Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by antibodies to complexes between platelet factor 4 (PF4) released from activated platelets and heparin or cellular glycosaminoglycans (GAGs). Remarkably, anti-PF4/heparin antibodies develop in most patients exposed to heparin in settings characterized by intense platelet activation and inflammation such as coronary bypass surgery, yet only ~1% of patients develop HIT. The reason why only a subset of anti-PF4/heparin antibodies is associated with HIT is unclear and is only partially explained by IgG isotype and titer. Antibody binding and procoagulant activity develops over a narrow range of PF4/heparin (PF4/H) concentrations that foster formation of stable ultralarge complexes (ULC). We hypothesize that the propensity to pathogenicity is determined by two interrelated aspects of antibody biology relating to ULC: 1) Composition ofthe complement determinant regions (CDRs) and 2) Epitope specificity. These features work in concert to permit access of antibody to antigenic epitopes within the complex charge field of PF4/H to foster formation of pathogenic ULCs. We have characterized two isotype matched murine anti-human PF4/H antibodies, KKO, which forms ULC in the presence of heparin or GAGs and causes HIT in an animal model, and RTO which binds less well to PF4 in the presence of heparin and does not cause HIT in vivo. We will systematically convert the CDRs of non- pathogenic RTO to simulate pathogenic KKO using molecular modeling and the crystal structures of their respective Fabs.
In Specific Aim 1, the impact of mutations of epitope specificity will be determined using a panel of existing and novel PF4 mutants and we will examine their capacity to induced oligomerization of PF4 teframers.
In Specific Aim 2, we will use scanning electron microscopy to assess the structure of ULC clusters on the surface of platelets, monocytes and endothelium stabilized by KKO, but not RTO, that we posit incorporate and promote activation of FcyRllas and we will characterize antibody-PF4 interactions in real time using optical trap-based force spectroscopy.
In Specific Aim 3, we will assess the impact of these changes in antibody on activation of an FcyRlla cell line, platelets and cultured endothelial cells in vitro and the induction of thrombocytopenia and thrombosis in vivo. Successful completion of this project will help elucidate the biologic basis of pathogenic autoantibodies, improve diagnosis by developing ELlSAs based on informative mutants and potentially ameliorate disease severity using specific PF4 antagonists.
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