Equinatoxin, isolated from Actinia equina, caused aggregation of washed rabbit platelets at a concentration as low as 0.01 ng/ml. ATP was released, but no formation of thromboxane B2 in challenged platelets. The aggregation was resistant to indomethacin or creatine phosphate/creatine phosphokinase or PAF antagonist. The aggregation was inhibited by imipramine, sodium nitroprusside, mepacrine, theophylline, prostaglandin E1 and EDTA. However, heparin and tetracaine were without any inhibitory effect. Verapamil suppressed both the aggregation and release reaction caused by equinatoxin in calcium concentrations from 0.01 to 15 mM. High concentrations of equinatoxin caused progressive cell lysis. It is concluded that equinatoxin-induced platelet aggregation is independent of ADP, thromboxane or PAF pathway. Phosphoinositide breakdown by phospholipase C is postulated to accomplish this phospholipase A2-independent platelet aggregation by equinatoxin.

Equinatoxin is a lethal protein isolated from a sea anemone, Actinia equina. The toxin (0.1-3 micrograms/ml) caused an initial inhibition followed by an augmentation of contractions and beating rates in the isolated guinea-pig atrium. The inhibitory phase was transient (30-60 sec), while the stimulant phase lasted for about 30 min. The treated atrium showed tachyphylaxis to the toxin. The inhibitory effect of the toxin was diminished by tetrodotoxin and atropine and abolished by 8-phenyltheophylline or mepacrine. Dipyridamole, which blocks the uptake of adenosine in the heart, enhanced the inhibitory effect. The stimulant effect of the toxin was inhibited by indomethacin or mepacrine and abolished by a combination of both, but was not inhibited by propranolol. Bioluminescent assay performed during the inhibitory phase showed an increased release of ATP and radioimmunoassay during the stimulant phase revealed an increased release of prostaglandin E2 from the treated atrium. These results suggest that the cardiac inhibitory effect of equinatoxin is mainly due to release of adenyl compounds, while the cardiac stimulant effect of the toxin may result from the liberation of arachidonic acid and subsequent formation of prostaglandins in the guinea-pig atrium.

On perfusion through isolated lungs from male Sprague-Dawley rats, equinatoxin caused a dose-dependent increase in the wet to dry weight ratio. Ratios were significantly elevated above control values at equinatoxin concentrations of 80–200 ng/ml. The increased ratios were accompanied by an increase in the permeability of the lung vasculature. When equinatoxin was perfused through isolated lungs at concentrations of 100 ng/ml or greater, significantly more [3H]polyethylene glycol (PEG; approximately 900 mol. wt) was retained in the extravascular space as compared to controls. Perfusion pressures of the lung were significantly elevated above controls at equinatoxin concentrations greater than 100 ng/ml. These effects of equinatoxin were not mediated by degranulation of mast cells, as preperfusion of the lung with 100 or 200 μM Na cromolyn or 1 μM lanthanum chloride did not modify the pulmonary response to equinatoxin. At concentrations of equinatoxin below 150 ng/ml the fluid movement appears to be restricted primarily to intracellular, or possibly interstitial, spaces, as no significant amounts of [3H]polyethylene glycol were recovered by tracheal lavage. At concentrations of equinatoxin equal to or greater than 150 ng/ml, significant amounts of PEG were washed from the trachea. As it is a potent inducer of pulmonary edema, equinatoxin may become an important probe to study fluid regulation in the lung.

Following the bite by Rhabdophis subminatus, preliminary studies were instituted on its venom to determine the mechanism of the clinical poisoining. A method for milking these snakes is presented. The i.v. ld50 in mice was 1.29 mg/kg. Three peaks were obtained on Sephadex G-100 and although none were lethal by themselves, a combination of two of the peaks produced immediate death in mice. The venom had no phosphodiesterase, fibrinolytic thrombin-like activities, but it did have some phospholipase activity. Further biochemical and pharmacological studies are in progress.