Factor VIIa-tissue factor (TF) complex formation in the presence of EDTA or divalent cations (Me(2+)) was investigated. The influence of Me(2+) on the amidolytic activity of factor VIIa and factor VIIa-TF complex was evaluated using low molecular weight synthetic substrates possessing substituted aminonaphthalenesulfonamides as detecting groups. Factor VIIa expressed low amidolytic activity in the presence of EDTA. In the presence of EDTA and saturating concentrations of TF, the amidolytic activity of factor VIIa was increased approximately 90-fold. Gel electrophoresis and sedimentation velocity studies demonstrated complex formation between factor VIIa and TF in the presence of EDTA. Substrate titration curves obtained at fixed factor VIIa and TF concentrations gave sigmoidal shapes, indicating that substrates influenced factor VIIa amidolytic activity in the presence of TF. In the absence of Me(2+), the K-D,K-app of the factor VIIa-TF complex was influenced by substrate structure and varied from 3.9 to 34 nM. All Me(2+) used increased the amidolytic activity of factor VIIa approximately 8-fold compared with experiments in the presence of EDTA. The K-D,K-app values for factor VIIa-Ca2+ complex and factor VIIa-Mn2+ complex were independent of substrate and were 270 and 40 mu M, respectively. The K-D,K-app for factor VIIa-Mg2+ complex varied from 3 to 12 mM and was substrate structure dependent. The presence of TF had no influence upon the K-D,K-app for the factor VIIa-Ca2+ complex. The amidolytic activity of factor VIIa was enhanced by TF significantly in the presence of Ca2+, and similar results were obtained with Mg2+ and Mn2+. The K-D,K-app observed for the factor VIIa-TF complex was influenced by metal ions. In the presence of Ca2+, this constant was found to be 1.1 nM, whereas in the presence of Mn2+ it was 5.1 nM. The K-D,K-app was independent of substrate structure for both metal ions. TF and Me(2+) had no significant influence on the K-M for substrate hydrolysis. These data indicate that (1) complex formation between factor VIIa and TF while Me(2+) dependent does not have an essential requirement for metal ions, (2) the amidolytic activity of the factor VIIa-TF complex is increased in the presence of Me(2+), and (3) cooperative interactions exist between factor VIIa, Me(2+), substrate, and TF.
