FIGURE 6.
Competitive binding of the BR peptide and FXa to FV-810. A, FXaS195A (●) or zymogen FXS195A (▴) was titrated into reaction mixtures containing 30 nm OG488-BR, 20 nm FV-810, and 50 μm PCPS in assay buffer at 25 °C. Changes in OG488-BR anisotropy were measured, and lines were drawn as described with the fitted constants Kd = 1.8 ± 0.2 nm for FXa and n = 1.1 ± 0.07 mol of FXa/mol of FV-810 assuming the binding constants for OG488-BR determined in Fig. 2A. B, reactions containing 1.4 μm prethrombin-2, 3 μm DAPA, 50 μm PCPS, 5 nm FV-810, and 0 nm (■), 125 nm (●), 250 nm (▴), 500 nm (♦), or 1 μm (▾) BR peptide were prepared in assay buffer at 25 °C. Reactions were initiated by the addition of 1–50 nm FXa, and thrombin generation was monitored as described under “Experimental Procedures.” Experimental data were fitted to a model for tight binding with calculated values of Kd = 2.0 ± 0.2 nm for FXa and Kd = 34.2 ± 3.6 nm for the BR.