ABSTRACT Heparin-induced thrombocytopenia (HIT) is an immune reaction caused by antibodies (Abs) that target platelet factor 4 (PF4) when bound to heparin (H) or other glycosaminoglycan-like polyanionic molecules. HIT usually results in a low platelet count, but is too often associated with devastating venous and/or arterial thrombosis and thromboembolism. While many heparin-exposed patients test positive for the presence of PF4-binding Abs, only those with HIT have "pathogenic" Abs that activate platelets. The complexity of current diagnostic tests limits their value for timely treatment decisions, which can lead to over-diagnosis of HIT. While failure to diagnose HIT creates a significant risk of catastrophic thrombosis, equally concerning is that an erroneous diagnosis of HIT not only prolongs hospital stay and increases costs, but also needlessly exposes a patient to treatments with potentially serious side effects. Hence, there is an urgent need for a rapid and precise diagnostic test for HIT. The molecular basis for the crucial functional differences between the PF4/H-binding Abs that are platelet activating (PA) from those that are not (NA) is unknown, however, and hinders this effort. Furthermore, the absence of monoclonal (m) Abs from actual HIT patients has also hampered our ability to dissect the pathogenesis of HIT. We have recently achieved a breakthrough in this regard by successfully cloning 54 IgG1+ PF4/H-binding mAbs from HIT patients. Among these clones, seven demonstrated platelet-activating properties. Notably, although the PF4/H-binding capability of both PA and NA mAbs was found to be similar, only the PA mAbs physically bound to PF4-coated platelets. Specific Aim 1 seeks to build upon these novel findings by further characterizing the molecular and structural biological interactions underlying the binding of platelet- activating HIT Abs to PF4 on the surface of platelets. A thematically related subject to be addressed in Project 2 deals with the etiology of HIT, and builds on our recent observations that pre-existing B cells that produce basal levels of PF4/H-reactive IgM Abs are constitutively present in both humans and mice. It remains unknown, however, how these pre-existing PF4/H-specific Abs and B cells are generated, or how they might contribute to the development of HIT. Studies in germ-free mice have linked gut microbiota to the presence of naturally occurring PF4/H-reactive IgMs and PF4/H-specific IgGs. Specific Aim 2, therefore, will identify the specific gut microbial species associated with the selection and development of PF4/H-specific B cells that predispose individuals to later development of HIT. Taken together, developing a more complete understanding of the interactions between HIT PA Abs and platelet surface antigens, coupled with elucidating the role of gut microbiota in generating these Abs, will provide novel insights into the pathogenesis of HIT, with significant implications for diagnosing, prevent...