Prothrombin activation: Ac-Globulin, lipid, platelet membrane, and autoprothrombin C (factor X a) requirements

Abstract

Thrombin formation was studied with the use of purified prothrombin, prothrombin complex, prethrombin, Ac-globulin, platelet factor 3, autoprothrombin C, autoprothrombin III (Factor X), and thrombin, With Auto-C alone, there was only a fractional yield of the potential thrombin from purified prothrombin complex or purified prothrombin. This low yield of thrombin was partly due to the simultaneous formation of prethrombin and autoprothrombin II-A, which is a competetitive inhibitor of Auto-C. Echis carinatus venom converted prothrombin completely to thrombin. In a standard reaction mixture consisting of prothrombin, Auto-C, Ac-G, calcium ions and crude “cephalin”, thrombin formation was rapid and complete. By progressively reducing the concentration of any of the five components, there was a corresponding reduction in yield and rate of thrombin formation. From the thrombin yield, the concentration of the limited reactant was determinable. The basic molecular biology, which applies to several clinical conditions, is outlined. With purified prothrombin as a substrate, Auto-C functioned most effectively in thrombin production in the presence of calcium ions, Ac-G, and lipids. The Km' was 9.50 × 10 −6 M prothrombin. The maximum rate of conversion of prothrombin to thrombin by Auto-C was an order of magnitude 120 times greater than that for prethrombin. Ac-G was regarded as a determiner protein becaue it modified the specificity of Auto-C. The lipid requirement was fulfilled equally well by platelet factor 3, cephalin, thromboplastin, and platelet membranes. One of the three basic reactions of blood coagulation may thus be related to the function of membranes. In a mixture consisting of prothrombin, Auto-III, thromboplastin, Ac-G and calcium ions, all of the prothrombin was converted to thrombin. No Factor VII was required.