A Highly Active Material that Promotes Conversion of Prothrombokinase Complex. ∗

Abstract

A procedure has been described 1 for analyzing blood coagulation in three experimental steps: 1. activation of prothrombokinase; 2. activation of prothrombin; 3. coagulation of fibrinogen. Crude thrombokinase, besides activating prothrombin, also accelerated conversion of prothrombokinase. Both activities where lost upon adsorption with barium sulfate.

It is now reported that a material, prepared by elution from barium sulfate, promotes the activation of prothrombin, and also the conversion of crude prothrombokinase. Beginning with a solution of bovine plasma globulins, fraction “A” was adsorbed on Hyflo (Johns-Manville) and eluted with phosphate-potassium chloride solution. The unadsorbed globulins were exposed to adsorption by barium sulfate. From the latter, two successive fractions were eluted, with O.2-0.3M phosphate, pH 6.6, and with 0.6M phosphate, pH 7.9. The pH 6.6 eluate had more prothrombin. From the pH 7.9 eluate, proteins soluble in 0.35, but precipitable by 0.45 saturated ammonium sulfate were selected as the “converter fraction.” This involved 3 extractions and 20 precipitations. Proteins soluble in 0.45, but precipitable by 0.60 saturated ammonium sulfate, constituted the “thrombin fraction” (3 extractions, 7 precipitations). Fifty-five liters of plasma yielded 10 ml of each fraction. Both were dialyzed simultaneously in the same beaker.

Activations were studied with calcium present, giving results summarized in Table I. At high dilution, the converter hastened both the activation of prothrombin and the conversion of crude prothrombokinase. In comparing different preparations, converter activities estimated by the 2-stage and 3-stage procedures were roughly parallel. This is compatible with the view that the tests measured two different effects of the same substance.

In contrast, converter activity did not parallel thrombin activity. The ratio of converter to thrombin activity was 100 times as great in the converter fraction as in the thrombin fraction. Conceivably, varieties of thrombin can exist with different converter activities, or some factor modifies the behavior of thrombin in one of the fractions. If, as must be contemplated, the converter is distinct from thrombin, the results suggest how difficult would be its elimination from thrombin by salting-out. Moreover, the converter is manifest in sufficiently minute amount to cause confusion, even as a small contamination in a highly purified thrombin.