FIGURE 2.

A, activity decay of WT and mutant factor VIII. Factor VIII (4 nm) was incubated at 55 °C and at the indicated times and aliquots were removed and assayed for activity by factor Xa generation assays as described under “Materials and Methods.” Results are shown for WT (dashed line, open circles), R282A (open triangles), S524A (open squares), N684A (open diamonds), R531A (closed circles), S650A (closed triangles), E287A (closed squares), and D302A (closed diamonds). Results for other variants are presented as supplemental data. B, activity decay of WT and mutant factor VIIIa in the presence of factor IXa. Thrombin-activated factor VIIIa (4 nm) in the presence of 40 nm factor IXa was incubated at 23 °C, aliquots were taken at the indicated time points and activity was measured by the factor Xa generation assay as described under “Materials and Methods.” Results are shown for WT (dashed line, open circles), R282A (open triangles), S524A (open squares), Y1792F (open diamonds), N684A (closed circles), Y1786F (closed triangles), R531A (closed squares), E287A (closed diamonds), and D302A (hatched circles). Results for other variants are presented as supplemental data. Results for selected fast decay variants are shown in an expanded scale in the inset. C, activity decay of WT and mutant factor VIIIa in the absence of factor IXa. Thrombin-activated factor VIIIa (4 nm) was incubated at 23 °C, aliquots were taken at the indicated time points and activity was measured by factor Xa generation assay as described under “Materials and Methods.” Results are shown for WT (dashed line, open circles), N684A (open triangles), Y1786F (open squares), Y1792F (open diamonds), R282A (closed circles), and S524A (closed triangles). The inset shows an expanded scale for the early time points. Data were fitted by non-linear least squares regression and each point represents the value averaged from three separate determinations.