-0.32) microg per mouse. The lethal toxin was pure according to disc- and SDS-PAGE as well as gel HPLC. Its apparent molecular weight determined by SDS-PAGE was 29 kDa. It is a basic protein consisting of two polypeptide chains having apparent molecular weights of 17 and 8 kDa, respectively. The toxin has PLA activity but is free of ACE activity.

Cross neutralization of the lethal activities of Myanmar and Thai Russell's viper venoms by Thai and Myanmar antivenoms.

The efficacy of homologous neutralization of the lethal activity of Myanmar Russell's viper venom (MRV) and Thai Russell's viper venom (TRV) by Myanmar antivenom (MAV) and Thai antivenom (TAV), respectively, were studied and compared with the heterologous neutralization of the lethal activity of MRV and TRV by TAV and MAV, respectively, in experimental mice. Although MRV and TRV were the same subspecies, their lethal activities and protective efficacy of MAV and TAV were apparently different from each other. However, there was some extent of cross reactivities between MRV and TAV, and TRV and MAV. The findings of this study suggested that it is necessary to administer heterologous antivenom in dosage of 1.7-4 times greater than that of homologous antivenom to achieve an equal potency.

Effects of Russell's viper venom on mediator production in cultured human umbilical vein endothelial cells.

Hemodynamic alterations in Russell's viper envenomation are the result of interactions of various vasoactive mediators and perhaps proinflammatory cytokines. Since vascular endothelium is likely to be exposed to high concentrations of the venom and the endothelial cell itself not only plays an important role in the physiologic control of the circulation, but also play a role in inflammation with the synthesis and secretion of proinflammatory cytokines. It was therefore, the objective of this study to determine the effects of Russell's viper venom (RVV) on proinflammatory cytokine production by cultured human umbilical vein endothelial cells (HUVEC) and the release of endothelium-derived substances. Endothelial cells were isolated from freshly obtained human umbilical cord vein and grown in tissue culture to confluence as a homogeneous population. Cells were then incubated at 37 degrees C under 5 per cent CO2 with RVV (0.2, 1.0, 5.0, and 25.0 microg/ml) or lipopolysaccharide (LPS, 10 microg/ml) for 3, 6, 12 and 24 hours. After an indicated time, the levels of endothelin-1 (ET-1); 6-keto-PGF1alpha (a stable metabolite of PGI2) tumor necrosis factor-alpha (TNF-alpha); interleukin-1beta (IL-1beta); and interleukin-6 (IL-6) in supernatants were measured by using ELISA or EIA. The effect of RVV or LPS on cell viability was also measured using MIT assay. The results showed copious amounts of ET-1 production irrespectively with the presence of RVV or LPS. Whereas, production of PGI2 (measured as 6-keto-PGF1alpha, a stable metabolite) was increased significantly higher in the RVV- and LPS-treated EC than in the control EC. However, TNF-alpha and IL-6 productions were not different among these groups. The levels of IL-1beta were very low, although IL-1beta was detectable in the group treated with RVV at a concentration of 25.0 microg/ml. In conclusion, RVV upto 25 microg/ml stimulated PGI2 production by cultured HUVEC. This effect was unlikely related to production of proinflammatory cytokines since LPS or RVV is not sufficient per se to elevate a substantial amount of EC-derived cytokines. The higher amount of IL-6 compared to TNF-alpha and IL-1beta may be produced through other pathways apart from production via a cascade of cytokines. This is the first report showing that RVV up to 25 microg/ml has no effect on prominent proinflammatory cytokine production by HUVEC. However, in blood circulation, the major source of cytokines production is monocyte-macrophage lineage cell. Thus, RVV in blood circulation may activate the production of proinflammatory cytokines mainly from those cells and subsequently induce toxicity.

Effectiveness of Thai cobra (Naja kaouthia) antivenom against sea snake (Lapemis hardwickii) venom: verification by affinity purified F(AB')2 fragments.

Commercial Thai cobra (Naja kaouthia) antivenom was found to be effective in neutralizing sea snake (Lapemis hardwickii) venom. Neurotoxin specific F(ab')2 fragments obtained from the antivenom by chromatography using Thai cobra neurotoxin or sea snake venom affinity columns were able to neutralize both venoms.

Development of reversed passive latex agglutination for detection of Thai cobra (Naja kaouthia) venom.

A simple, rapid, and sensitive diagnostic kit for detecting Thai cobra (Naja kaouthia) venom was developed using latex particles sensitized with venom specific immunoglobulin. The kit is capable of detecting 25-50 ng/ml of Thai cobra venom. The capability was not affected by human plasma. Specificity of the kit was proven using snake venoms from Vipera russelli, Calloselasma rhodostoma, Trimeresurus albolabris, Naja siamensis, Ophiophagus hannah, and Bungarus fasciatus. The diagnostic kit does not lose its capability under refrigeration for two months and by lyophilization.

The effects of green pit viper (Trimeresurus albolabris and Trimeresurus macrops) venom on the fibrinolytic system in human.

Green pit viper (Trimeresurus albolabris and Trimeresurus macrops) venom was found to have a thrombin-like effect in vitro but cause a defibrination syndrome in vivo. The effects of venom on fibrinolytic system have not been well characterized. This knowledge can help to define the roles of antifibrinolytic therapy, give insights in fibrinolytic system regulation and potentially lead to identification of a new profibrinolytic agent from this venom. Forty-six cases of green pit viper bites were studied for various coagulation and fibrinolytic parameters and correlated with serum venom levels measured by ELISA. Fibrinolytic system activation is very common as indicated by low plasminogen (50%), low antiplasmin (56.5%) and elevated fibrin-fibrinogen degradation products (FDPs, 97.4%) levels. FDP test is very sensitive and a normal level is useful for exclusion of systemic envenomation. In contrast to some other models of defibrination syndrome, such as Russell viper (Daboia russelli siamensis), elevation of plasminogen activator activity (PA) was found indicating a hyperfibrinolytic state. Definite increase in tissue-type plasminogen activator (t-PA) antigen (p = 0.00075) with a modest elevation of its inhibitor plasminogen activator inhibitor-1 (PAI-1) (p = 0.27) probably contributes to this effect. This supports the idea that the balance between plasminogen activators and inhibitors can determine fibrinolytic responses in pathologic states. Fibrinopeptide A levels were markedly elevated (68.43 +/- 51.57 ng/ml in cases and 2.83 +/- 3.80 ng/ml in control, p < 0.0001) and correlated well with clinical severity suggesting that the fibrin deposition from the thrombin-like effect is the main mechanism of fibrinolysis. Therefore, antifibrinolytic agents probably have no role in treatment. However, the components of green pit viper venom that have these profibrinolytic effects in human are interesting and should be further identified.

Genus specific neutralization of Bungarus snake venoms by Thai Red Cross banded krait antivenom.

Thai commercial antivenom raised to Bungarus fasciatus venom neutralized the lethal activity of all Thai Bungarus venoms tested in in vitro neutralization experiments. The neutralizing capacities against B. fasciatus and B. candidus venoms were almost the same, but that against B. flaviceps venom was significantly greater. The efficacy of the antivenom was confirmed in in vivo neutralization experiments also. Results of immunochemical analyses supported results of the animal experiments suggesting the presence of genus specific neutralization.

Envenomation of mice by Thai cobra (Naja kaouthia) venom: tolerable venom concentration and exposure time.

Naja kaouthia venom appeared in circulation rapidly after intramuscular injection into mice. The venom concentration attained a maximum level with all doses examined after 20 min. The half value of the maximum level was obtained 1 min after injection when a dose of 4LD50 was used. A critical venom concentration endangering mice was assessed from venom concentration in the sera of mice envenomed with sublethal dose (LD50). A fatal condition was produced within 30 min at a venom concentration of 200-300 ng/ml or within 50 min at a venom concentration of 100-150 ng/ml.

Efficacy and cross reactivity of Thai green pit viper antivenom among venoms of Trimeresurus species in Thailand and Japan.

The efficacy of the Thai green pit viper antivenom to neutralize lethal, hemorrhagic, and enzyme activities of Trimeresurus venoms was examined using venoms of Trimeresurus albolabris, T. macrops, and T. flavoviridis (Japanese Habu). Antivenom against Japanese Habu venom was also used to study immunological cross reactivity among Trimeresurus venoms. Thai green pit viper antivenom was comparably effective to Habu antivenom to neutralize all activities. Distinct cross neutralization was demonstrated indicating the presence of genus specific protection by Thai and Japanese Trimeresurus antivenoms. Results of immunoblotting analyses indicated that Thai and Japanese Trimeresurus venoms contain many cross-reactive protein components.

Production of highly potent horse antivenom against the Thai cobra (Naja kaouthia).

Naja kaouthia (NK) causes the highest fatality due to snake venom poisoning in Thailand. The specific antivenom produced is of low potency and in short supply. The aim of this study was to improve the antivenom potency. Bentonite and complete Freund's adjuvants (CFA) and various immunogens were compared. Six groups of three to five horses were immunized as follows: Group 1, NK venom adsorbed on bentonite; Group 2, NK venom in CFA; Group 3, NK venom in CFA in multi-emulsion formulation; Group 4, NK venom in 25% CFA; Group 5, NK neurotoxin 3 (NK3) conjugated with tetanus toxoid (NK3-TT) in CFA; Group 6, NK3 conjugated with diphtheria toxoid (NK3-DT) in CFA. Horses in Group 2-6 produced antivenom of very high neutralizing activity, up to four times higher than that of horses of Group 1. CFA (100 or 25%) or as a multi-emulsion formulation, induced comparable neutralizing antibody production with all three immunogens. All horses showed normal weight gain during the course of immunization. Group 1 horses exhibited minimal local reactions while horses in the other five groups had mild and comparable local reactions at the injection sites. No significant differences in the reactions caused by CFA in different formulations or different immunogens were observed. The production of highly potent antivenom against N. kaouthia from these horses should help solve problems associated with the currently available antivenom.

Cross-neutralization of Thai cobra (Naja kaouthia) and spitting cobra (Naja siamensis) venoms by Thai cobra antivenom.

The neutralizing capacity of antivenom prepared against Thai cobra (Naja kaouthia) venom was compared in mice using the homologous venom and that of spitting cobra (Naja siamensis). The amounts of antivenoms neutralizing a dose of 4 LD50 of the test venom were determined. Four antivenom preparations were used: three purified antivenoms and a crude antivenom, which were made using N. kaouthia venom only. Almost the same neutralizing capacity was obtained with the purified antivenoms, whereas a slightly lower capacity was seen with the crude antivenom. However, ratios of the amounts of four antivenoms neutralizing the homologous and heterologous test venoms were almost constant. These results indicated that Thai cobra antivenom possesses neutralizing capacity against spitting cobra venom.