The effect of plasmin or F.XIa on F.IX complexation with AT in the presence of heparin and aPTT coagulant activity.

The different F.IX derivatives were produced as described in “Materials and Methods.” Aliquots of the samples (containing approximately 3 μg of F.IX protein) were prepared for nonreducing SDS-PAGE without and with incubation with AT (5 μM) and heparin (2 mg/mL) for 30 minutes at 37°C (A and B, respectively). The different F.IX samples were also diluted in HBS/BSA and assayed for their F.IX aPTT coagulant activity (C). A and B illustrate the nonreducing SDS-PAGE analysis of the different F.IX samples without and with incubation with AT and heparin, respectively: F.IX (lane 1), F.IXa (lane 2), plasmin-treated F.IX (lane 3), plasmin-treated F.IXa (lane 4), F.IX treated with plasmin then F.XIa (lane 5). B also shows AT and heparin alone (lane 6). The migration positions of the molecular weight standards (in kd) are shown to the left of A and B. C illustrates the log F.IX aPTT clot time (in seconds) versus the log F.IX derivative concentration (in pmol/L): HBS/BSA buffer control (closed circles); F.IX (open circles); plasmin-treated F.IX (open downward triangles); F.IX treated with plasmin then F.XIa (open squares); F.IXa (closed downward triangles); plasmin-treated F.IXa (closed squares).