Ewa Marciniak

Isolation and some properties of prethrombin and autoprothrombin III

Abstract Purified bovine prothrombin preparations were dissociated by digestion with thrombin. The reaction mixture was separated to obtain an inhibitor, the precursor of autoprothrombin C called autoprothrombin III, and the precursor of thrombin called prethrombin. Prethrombin was purified and found to have an isoelectric point at pH 5.5 and in the ultracentrifuge s020,w = 3.9. It contained 75 more amino acid residues than purified thrombin. Autoprothrombin III was also purified. The observed isoelectric point was pH 4.7, and s020,w = 3.4. After the separation and purification of prethrombin and autoprothrombin III, it was possible to study the activation requirements of each one independently of the other. Autoprothrombin III readily converted to autoprothrombin C in the presence of tissue thromboplastin and calcium ions, but other suitable conditions for activation were also found. The formation of autoprothrombin C was observed to occur slowly in the presence of calcium ions and platelet cofactor I. It is therefore concluded that rapid clotting of blood with tissue extracts as compared with platelets plus plasma is due to the vastly greater quantity of autoprothrombin C produced with tissue extracts than with platelets plus plasma. The conversion of prethrombin to thrombin required only autoprothrombin C; however, the reaction was accelerated by the simultaneous addition of purified Ac-globulin, lipids, and calcium ions. A sequence in blood coagulation occurs in three major steps; namely, (a) the formation of autoprothrombin C, (b) the formation of thrombin, and (c) the formation of fibrin. Purified autoprothrombin III corrected the prolonged one-stage prothrombin time of Stuart plasma and also of “factor VII” deficient plasma.

Some activation characteristics of the prethrombin subunit of prothrombin

Abstract Prethrombin, autoprothrombin III and an inhibitor were separated from purified prothrombin by degradation with thrombin and subsequent chromatography. Prethrombin was refractory to activation with tissue thromboplastin, purified Ac-globulin and calcium ions. Prethrombin activated in physiological saline solutions with only trypsin or purified autoprothrombin C. In 25% sodium citrate solution activation was very slow. This was, however, accelerated with either autoprothrombin C or autoprothrombin III, but not with purified thrombin. The specific activity and amino acid composition of prethrombin preparations was found to be about the same as that of purified thrombin. The autoprothrombin III fraction yielded autoprothrombin C with trypsin, Russells viper venom, and with tissue extracts plus calcium ions, but not in 25% sodium citrate solution.

SENSITIVITY OF THROMBIN AND AUTOPROTHROMBIN C TO SELECTED ENZYME INHIBITORS.

Abstract The enzyme thrombin is sensitive to diisopropylfluorophosphate and phenylmethanesulfonyl flouride, but not to p-toluenesulfonyl-phenylalanyl-chloromethylketone. It is relatively resistant to -SS- reducing agents. Under appropriate conditions the enzyme activity regenerates after reduction. Autoprothrombin C activity is easily destroyed 3y -SS- reducing agents, but resistant to the other three reagents cited above.

The coagulation of blood: Preliminary survey of thrombin and autoprothrombin zymogen structure☆

Abstract Numerous facts are consistent with the conclusion that bovine thrombin zymogen is a dimer of thrombin and is easily dissociated from autoprothrombin III (Stuart factor, F-X, autoprothrombin, prothrombokinase). The latter comprises about 4.4% of the total protein in our preparation. When thrombin forms, at least two peptides are removed. Prethrombin has one N-terminal amino acid in common with thrombin. Autoprothrombin C has an amino acid composition and specific activity similar to autoprothrombin III, but has only half the molecular weight. A likely possibility is that autoprothrombin III is a dimer of autoprothrombin C. Alternatively it could represent one mole of autoprothrombin C and one of F-VII.

Toxicity of Blood Clotting Factors

Pure bovine thrombin was separated from autoprothrombin C, and the lethal intravenous dose for mice weighing 25 g was 0.4 ml of a solution containing 50 units of thrombin per milliliter. Autoprothrombin C was not toxic alone, but with crude cephalin it was fatal. The clotting time of human plasma was only slightly accelerated by autoprothrombin C alone; the clotting time was as short as 5 seconds with a combination of autoprothrombin C and cephalin.