South American rattlesnake venom: its hemolytic power

Foi estudado o poder hemolítico do veneno da cascavel (Crotalus durissus terrificus). Encontrou-se grande número de suas fraçöes sem capacidade de hemolisar eritrócitos de carneiro. O veneno "in cura", recentemente extraído, e em estado líquido tem pouca atividade lítica. A cristalizaçäo do veneno aumenta sua concentraçäo e poder lítico. Os resultados de hemólise do sangue de carneiro obtidos em placas e tubos foram comparados evidenciando um grande número de animais com venenos com alto poder heomlítico. Os valores näo foram proporcionais quando os mesmos venenos foram examinados com hemáceas de homem. Neste caso os percentuais de hemólise foram mais baixos. Pode-se verificar que o poder hemolítico do veneno se relaciona com a concentraçäo e pureza de suas fraçöes

South American rattlesnake venom: its hemolytic power.

The hemolytic power of rattlesnake venom (Crotalus durissus terrificus) was studied. A high percentage of sample with negative hemolytic power was detected when sheep red blood cells were used. A large number of venoms with hemolytic power, though with a low hemolysis percentage, were detected when liquid, recently extracted venom was used. When crystallized venom was used under the same experimental conditions, a higher percentage of positivity for hemolysis was obtained. When the results obtained on agar plates were compared to those obtained in test tubes, a large number of animals with a higher percentage of hemolysis were detected, though this value was not proportional to the number of animals showing positive plate hemolysis. When the hemolytic power of these venoms was tested on human red blood cells, a large percentage of animals with venoms having a low hemolytic power was also detected. Hemolytic power was much greater when human red blood cells were tested with crystallized venom. The preparation of red blood cells also had an important effect and the use of red blood cells from defibrinated blood is recommended. We conclude that rattlesnake venom has hemolytic power that increases when the venom is crystallized. Red blood cells should be properly prepared for the lysis reactions. We suggest that the lytic power of the venom is related to venom concentration and to the purity of its fractions.

Role of botrocetin in platelet agglutination: formation of an activated complex of botrocetin and von Willebrand factor.

Botrocetin (venom coagglutinin) induces binding of von Willebrand factor (vWF) to platelet glycoprotein Ib (GPIb), resulting in platelet agglutination. A mechanism whereby botrocetin causes vWF to change to an active platelet-agglutinating form is proposed. Incubation of native vWF with botrocetin yielded an increasingly active vWF with slower migration in two-dimensional immunoelectrophoresis but with no apparent change in vWF multimer pattern. The "activated" vWF eluted mainly in the void volume (Vo) (Bio-Gel A-15m column chromatography). Botrocetin eluted in the included volume (Vi). Vo peaks appeared to contain a vWF-botrocetin complex, based on bioassays and immunoassays. 125I-Botrocetin mixed with vWF eluted in two peaks: in the Vo, coincident with active vWF, and in the Vi. With von Willebrand disease (vWD) plasma lacking vWF, 125I-Botrocetin eluted in the Vi only. It did not bind to platelets without vWF. In aggregometric studies, antibodies (Ab) against botrocetin, vWF, and GPIb prevented botrocetin-induced platelet agglutination and caused dissolution of preformed platelet agglutinates. Immunostaining of aggregates with antibotrocetin Ab revealed a positive reaction. Botrocetin appears to act in a two-step manner, first binding with vWF to form a complex, which then binds to GPIb to cause agglutination. All three components, vWF, botrocetin, and GPIb, appear to be required for maintenance of stable platelet agglutinates.

Two toxins from the venom of Trimeresurus tokarensis.

1. Two toxins, Tokaratoxin-1 (TT-1) and Tokaratoxin-2 (TT-2), were isolated from the venom of Trimeresurus tokarensis using gel filtration on a Sephadex G-100 column, followed by chromatography on DEAE-Sephacel and carboxymethyl-cellulose. TT-1 possessed both hemorrhagic and proteolytic activities. However, TT-2 did not show hemorrhagic activity. 2. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing and SDS-PAGE. 3. Molecular weights of TT-1 and TT-2 were 71,000 and 25,400, respectively. Although TT-2 is a basic protein, TT-1 is an acidic protein. 4. Biological activities of TT-1 and TT-2 were inhibited by EDTA, EGTA and o-phenanthroline, suggesting that the toxins are metalloproteins. Atomic absorption analyses indicated that TT-1 contains 2.79 mol Ca/mol protein and TT-2 contains 1.04 mol Ca/mol protein and 1.07 mol Zn/mol protein, respectively. 5. The two toxins degraded the A alpha and B beta chains of fibrinogen. 6. TT-1 induced necrosis in addition to its hemorrhagic activity while TT-2 induced necrosis only.