The effect of HNE on F.IX and F.IXa as determined by aPTT clotting assay. Purified F.IX or F.IXa (4 μmol/L of each) was incubated either alone (control) or with HNE (400 nmol/L) in HBS/Ca at 37°C. After various times, two simultaneous aliquots of the same reaction mixtures were withdrawn and assayed for either aPTT clotting activity after the addition of 10 μmol/L AAPV-CMK (A and B) or analyzed by reducing SDS-PAGE (see Fig 2 below). The relative F.IX and F.IXa aPTT clotting activity (normalized to the time = 0 sample in each case) versus incubation time with (•) or without (○) added HNE is plotted in (A) and (B), respectively.