Kinetics of Ixolaris/FXa and FVIIa/TF interaction.

(A) Amidolytic assays. Ixolaris (10 nM) and DEGR-FXa (0-10 nM) were incubated for 15 minutes in the presence of S2288 (1 mM) followed by the addition of FVIIa/TF (1 nM). a, Ixolaris; b, Ixolaris plus 0.1 nM DEGR-FXa; c, Ixolaris plus 1 nM DEGR-FXa; d, Ixolaris plus 10 nM DEGR-FXa. (B,C) FIX activation assays: (B) Lane 1, buffer; lane 2, Ixolaris only (10 nM); or lane 3, Ixolaris/DEGR-FXa (10 nM each) were incubated for 15 minutes, followed by addition of FIX (1.2 μM). Five minutes later reaction was initiated by FVIIa/TF (1 nM) (except lane 1, which was not allowed to proceed) and followed as described in Figure7A. (C) Lane 1, buffer; lane 2, Ixolaris (10 nM); or lane 3, Ixolaris/DEGR-FX (10 nM each) were incubated for 15 minutes, followed by addition of FIX (1.2 μM). Five minutes later reaction was initiated by FVIIa/TF (1 nM) (except lane 1, which was not allowed to proceed) and followed as described in Figure 7A. In panels B and C reactions were stopped as described in Figure 7A. 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 gels are shown (n = 3).