Mass spectrometry (MS)-based characterization of PF4-specific clonotypic antibodies. (A) Proteomics workflow to identify molecular signatures of anti-PF4 antibodies. PF4-specific immunoglobulins are purified from serum of VITT patients using PF4-coupled magnetic beads. Heavy (H) and light (L) chains are separated by reduced sodium dodecyl–sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and excised and digested with enzymes to generate peptides for liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS). IgV region peptide sequences are analyzed by combined de novo sequencing and IMGT database matching. (B) Specificity of purified anti-PF4 antibodies. Monospecificity of magnetic bead–purified anti-PF4 IgGs is verified by testing starting serum, bead-purified anti-PF4 antibody fraction, and unbound fractions using ELISAs coated with individual PF4, SARS-CoV-2 spike S1, and S2 proteins. Data are shown as mean ± standard deviation (n = 2). (C) Clonotypic H- and L-chain third complementarity-determining region (CDR3) signatures. IgV region subfamilies of PF4-specific antibodies as highlighted in yellow are assigned by IMGT database matching. HCDR3 and LCDR3 amino acid sequences from 5 individual VITT patients are identified by de novo sequencing. Bold amino acids in HCDR3s and LCDR3s denote shared motifs across unrelated patients, and amino acids in color denote amino acid replacement mutations found in individual patient HCDR3 and LCDR3 regions.