Cloned antibodies from patients with HIT provide new clues to HIT pathogenesis

•PF4/H-binding antibodies were cloned from patients with HIT, a subset of which were platelet activating and presumptively pathogenic.•Specific structural motifs appear to be responsible for platelet-activating properties of “pathogenic” clones. [Display omitted] Heparin-induced thrombocytopenia (HIT) is a serious adverse drug reaction characterized by antibodies that recognize platelet factor 4/heparin complexes (PF4/H) and activate platelets to create a prothrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset also makes antibodies that are platelet activating (PA). A close correlation between PA antibodies and the likelihood of experiencing HIT has been demonstrated in clinical studies, but how PA (presumptively pathogenic) and nonactivating (NA) (presumptively benign) antibodies differ from each other at the molecular level is unknown. To address this issue, we cloned 7 PA and 47 NA PF4/H-binding antibodies from 6 patients with HIT and characterized their structural and functional properties. Findings showed that PA clones differed significantly from NA clones in possessing 1 of 2 heavy chain complementarity-determining region 3 (HCDR3) motifs, RX1-2R/KX1-2R/H (RKH) and YYYYY (Y5), in an unusually long complementarity-determining region 3 (≥20 residues). Mutagenic studies showed that modification of either motif in PA clones reduced or abolished their PA activity and that appropriate amino acid substitutions in HCDR3 of NA clones can cause them to become PA. Repertoire sequencing showed that the frequency of peripheral blood IgG+ B cells possessing RKH or Y5 was significantly higher in patients with HIT than in patients without HIT given heparin, indicating expansion of B cells possessing RKH or Y5 in HIT. These findings imply that antibodies possessing RKH or Y5 are relevant to HIT pathogenesis and suggest new approaches to diagnosis and treatment of this condition. Heparin-induced thrombocytopenia (HIT) is associated with antibodies that recognize platelet factor 4/heparin (PF4/H) complexes and activate platelets. However, not all PF4/H antibodies are platelet activating (PA), and the development of HIT correlates with PA antibodies. Zhu et al cloned platelet-activating and nonactivating antibodies from 6 patients with HIT and identified 2 immunoglobulin heavy-chain motifs that characterize PA antibodies, providing potential new approaches to PF4/H analysis to improve HIT diagnosis.