The basic amino acids, but not the uncharged amino acids, mediate binding of the TFPIα basic region to the FVa acidic region. (A) Rhod-LIKTKRKRKK (30 nM) was incubated with FV810 (30 nM) and the indicated concentrations of LIKTKRKRKK (LIKT; filled square), AAKAKRKRKK (AAKA; open square), AIKTKRKRKK (AIKT; filled circle), LAKTKRKRKK (LAKT; open circle), or LIKAKRKRKK (LIKA; filled triangle). Fluorescence anisotropy was measured and curve fits generated as described in Materials and methods. (B) FV810 (0.5 nM), phospholipid vesicles (20 µM), and the thrombin inhibitor DAPA (3 µM) were incubated with LIKTKRKRKK (LIKT; 3 µM) and varying concentrations of AAKA, and thrombin generation was measured as in Figure 2A. The initial rate of thrombin generation is shown as a percentage of control (no peptide; mean ± standard deviation; n = 3).