Inhibitory activities of wild-type and mutants of Inf4 on FXIIa activity
(A) Sequence alignment of the reactive-site loops of AaTI and Inf4 from residues P5 to P5′. Residues in Inf4 that are different from those in AaTI are underlined. Mutations to these residues were made in Inf4 to determine the relative contributions of these residues in prohibiting the activity of AaTI. (B) Plots showing the relative inhibition of FXIIa by wild-type Inf4 (open circles), Inf4_F10P (closed squares), Inf4_N12I (closed circles) and Inf4_V14M (open squares). The IC50 values derived from curve fittings are 3.1 ± 0.5 nM for wild-type Inf4, 11.6 ± 1.1 nM for Inf4_F10P, 8.4 ± 0.9 nM for Inf4_N12I and 9.9 ± 1.6 nM for Inf4_V14M. Data represent the mean ± SEM for triplicate values. (C) Plots showing the relative inhibition of FXIIa by wild-type Inf4 (open circles), Inf4_N12I+V14M (closed circles), Inf4_F10P+N12I (open squares), Inf4_F10P+V14M (closed squares) and Inf4_F10P+N12I+V14M (open triangles) on FXIIa. The IC50 values derived from curve fittings are 3.1 ± 0.5 nM for wild-type Inf4, 7.9 ± 2.1 nM for Inf4_N12I+V14M, 22.8 ± 4.7 nM for Inf4_F10P+N12I, 42.1 ± 8.4 nM for Inf4_F10P+V14M and 70.5 ± 9.7 nM for Inf4_F10P+N12I+V14M. Data represent the mean ± SEM for triplicate values.