Inhibition of Prothrombin, Thrombin and Autoprothrombin C with Enzyme Inhibitors

Abstract

SummaryNumerous chemical enzyme inhibitors were employed to determine the active polar groups in the prothrombin, thrombin and autoprothrombin C molecules. Prothrombin and autoprothrombin C activities were decreased by the reduction of disulfide bonds, but thrombin activity was affected only at higher concentrations of reducing agent. Amino groups were not essential for the activity of autoprothrombin C; however, the blocking of such groups decreased the activities of prothrombin and thrombin. The blocking of carbonyl groups decreased the activity of both prothrombin and autoprothrombin C, but thrombin was not inactivated by hydroxylamine. The activities of none of the three were decreased by sulfhydry] blocking agents. Nonspecific oxidizing agents reduced the activities of all three molecules ; however, oxidizing agents which specifically oxidize sulfhydryl groups, were incapable of decreasing the activities of prothrombin, thrombin or autoprothrombin C. The sulfhydryl groups are not essential for the activities of prothrombin, thrombin or autoprothrombin C.

Enzymology and the Blood Clotting Mechanism

“…X, is rapidly inactivated by 2,3-dimercaptopropan-1-01 (39,128), and by DFP in the cold at 4 : l molar ratio of inhibitor to protein, inactivation being complete in 1 hr, as judged by the rate of hydrolysis of carbobenzoxy phenylalanine nitrophenol ester (128). The coagulant and esterase activity are inhibited by phenylmethylsulfonyl fluoride (PMSF) and by DFP, the rate of inactivation being a function of the protein concentration, and is greatest at high concentrations of X,, PMSF inactivating X, more rapidly than DFP (50).…”

The Reaction of Thiol Compounds with the Vitamin-K Dependent Clotting Factors

“…Carter and Warner (1953) oxidised reduced prothrombin with hydrogen peroxide or potassium ferricyanide to active prothrombin. The inactivation of autoprothrombin C presumably identical with activated factor X with BAL was observed by Caldwell and Seegers (1965). The influence of thiols on factor V activity was not investigated because according to Koppel, Mueller and Olwin (1956) free SH groups are required for this activity.…”

The Inhibition of Activated Factor X with Di:isopropyl Fluorophosphate

“…Similar results were obtained by Marciniak and Seegers (1966). Caldwell and Seegers (1965) tested the effect of various reagents on the coagulant activity of Factor Xa, and one can conclude from their results that Factor Xa was only inhibited by reagents which caused gross modification to its structure. The failure to find inhibition by agents which are known to be specific in their action indicates that not only is this enzyme very different from thrombin but also that it is unique among proteolytic enzymes.…”