Outstanding Characteristics of Thrombokinase Isolated from Bovine Plasma

Abstract

Thrombokinase has been isolated from bovine plasma by a procedure which begins with the highly purified product of a previously described method, chromatographs it on DEAE-cellulose, and then fractionates it by continuous flow electrophoresis, yielding 0.2 mg per liter of oxalated plasma. The electrophoretic fraction has shown a single boundary in the ultracentrifuge; and its esterase activity on toluenesulfonylarginine methyl ester has been about the same as that of thrombokinase previously isolated by repeated electrophoretic fractionations. Thrombokinase is a euglobulin with minimum solubility near pH 5.0. It is most stable within the pH range 7.5 to 9.5; but there is also a peak in the stability curve near pH 1.8. A few micrograms of thrombokinase per milliliter can activate prothrombin in the presence of EDTA. A few thousandths of a microgram causes rapid production of thrombin in the system: prothrombin, thrombokinase, calcium chloride, phosphatide, "accelerator." But, thrombokinase has less than 1/175 the proteolytic activity of crystallized trypsin.Thrombokinase was first isolated from bovine plasma by a series of three electrophoretic fractionations (1). Later, it was reported (2, 3) that chromatography on DEAE-cellulose offered a less arduous means for attaining a comparable specific activity. However, it has been found that the chromatographic preparation is not homogeneous electrophoretically. Now, one chromatographic step and one electrophoretic step have been incorporated into the procedure for isolation of thrombokinase.Thrombokinase so prepared, has about the same esterase activity on toluenesulfonylarginine methyl ester (TAMe) as thrombokinase prepared by the more laborious procedure; and it sediments in the ultracentrifuge with a single, grossly symmetrical boundary. A few of its outstanding enzymatic and physicochemical properties are herein delineated.

The Role of Proaccelerin in Human Blood Coagulation. Evidence that Proaccelerin Is Converted to a Prothrombin-converting Principle by Activated Stuart Factor: With Notes on the Anticoagulant Action of Soybean Trypsin Inhibitor, Protamine Sulfate, and Hexadimethrine Bromide*

“…The characteristics of the prothrombin-converting principle generated by the interaction of activated Stuart factor and proaccelerin are similar to those proposed for thrombokinase by Milstone and associates (25,27,34). The substance generated converted prothrombin to thrombin directly without cofactors other than calcium and in low concentration.…”

“…Through the kindness of Dr. Milstone we were able to perform experiments with the clot-promoting substance he has isolated from bovine plasma (34). This substance corrected the defect in the plasma of a patient congenitally deficient in Stuart factor.…”

The preparation of activated Factor X and its action on prothrombin

“…The action of coagulant protein of Russell's-viper venom on Factor X appears to be one of progressive proteolysis, as is suggested by the range of molecular weights obtained for different preparation of Factor Xa. A similar progressive degradation of human Factor X was observed by Aronson & Mustafa (1971), and Milstone et al (1971) have found that chymotrypsin degrades bovine thrombokinase in such a way that its coagulant activity is lost before its esterolytic activity. The activation of the Factor X by the venom involves cleavages of both the heavy and the light chain of the zymogen; this is in contrast to the relatively minor changes caused by the activation of Factor X by the intrinsic system (Fujikawa et al, 1971).…”

XIV. Prothrombinase Complex

“…Our conclusions were based on several considera tions in the same year Seegers, Mar ciniak and Cole 1962;Marciniak and Seegers 1962;. One year later, Milstone (Milstone 1963) had the experimental data for arriving at the same conclusion. He called pri mary factors prothrombin and thrombokinase and accessory factors calcium ions, phospholipids, and accelerator.…”