Activity decay of WT and mutant factor VIIIa in the absence and presence of factor IXa. (A) Thrombin-activated factor VIIIa (4 nM) was incubated at 23°C, aliquots were taken at indicated time points, and activity was measured by factor Xa generation assay. (B) Activity decay of WT and mutant factor VIIIa in the presence of factor IXa. Factor VIII (4 nM) was activated with thrombin in the presence of 40 nM factor IXa, aliquots were taken at indicated time points, and activity was measured by factor Xa generation assay. Results are shown for WT (, ×), Glu272Ala (□), Glu272Val (■), Asp519Ala (○), Asp519Val (●), Glu665Ala (△), Glu665Val (▲), Glu1984Ala (◇), and Glu1984Val (♦). Data were fitted by nonlinear least squares regression, and each point represents the value averaged from 3 separate determinations.