PROJECT SUMMARY Heparin is arguably the most versatile biopolymer, capable of interacting with and modulating the behavior of an impressive variety of biomolecules even outside of its native environment (mast cell granules). A large fraction of heparin interactome in the human body are key players in a variety of physiological processes (including several pathologies) and are considered high-value therapeutic targets, although at present antithrombin remains the only protein whose interaction with heparin has been successfully exploited in medicine. Successful exploitation of heparin’s unique versatility for other therapeutic purposes critically depends on the ability to characterize its interactions with relevant proteins; however, specific molecular mechanisms remain elusive outside of the very few extensively studied systems. In the previous period of support, we developed powerful analytical tools capable of providing information on protein/heparin interactions at the whole molecule level, as well as identifying specific structural motifs within highly heterogeneous heparin macromolecules that enable their association with specific proteins. Building upon this success, we now propose to focus our inquiry on understanding the molecular mechanisms underlying etiology of heparin-induced thrombocytopenia (HIT), a serious (and potentially fatal) immune disorder affecting up to 5% of patients receiving heparin as an anticoagulant. Despite the central role played by heparin in formation of antigenic aggregates that may lead to development of HIT, relatively little is known about the specific molecular mechanisms governing these interactions. This not only creates a tremendous challenge vis-a-vis the ability to design efficient anti-HIT therapeutic strategies, but also prevents a reliable prognosis of the occurrence of this pathology. Recognizing the importance of multiple factors that may modulate interaction of heparin with relevant proteins (mostly platelet factor 4, PF4), we will use a multi-level strategy to study the mechanism of PF4/heparin association, architecture of these aggregates and specific features that make them immunogenic and trigger the onset of HIT. The work will be carried out in close collaboration with the Hematology team at McMaster University Health Centre headed by Dr. I. Nazy, a leading expert in the field of thrombocytopenia and thrombosis. This knowledge will catalyze efforts to develop reliable diagnostic and prognostic tools for HIT, and will be critical for designing safe and effective therapeutic intervention strategies.