A STUDY OF THE EFFECT OF THE AFLATOXINS ON BLOOD CLOTTING

Abstract

The structural configurations of the synthetic coumarins have been shown to be of major importance in their action on blood clotting. In view of the similarities in the structures of the synthetic coumarins and the aflatoxins, attempts have been made to compare the blood clotting activities exhibited by 4-hydroxycoumarin with similar effects obtained with the aflatoxins. The effect of a balanced diet, which had been infected by a toxic strain of Aspergillus flavus, was fed to a set of rats. The blood clotting times of these animals and their controls were determined. A "Thrombotest" reagent, which estimates simultaneously quantities of blood clotting factors II, VII, IX and X present in a known values of blood, was used. The clotting time of blood withdrawn from the poisoned animal was prolonged by approximately 85% of the normal time. There was a suggestive evidence that the increase in blood clotting time was due to the action of aflatoxins which are metabolites of Aspergillus flavus. The mouldy diet was therefore extracted with methanol and the mixture of aflatoxins purified using chloroform in thin layer chromatography. The different effects on blood clotting time of (a) the infected diet (b) the aflatoxin mixture, and (c) pure aflatoxin B₁, were compared. The average percentage increase in clotting time in the three cases were the same. The antagonism of therapeutic amounts of 4-hydroxycoumarin by vitamin K preparations was studied in rats, and comparison was made with the effect of these vitamin K preparations on the blood clotting of Aflatoxin B₁-treated animals. Vitamin K₁ and, to a lesser degree, 2-methyl-1:4-naphthoquine (Konadione) were effective in decreasing the prolonged clotting times which were induced by 4-hydrocoumarine and Aflatoxin B₁. By using the "thromboplastin" reagent, factors II and VII were found to be deficient in the plasma obtained from the blood of the aflatoxin-poisoned animal. A study of the "In vitro" synthesis of these two factors by rats liver slices was attempted. Inhibition of the syntheses of these factors was caused by the process of aflatoxin and also by the administration of 4-hydoxycoumarine. In each case, reversal of inhibition with vitamin K₁ has also been demonstrated. It was necessary to investigate whether the deficiency of one or both of factors II and VII was responsible for this prolongation of blood clothing. For this purpose, thromboplastin was replaced with viper venom in the clotting time determinations of plasma. Only the depression of prothrombin content of the plasma was measured. Some liver function tests were performed on the experimental rats when the effects of aflatoxin B₁ and 4-hydroxycoumarin on blood clotting were maximal, side by side with their controls, in order to determine whether prothrombin was being destroyed in the parenhymal cells of the liver or whether the aflatoxins were setting like the coumarins by competing with Vitamin K the latter being an essential co-factor in the production of prothrombin in the liver cells.