Three-stage analysis of blood coagulation.

1. Blood-clotting mechanism has been analyzed by a procedure which devotes a separate experimental step to each of the three primary reactions: 1. Prothrombokinase --> thrombokinase 2. Prothrombin --> thrombin 3. Fibrinogen --> fibrin 2. Activation of prothrombin by thrombokinase followed the course of a unimolecular reaction, and the concentration of thrombokinase determined the initial rate. By this relation thrombokinase was measured, and the activation of its precursor was charted. 3. When the activation of prothrombokinase was plotted against time, the experimental points fell close to the theoretical curve for a simple autocatalytic reaction. Moreover, the process was accelerated by seeding with a small amount of crude thrombokinase. It was concluded that the activation of prothrombokinase involves an autocatalytic or chain reaction. 4. The three-stage procedure made possible the separate estimation of the power to activate prothrombin, on one hand, and the capacity to accelerate the transformation of prothrombokinase on the other. Drastic losses of both activities occurred when crude thrombokinase solutions were heated at 60 degrees C., or adsorbed with barium sulfate. 5. The concentration of calcium was important for the normal progress of prothrombin activation, and also for the transformation of prothrombokinase.