ABSTRACT Heparin-induced thrombocytopenia (HIT) is the most common drug-induced immune thrombocytopenia and can have devastating consequences for an affected patient. Antibodies that recognize the platelet alpha- granule protein, platelet factor 4 (PF4), in a complex with heparin (“HIT antibodies”) are central to HIT pathogenesis. The kinetics of HIT antibody production, characterized by rapid onset and apparent lack of immunologic memory, suggest T-cell independence, but IgG antibodies typical of HIT argue for T cell involvement. These ambiguous features of the HIT antibody response have confounded efforts to characterize HIT immune- pathogenesis. There are three subsets of mature B cells, follicular (FO), marginal zone (MZ) and B1 B cells. Each subset has a distinct role in the humoral immune response and is regulated differently. Signals generated through the B cell receptor (BCR) are required for B cell development, function and establishment of self-tolerance and dysregulation of BCR signaling can lead to autoimmunity. In previous studies under this grant, we showed in a mouse model that MZ B cells play a critical role in HIT antibody production, that breakdown of immunologic tolerance appears to be involved in HIT antibody production in mice and humans, and that T helper cells control heparin-induced production of HIT antibodies in mice. We also identified important signaling molecules and pathways that are critical for BCR-mediated B-cell function and tolerance. Based on these findings, we hypothesize that PF4/heparin-specific B cells originate from the distinct MZ B cell subset in human HIT patients and that activation and proliferation of pathogenic B cells is controlled by T cells and the major tolerance mechanism, anergy. To test this hypothesis, three specific aims are proposed. Specifically, we will 1) define the cellular source and molecular properties of anti-PF4/heparin antibodies in human HIT, 2) investigate mechanisms by which T cells help PF4/heparin-specific MZ B cells, and 3) characterize molecular mechanism(s) that regulate PF4/heparin antibody production. Defining the cellular and molecular mechanisms responsible for HIT antibody production will provide new clues to the pathogenesis of HIT, suggest novel tools for HIT diagnosis, prophylaxis and treatment and may contribute to the understanding of other autoimmune diseases relevant to transfusion medicine.