Characterization of GPVI-Affimer complexes by native mass spectrometry and molecular modeling. (A-E) The native mass spectra of D18 (dimeric, measured molecular weight is at 21 760 Da, approximately two-fold larger than that calculated based on the monomeric protein sequence, at 11 013 Da) (A), GPVI monomer N72Q (monomeric, measured molecular weight is at 21 380 Da, similar to that based on the monomeric protein sequence, at 21 249 Da) (B), GPVI monomer N72Q with D18 (no complex formation detected, measured molecular weight is at 21 380 and 21 810 Da for GPVI and D18, respectively) (C), GPVI-Fc N72Q (monomeric, measured molecular weight is at 98 040 to 98 530 Da due to heterogeneous glycosylation in Fc, similar to that based on the sequence of the monomeric protein, at 95 154 Da) (D), and GPVI-Fc N72Q with D18 (1:1 complex formation detected, measured molecular weight is at 120 641 Da, similar to that based on the protein sequence of the 1:1 complex, at 117 181 Da) (E). Orange diamonds and blue spheres represent GPVI and Affimer D18, respectively. (F) Molecular model of D18 dimer generated using AlphaFold. Molecular docking model of M17 (G), D22 (H), and D18 (I) interacting with GPVI generated using high ambiguity driven protein-protein docking. GPVI is colored in orange and brown. The regions interacting with Affimers predicted by HDX-MS are colored in cyan. Affimers are colored in magenta and green. The variable loops that are crucial for interacting with GPVI are colored in red.