The effect of Affimer A11 in plasmin activity and plasmin generation rate and interaction with plasminogen. (A) Fluorometric plasmin activity assay investigating the effects of α2AP on plasmin activity in the absence and presence of Affimer A11. Data are presented as the mean ± SD of 3 independent experiments. One-way ANOVA test was used for statistical analysis to compare A11 with buffer only control. (B) Chromogenic substrate S2251 hydrolysis assay analyzing plasminogen conversion to plasmin in the absence and presence of Affimer A11. Data are presented as the mean ± SD of 3 independent experiments. One-tailed Mann Whitney test was used for statistical analysis to compare A11 with buffer only control. (C) Competitive inhibition of plasminogen binding to α2AP in the presence of increasing concentrations of Affimer. The absorbance units were converted to the percentage of α2AP-bound plasminogen, with results normalized to the binding of plasminogen in the absence of Affimer. Data are presented as the mean ± SD of 3 independent experiments. (D) Predicted binding pose of Affimer A11 (magenta) with α2AP (green). The 2 loops of A11 are highlighted as sticks; the residues of α2AP interacting with the Affimer are shown as coral sticks, and RGD domain is highlighted blue. Figure produced using PyMOL version 2.5.2.