Arg¹⁶²is inactive and not an obligate intermediate necessary for the generation of chem158K by FXIa. (A) Ten mM 6his-chem163S and 10 nM 6his-chem163S_R162A were incubated in assay buffer in the presence of 30 nM FXIa. Aliquots of reactions were removed and stopped at the indicated time and analyzed by 6his-chem163S ELISA. Each experiment was repeated ≥3 times. (B) FIX-10mer, chem-15mer, and chem-15mer_R162A were incubated with 30 nM FXIa for 30 minutes before analysis by HPLC. The product concentration was calculated by interpolation from a standard curve, and the velocities of product generation were determined. (C) Chem163S full-length protein was incubated with 30 nM FXIa for 30 minutes, and products were analyzed by ELISA. (D) Ca⁺⁺ flux in hCMKLR1/L1.2 cells was followed after the addition of the peptides chem-9mer, chem-14mer and chem-15mer, representing the C-terminal sequence of chem157S, chem162R, and chem163S, respectively. The maximum fluorescence intensities triggered by the addition of each peptide were plotted and used to determine the EC₅₀ value. Data from 4 independent experiments were pooled, and the data are presented as the mean ± standard error of the mean.