Fig 3. Binding of thrombin to immobilized GpIbα269-286ppp.

A. Increasing concentrations of thrombin up 4 μM were injected at a rate of 25 μl/min into flow cells of a streptavidin-coated CM5 biosensor chip containing adsorbed biotinlylated GpIbα269-286ppp. Response units (RU) are plotted versus time for the indicated thrombin concentrations (in nM) in triplicate (inset). RU values at equilibrium (Req) were determined and plotted versus the input thrombin concentration. The data were analyzed by nonlinear regression (line) to a rectangular hyperbola to determine K_d. Symbols represent mean ± SD for from 3 runs. B. Thrombin (250 nM) was injected into flow cells containing immobilized biotinlylated GpIbα269-286ppp in the presence of 0–5000 nM dabigatran (circles), argatroban (triangles), or DAPA (squares). Req values were determined, converted to a fraction of that measured in the absence of inhibitor (Bound/Bound₀), and plotted versus the inhibitor concentration. Data points indicate mean ± SD for 3 determinations, and the lines represent nonlinear regression analyses.