Figure 4.

(A) Effect of 2007–2016 peptide on FVIII LC binding to FX. FVIII LC (200 nM) was mixed with various concentrations of the 2007–2016 peptide (open circles) or scrambled sequence peptide (closed circles) and mixtures were then incubated with immobilized FX (50 nM). Bound FVIII LC was detected using a biotinylated anti-FVIII LC antibody. The absorbance value corresponding to FVIII LC binding to FX in the absence of competitor was defined as 100%. The data were fitted (dashed line) by non-linear least squares regression using Equation 2 (competitive inhibition model). Experiments were performed at least three separate times and mean values are shown. (B) Direct binding of 2007–2016 peptide to FX as determined by SPR. Various amounts of the 2007–2016 peptide (open circles) or scrambled sequence peptide (closed circles) were injected onto a sensor chip containing immobilized FX for 2 minutes, followed by a change to running buffer for 2 minutes. Maximum values for association of the peptide were plotted as a function of peptide concentration, and the data were fitted using the Equation 1 (single-site binding model). Experiments were performed at least three separate times and mean values are shown.

(C) EDC cross-linking of 2007–2016 peptide and FX. Panel a) FX (200 nM) was incubated with biotinylated 2007–2016 peptide (200 μM) in the presence of indicated concentrations of EDC at 23 °C for 2 hours, followed by immunoblotting using streptavidin. Panel b) Cross-linked products formed with FX (200 nM) and indicated concentrations of the 2007–2016 peptide with EDC (2 mM). Panel c) FX (150 nM) and biotinylated 2007–2016 peptide (100 μM) reacted with EDC (2 mM) in the presence of indicated concentrations of the unlabeled 2007–2016 peptide and were immunoblotted. Experiments were performed at least three separate times and typical results are shown.