Alanine scanning mutagenesis was used to map epitopes of amino acids on PF4 that are critical for binding antibodies from patients 1, 2, and 3. (A) Patient 1, (B) patient 2, and (C) patient 3 showed that there is a binding region (red) that aligns with (D) the previously described VITT binding region for ChAdOx1 nCoV-19 (AstraZeneca) vaccination (red, R22, H23, E28, K46, N47, K50, K62, K66) and within (E) the heparin-binding region on PF4 (purple). All 3 patients also have additional amino acids that are important in binding that correspond to the PF4 binding site of typical HIT (blue). However, patient 2 only had 3 additional amino acids outside the typical VITT binding site. Antibody epitopes usually comprise 5 to 8 amino acids; therefore, although important binding amino acids were found in the typical HIT site, there are not enough amino acids to constitute a full epitope. Because patient antibodies are polyclonal, it is possible that the pool of antibodies that binds outside the typical VITT site has either a lower titer or a weaker affinity that cannot be differentiated in the assay. Images are modified from Protein Data Bank file 1RHP.