FX and FXa, but not DEGR-FXa, are scaffolds for Ixolaris: recombinant FIX activation assays.

(A) Ixolaris (10 nM) was incubated with DEGR-FX derivatives (0-10 nM) for 15 minutes at 37°C followed by addition of FVIIa/TF (1 nM) and incubated for additional 15 minutes at 37°C. Reactions were initiated with recombinant FIX (BeneFIX, 1.2 μM). Ninety minutes later reactions were stopped with Laemmli buffer and proteins were separated by 4% to 12% SDS-PAGE. Lane 1, after addition of FVIIa/TF reactions were not allowed to proceed (0 minutes incubation time); lane 2, buffer (no inhibitor/no scaffold); lane 3, Ixolaris plus DEGR-FX (0.1 nM); lane 4, Ixolaris plus DEGR-FX (1 nM); lane 5, Ixolaris plus DEGR-FX (10 nM); lane 6, Ixolaris plus DEGR-FXa (0.1 nM); lane 7, Ixolaris plus DEGR-FXa (1 nM); lane 8, Ixolaris plus DEGR-FXa (10 nM); lane 9, Ixolaris plus des-Gla-DEGR-FXa (1 nM); lane 10, Ixolaris plus des-Gla-DEGR-FXa (10 nM). The bands correspond to (from the top): uncleaved FIX (FIX), the heavy chain of FIXα (FIXα HC), the heavy chain of FIXaβ (FIXa HC), and the light chain of FIXa (FIXa LC). The activation peptide is not detected. Typical gel is shown (n = 8). FX, FXa, and des-Gla-FXa only (tested up to 20 nM each), or Ixolaris (tested up to 200 nM) did not affect FIX activation by FVIIa/TF (not shown). (B) Results of the band densitometry for FIXa HC (A), showing inhibition of FVIIa/TF activity by Ixolaris/DEGR-FXa (●), Ixolaris/DEGR-FX (▪), but not Ixolaris/des-Gla-DEGR-FXa (▴).