IC50 determination of hit molecules as selected by the SPR-based assay. Increasing concentrations of compound are incubated with 4 nM of FVIII, which is next injected in a Biacore T100. The residual binding of FVIII to a 10/90 M/M PS/PC membrane surface in the presence of small-molecule inhibitors (flowcell 2), corrected for binding to a 100% PC (flowcell 1), relative to the FVIII binding in the absence of small-molecule inhibitors is given here. Lines indicate a nonlinear fit of the data points (sigmoidal dose-response fit). Shown are averages ± standard deviation for at least 2 independent experiments per point. ◆, A11-8;▲, D7-5; ●, H5-8; ▪, C11-10; ⋄, C8-4; △, D11-5; ○, C8-5; □, B7-5.