A number of high-potency purified prothrombin preparations,61 in 0.2-0.5 per cent solution in borate buffer (pH = 7.7), maintain for days or weeks a stable thrombin-forming ability, whether (a) with buffer alone, (b) with brain thromboplastin suspension, (c) with CaCl2. Nevertheless, they all contain a trace of thrombin and continue to activate "spontaneously" at a very slow rate. Optimal addition of Ca-salt somewhat accelerates this and usually leads to maximal (complete) thrombin formation in 2-11 days at room temperature. Except in a few cases, where ionized Ca++ is demonstrable, thromboplastin, alone, is without effect, but added with calcium, it completes the activation in a matter of minutes or hours depending principally upon the concentration used.

Experimental analysis of the activation process stresses the participation of (1) Ca-ions, (2) thromboplastic P-lipid factor (cephalin), (3) plasma and tissue tryptase (proteolytic enzyme). Each of these factors is studied in detail with reference to mode of action, optimal concentration, side-effects, and relation to inhibitors.

Inhibition of prothrombin activation may be considered under the following heads: (1) "decalcifying" agents (e.g., oxalates, citrates, etc.), which (a) depress Ca-ionization and thus prevent thrombin formation, and (b), under special circumstances, reverse the process of activation; (2) "antithromboplastic" (? anticephalin) agents, (e.g., heparin, and probably soybean trypsin-inhibitor, to some extent); (3) "antitryptase" agents (e.g., crystalline trypsin-inhibitors from pancreas and soybean), which inhibit the thromboplastic enzyme (accessory factor). Excess Ca++ slows rate of thrombin formation.

The evidence suggests that thrombin formation proceeds via an "intermediary" calcium-prothrombin-cephalin (thromboplastic phosphatide) complex or compound. The amounts of (a) thromboplastic P-lipid (cephalin or "thromboplastin") and (b) Ca++ determine both the rate of activation and the final thrombin yield. However, the ultimate ("ripe") thrombin owes none of its activity to the presence of any calcium or phospholipid.

The three types of activator (Ca, thromboplastin, and thromboplastic enzyme) occur as "trace impurities" in prothrombin preparations, but Seegers’ most purified materials are tryptase-free. Tryptase (and trypsin) are "thromboplastic" only in the presence of adequate calcium and phospholipid factors, which may, however, be "mobilized" from protein combination, including prothrombin. In this way the two basic activators are, in a sense, "catalyzed" in their prothrombin-activating reactions.

The significance of proteolytic actions (fibrinolysis, fibrinogenolysis, prothrombinolysis, and sometimes thrombinolysis) by natural tryptase of plasma and tissue origin is investigated and discussed in relation to the broader aspects of the blood-coagulation problem.

ACKNOWLEDGMENTS We are especially indebted to Dr. W. H. Seegers (Wayne University, Detroit) for his help and advice with regard to prothrombin preparations. The project owes much also to the workers of a number of other laboratories who have assisted in supplying materials (see Reagents) and to whom we render a sincere appreciation. The human plasma fractions used in this work were prepared from blood collected by the American Red Cross under contract between Harvard University and the Office of Scientific Research and Development. The figures are reprinted by permission of the original publishers cited. This paper is the fourth of a series of studies on "Enzymes and enzyme-inhibitors in relation to blood-coagulation and hemorrhagic diseases," aided by a grant from the John and Mary R. Markle Foundation.