Studies in rats on in vitro inhibition and in vivo activity of vitamin-K-dependent carboxylation.

Vitamin-K-dependent procoagulant activity was studied in vitro by characterizing vitamin-K-dependent carboxylation and in vivo by assessing prothrombin complex activity (PCA). The kinetics of endogenous substrate carboxylation were apparently first order. Inhibition of vitamin-K-dependent carboxylation versus antagonist concentration was determined for 2,3,5,6-tetrachloropyridin-4-ol (TCP), phenindione, 2,6-dichloroindophenol sodium (2,6-DIP), 2-chloro-1,4-naphthoquinone (chloro-K3), 2-chloro-3-phytyl-1,4-naphthoquinone (chloro-K1) and warfarin. These compounds represent different chemical classes of anticoagulants that exert their effects via vitamin K antagonism. The percent inhibition versus concentration plots exhibited a sigmoidal shape and were described by the logistic function. The following concentrations were associated with 50% inhibition of vitamin-K-dependent carboxylation: TCP = 1.23 +/- 0.238 microM (mean +/- SD), phenindione = 19.0 +/- 11.2 microM, 2,6-DIP = 116 +/- 39.2 microM, chloro-K3 = 146 +/- 62.1 microM, chloro-K1 = 285 +/- 89.3 microM and warfarin = 6.63 +/- 2.82 mM. The slope parameters of the percent inhibition versus concentration plots for chloro-K1 and phenindione were different from those for the other antagonists, suggesting a mechanism of action consistent with other data in the literature, i.e. competitive antagonism of the vitamin-K-dependent carboxylase. The relationships between in vitro parameters of vitamin-K-dependent carboxylation of precursor proteins and in vivo indices of rate of production of vitamin-K-dependent coagulation factors were studied in control animals and animals pretreated with compounds perturbing hepatic function. The in vivo rate of synthesis of prothrombin complex activity and the circulating levels of PCA were correlated with the in vitro first-order rate constant of vitamin-K-dependent carboxylation, but not with the amount of precursor proteins present. The results of these studies suggest that the rate of vitamin-K-dependent carboxylation is intimately involved in the regulation of levels and activity of vitamin-K-dependent coagulation factors.