The role of proteoglycans in Heparin-induced thrombocytopenia: Implications for pathophysiology, diagnosis and treatment - PROJECT SUMMARY/ABSTRACT Heparin is administered as an anti-coagulant to approximately 10 million patients annually, placing it among the most frequently utilized clinical interventions in the US. About 3% of patients that receive heparin generate antibodies that cause heparin-induced thrombocytopenia (HIT). Antibodies that cause HIT are especially dangerous due to their ability to bind platelet/PF4 complexes on the surface of platelets and stimulate platelet activation and thrombosis. Unfortunately, patients with HIT have a very high mortality rate (~10-25%), even with treatment. The crucial knowledge gap limiting the optimal diagnosis and treatment of HIT is that the platelet components that recruit PF4 to the platelet surface to elicit the epitopes recognized by HIT antibodies have yet to be discovered. Aim 1 of this proposal will identify the platelet components that recruit PF4 to the platelet surface and quantify differences in their expression based on age, sex, and ethnicity to better risk stratify patients and identify candidates that can be targeted to improve HIT treatment. Identifying the platelet molecules that recruit PF4 to platelets will also define a set of antigenic targets to improve diagnostic enzyme-based immunoassays (EIAs) for HIT. EIAs are used as the frontline diagnostic tool for HIT but have a poor positive predictive value (~50%), necessitating the use of platelet-based assays to confirm the presence of platelet- activating antibodies. Improving the accuracy of HIT EIAs would eliminate the need to perform platelet-based confirmatory assays and significantly shorten the time to diagnosis and therapeutic intervention. Because only approximately 50% of HIT patients develop thromboses, we hypothesize an inhibitory platelet mechanism limits HIT antibody-mediated thrombosis. As a proof-of-principle, we fractionated platelet granule components and found that glycosaminoglycan-rich proteoglycan fractions specifically inhibit HIT antibody- mediated platelet activation. Aim 2 of this proposal will identify the platelet components that selectively inhibit antibody-mediated platelet activation and will leverage this discovery to develop improved HIT therapeutics. Our preliminary data also identified a subclass of HIT patients who test negative in diagnostic EIAs but positive in platelet activation assays. Because platelet activation assays are typically only performed after a positive EIA, this subclass of patients may be significantly underdiagnosed using current HIT diagnostic workflows. Aim 3 of this proposal will assess the efficacy of current HIT diagnostic strategies by defining the frequency of HIT- suspected patients that test serologically negative in EIAs but positive in platelet activation assays. Aim 3 will also establish the pathogenicity of this HIT antibody subclass in a humanized murine model of HIT. The long-term goals of this study are to better understand HIT pathogenesis, including the age, sex, and ethnicity-driven factors impacting HIT severity, and improve the diagnosis and treatment of HIT by identifying platelet components that 1) recruit PF4 to the platelet surface to elicit the epitopes recognized by HIT antibodies, and 2) participate in an inhibitory feedback mechanism that attenuates HIT antibody-mediated platelet activation.
