PROTEOLYTIC ACTIVATION OF HUMAN FACTOR-IX AND FACTOR-X BY RECOMBINANT HUMAN FACTOR-VIIA - EFFECTS OF CALCIUM, PHOSPHOLIPIDS, AND TISSUE FACTOR

Previous studies indicated that factor VIIa, in complex with tissue factor, readily activates either factor X or factor IX in the presence of calcium ions. In order to assess the relative physiological importance of the activation of factor IX versus the activation of factor X by recombinant factor VIIa, we have obtained steady-state kinetic parameters for the factor VIIa catalyzed activation of factor IX and factor X under a variety of cofactor conditions that include calcium alone, calcium and phospholipids, calcium, phospholipids, and tissue factor apoprotein, and calcium and cell-surface tissue factor. Calcium alone stimulated the activation of factors IX and X by factor VIIa maximally at 1 and 2.5 mM, respectively. In the presence of 25 μM phospholipids, maximal rates of factor IX and factor X activation were achieved at 2.5-5 mM calcium. With calcium alone, or with phospholipid and calcium, the initial rates of factor IX activation by factor VIIa were significantly higher than that observed for factor X. Kinetic studies revealed that the Km for the factor VIIa catalyzed activation of factor IX was essentially constant in the presence of 5 mM calcium and 1-500 μM phospholipid, whereas the Km for factor X activation varied with phospholipid concentration, reaching a minimum at 7-20 μM phospholipid. At all concentrations of added phospholipid, the kcal/Km ratio for the activation of factor IX by factor VIIa appeared to be considerably greater than that observed for the activation of factor X. In the presence of 5 mM calcium and varying concentrations of factor VIIa-relipidated tissue factor apoprotein complex (9-600 pM), kcat/Km values for factor IX activation were 2-9-fold greater than that obtained for factor X activation. In addition, in the presence of 5 mM calcium, factor IX activation by a complex of factor VIIa and endothelial cell tissue factor was approximately 3-fold more efficient than factor X activation. In contrast, factor VIIa bound to cell-surface tissue factor on monolayers of a human bladder carcinoma cell line activated factor X 4-fold more efficiently than factor IX in the presence of 5 mM calcium. These results indicate that factor IX appears to be the preferred substrate for factor VIIa under a variety of cofactor conditions and suggest that the activation of factor IX by factor VIIa may be an important reaction in normal hemostasis. © 1990, American Chemical Society. All rights reserved.