ACTIVATION OF FACTOR-X BY FACTOR-IXA AND FACTOR-VIII SPECIFIC ASSAY FOR FACTOR-IXA IN PRESENCE OF THROMBIN-ACTIVATED FACTOR-VIII

Abstract

The activation of [human] factor X by the intrinsic pathway of blood coagulation using a new assay of factor X activation was studied. When factor X tritiated in its sialic acid residues was activated, activation could be measured by the release of tritiated activation peptide and the initial rate of activation could be determined under varying conditions. In the presence of phospholipid and Ca ions, factor IXa activated factor X slowly without factor VIII, and this activation was blocked by a specific factor IX inhibitor. Strong evidence that factor IXa is the enzyme responsible for factor X activation by the intrinsic pathway was provided. The role of factor VIII was also investigated. Factor VIII could be reproducibly thrombin activated and then stabilized by the addition of 2 mM benzamidine hydrochloride. Inactivation may be due to proteolysis. Neither unactivated nor thrombin-activated factor VIII produced factor X activation without factor IXa. With a constant level of factor IXa, factor X activation was directly proportional to the level of activated factor VIII. With a constant level of activated factor VIII, factor X activation was proportional to the factor IXa concentration. This observation was exploited to develop a specific, sensitive assay for factor IXa.