FIGURE 7.
SPR analysis of the effect of exosite ligands on thrombin binding to immobilized γ′-peptide. Biotinylated γ′-peptide was adsorbed to the flow cell of a streptavidin-CM5 chip to ∼700 RU. To control for nonspecific binding, a flow cell containing only streptavidin was used. A, 1 μm FPR-α-thrombin was injected in the presence of increasing concentrations of HD1 (○), HD22 (●), or hirudin-(54–65) (▵) at a rate of 20 μl/min, and maximal RU values determined at each concentration of exosite ligand were corrected by subtracting the background RU from the control flow cell. Symbols represent the means ± S.E. of three experiments, while the lines represent non-linear regression analysis of the data. HD22, HD1, and hirudin-(54–65) inhibit FPR-α-thrombin binding with Kiobs values of 2.6 ± 0.4, 0.7 ± 0.1, and 1.3 ± 0.3 μm, respectively. B, the experiment was repeated using 2 μm γ-thrombin in place of FPR-α-thrombin. Hirudin-(54–65) has no effect on γ-thrombin binding, whereas the Kiobs values for HD1 and HD22 are 20.8 ± 2.1 and 3.2 ± 0.6 μm, respectively.