Effect of HNE or F.XIa on F.IX complexation with ATIII in the presence of heparin and aPTT activity. The different F.IX samples were generated as described in the Materials and Methods. Aliquots of the samples were solubilized before and after incubation with ATIII (5 μmol/L) and heparin (2 mg/mL) for 30 minutes at 37°C for nonreducing SDS-PAGE (A and B, respectively). In addition, these same F.IX samples were also diluted in HBS/BSA and assayed for aPTT clotting activity (C). (A and B) Nonreducing SDS-PAGE analysis of F.IX derivatives before and after incubation with ATIII in the presence of heparin, respectively: F.IX alone (lane 1), F.IXa (lane 2), F.IX treated with HNE (lane 3), F.IXa treated with HNE (lane 4), and F.IX treated with HNE then F.XIa (lane 5). Also shown in (B) is ATIII and heparin alone (lane 6). A 5% to 15% linear polyacrylamide gradient gel was used and the positions of the molecular weight markers (in kilodaltons) are indicated at the left of (A) and (B). (C) Log aPTT clot time (in seconds) versus log F.IX derivative concentration (in picomoles per liter): HBS/BSA buffer alone (•); F.IX (○); HNE-treated F.IX (▿); F.IX treated with HNE then F.XIa (□); F.IXa (▾); F.IXa treated with HNE (▪).