ABSTRACT Despite decades of research into the immunopathology of heparin-induced thrombocytopenia (HIT) anunpredictable life-threatening immune-mediated adverse reaction to heparin treatment a fundamentalknowledge gap regarding the cause of HIT remains. The inability to predict HIT represents a considerable liabilityassociated with heparin which is given to 12 million individuals or one third of all hospitalized patients everyyear. Because the exact cellular and molecular mechanisms underlying HIT have yet to be identified includingintrinsic immune cell roles and the difference between pathogenic and non-pathogenic antibodies there is anessential need to apply alternative approaches to understand the biological basis for HIT and to identify clinicallyimplementable biomarkers. The PIs central hypothesis is that immunogenomic variation impacts HITpathogenesis and can be used to differentiate non-pathogenic and pathogenic PF4/heparin antibodies whichare produced by immune cell populations intrinsic to HIT. Based on strong preliminary data that constitute thelargest genome-wide association study (GWAS) for HIT the working hypothesis is that the presence of theHLA-DRB3*01:01 allele combined with proliferating V 5.1 family T-Cell receptor (TCR) clonotypes in Th2 cellspredisposes patients to pathogenic PF4/heparin antibody production and these antibodies result in high HIT riskin patients with the ABO O blood group. We will pursue three Specific Aims (SAs) to test the central hypothesis:(SA1) Determine the role of ABO variation in HIT; (SA2) Determine the influence of genomic variation onPF4/heparin antibody production; and (SA3) Identify intrinsic immune cell involvement in HIT. In SA #1 deepABO sequencing data will be utilized in our large cohort of functional assay-confirmed HIT patients to elucidatethe role of ABO variability in HIT. In SA #2 we will perform several GWAS to determine genetic influences ofPF4/heparin antibody production including diverse populations and differentiate genetic influences onpathogenic versus non-pathogenic antibodies. In SA#3 we will sample paired cell populations during the acutephase of HIT and after HIT resolution and conduct focused single cell (sc) studies to determine proliferation ofTCR clonotypes and activated cell populations using sc-RNA-TCR-CITE-seq. Our studies overcome majorlimitations of previous genomic studies of HIT by incorporating large diverse cohorts a PF4/heparin antibody-positive case group and functional assay confirmation of HIT cases. This work is technically and conceptuallyinnovative as it leverages a sc-RNA-TCR-CITE-seq approach utilizes large unique HIT cohorts and advancesan original model of HIT immunopathogenesis. We expect to provide mechanistic insights into HIT pathologyand advance a framework for clinical translation of HIT biomarkers with direct application to other adverse drugreactions. The proposed studies leverage large-scale biological data to distinguish patients pre-disposed to HITpotentially shifting clinical practice from treatment to prevention through biomarker-guided heparin treatment.