Cloned antibodies from patients with heparin-induced thrombocytopenia (HIT) provide new clues to HIT pathogenesis

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 pro-thrombotic state. While a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset also makes antibodies that are platelet-activating. A close correlation between platelet-activating antibodies and the likelihood of experiencing HIT has been demonstrated in clinical studies but how platelet-activating (presumptively pathogenic) and non-activating (presumptively benign) antibodies differ from each other at the molecular level is unknown. To address this issue, we cloned seven platelet-activating (PA) and 47 non-activating (NA) PF4/H-binding antibodies from six HIT patients and characterized their structural and functional properties. Findings made showed that PA clones differed significantly from NA clones in possessing one of two heavy chain complementarity-determining region 3 (HCDR3) motifs - RX1-2R/KX1-2R/H (RKH) and YYYYY (Y5) - in an unusually long CDR3 region (≥20 residues). Mutagenic studies showed that modification of either motif in PA clones reduced or abolished their platelet-activating activity and that appropriate amino acid substitutions in HCDR3 of NA clones can cause them to become platelet-activating. Repertoire sequencing showed that the frequency of peripheral blood IgG+ B cells possessing RKH or Y5 was significantly higher in HIT than in non-HIT patients 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.