The location of Affimer binding sites on GPVI for M17, D22, and D18. The residues involved in Affimer binding on GPVI determined by HDX-MS for M17 (A), D22 (B), and D18 (C) are displayed and colored on the crystal structure of GPVI (gray, PDB code: 2GI7). The residues that had strong and weak protection effect upon Affimer binding are colored blue and light blue, respectively. The residues that had strong and weak deprotection effect upon Affimer binding are colored red and light red, respectively. Red, blue, and gray bars shown in each graph below the GPVI structure represent different GPVI peptide fragments generated by proteolysis in the presence and absence of Affimers. The peptide fragment represented by red and blue bars have positive and negative differences in deuterium uptake, respectively. The peptide fragments represented by gray bars have no significant changes in deuterium uptake. (D) Representation and comparison of the binding site residues of M17, D22, and D18 with collagen/CRP (cyan), glenzocimab (green) and Nb2 (gray). The amino acids colored in blue and red had the strong protection and deprotection effect, respectively, upon the binding of Affimers.