Proteolytic activation of human factors IX and X by recombinant human factor VIIa: effects of calcium, phospholipids, and tissue factor

• Published in: Biochemistry (Easton) Vol. 29; no. 40; pp. 9418 - 9425
• Main Authors: Komiyama, Yutaka, Pedersen, Anders H, Kisiel, Walter
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-10-1990
• Subjects: Biological and medical sciences; Blood coagulation. Blood cells; Calcium - pharmacology; Coagulation factors; Factor IX - metabolism; Factor VIIa - metabolism; Factor VIIa - pharmacology; Factor X - metabolism; Fundamental and applied biological sciences. Psychology; Humans; In Vitro Techniques; Kinetics; Molecular and cellular biology; Phospholipids - pharmacology; Thromboplastin - pharmacology; Tumor Cells, Cultured - drug effects; Tumor Cells, Cultured - metabolism; Human; Calcium; Tissue factor; Liposome; Phospholipid; Activation; Ultracentrifugation; Factor VII; Factor X; Factor IX; Cell line; Tumor; Kinetic parameter; Coagulation factor; ELISA assay
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00492a016

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 microM 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 microM phospholipid, whereas the Km for factor X activation varied with phospholipid concentration, reaching a minimum at 7-20 microM phospholipid. At all concentrations of added phospholipid, the kcat/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.