Molecular Basis of the Humoral Immune Response in Heparin-Induced Thrombocytopenia

Versiti Blood Health, Inc., Milwaukee WI

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Abstract

Abstract Heparin-induced thrombocytopenia (HIT) is a severe immune reaction caused by heparin treatment, reducing platelets and increasing life-threatening thrombosis. It is thought that the administered heparin forms a complex with platelet factor 4 (PF4) and triggers B cells to produce PF4/H-reactive IgGs. While many patients receiving heparin develop PF4/H-reactive antibodies (Abs), only those with the ability to activate platelets, determined by serotonin release assay (SRA) or P-selectin expression assay (PEA), are "pathogenic" Abs associated with HIT. A recent case report highlighted a patient receiving heparin who displayed clinical signs of HIT, despite testing negative in the ELISA assay for PF4/H IgG (ELISA-). While a negative ELISA result excluded HIT diagnosis, the patient experienced cardiac arrest upon resuming heparin treatment. We identified four ELISA-SRA+ HIT patients, representing around 0.7% of suspected HIT cases. With an estimated one million HIT-suspected cases annually in the US, an alarming 7000 ELISA-SRA+ HIT cases could go undetected. Preliminary study demonstrated prevalent presence both serologically and clonally of ELISA-PEA+ Abs in HIT patients with an ELISA+SRA+ test profile. Notably, patient-derived ELISA-PEA+ monoclonal Abs (mAbs) bind to PF4-coated platelets, suggesting potential detection methods. Our findings challenge the conventional knowledge of HIT pathogenesis and emphasize the need to study the role of ELISA-SRA+ Abs in HIT. In healthy individuals, over 20% of mature naive B cells are autoreactive and controlled through an anergy process. These autoreactive anergic B cells can be activated upon exposure to antigens. A tolerance mechanism prompts these B cells to undergo immunoglobulin (Ig) gene mutations, reducing autoreactivity and allowing B cell differentiation through autoantibody redemption. Preliminary study demonstrated autoreactivity of ELISA+PEA+ mAbs cloned from HIT patients. Notably, we observed an increase in the binding of these mAbs to PF4/H complexes with varying changes in platelet activation when we reversed the mutations to restore their germline sequences. We thus propose a model that large PF4/H complexes formed upon heparin administration activate ELISA+PEA+ B cells in patients with trauma and inflammation. However, the process of autoantibody redemption leads to Ig mutations in ELISA+PEA+ B cells that reduce their reactivity to PF4/H complexes, enabling B cell survival, differentiation, and eventual production of ELISA-PEA+ Abs. Thus, we hypothesize that ELISA-PEA+ Abs, originating from anergic ELISA+PEA+ B cells through autoantibody redemption, constitute a substantial proportion of platelet-activating Abs in HIT. Specifically, we will (1) investigate the serological and clonal prevalence of ELISA-PEA+ Abs in HIT, (2) study the binding of ELISA-PEA+ Abs to PF4-coated platelets and PF4/chondroitin sulfate complexes, and (3) determine the molecular mechanism underlying the emergence of ELISA-PEA+ Abs in HIT. These studies aim to understand the role and molecular mechanism of ELISA-PEA+ Ab generation in HIT, facilitating their detection and control of the pathogenic B cell response.