Effect of DHG on the affinity of the factor IXa–factor VIIIa complex on phospholipid vesicles. The apparent affinity (K_d(app)) of the factor IXa–factor VIIIa interaction was determined using enzymatic activity to detect complex formation in the absence (•) and presence of 4 (○) or 6 (▪) nM DHG. The rate of factor X activation by factor IXa–factor VIIIa was determined by adding increasing concentrations of factor IXa (0 to 30 nM) into a reaction containing 0.15 nM factor VIIIa, 200 nM factor X, and 50 μM PC/PS vesicles. Mean values were plotted versus factor IXa concentration with error bars representing ± SD (n = 4 to 6). The K_d(app) was determined by fitting the data to a single site–binding model. The K_d(app) and B_max values ± SE were 1.2 ± 0.1 nM and 21.9 ± 0.3 nM/min, 10.0 ± 0.3 nM and 24.4 ± 0.3 nM/min, and 16.9 ± 1.4 nM and 23.2 ± 0.9 nM/min in the presence of 0, 4, and 6 nM DHG, respectively.