Project Summary Atherosclerosis is the leading cause of cardiovascular disease, accounting for nearly 30% (859,125) of deaths in the United States, with an estimated direct healthcare cost of $218.7 billion every year. Patients with late stage atherosclerosis suffer from life-threatening complications, such as myocardial infarctions, ischemic strokes, aneurysms, and multi-organ failure. Many current treatment options, such as anti-platelet and anti- coagulant therapies, although successfully alleviating or preventing thrombotic events of atherosclerosis, carry a risk for bleeding and hemorrhagic complications. Thus, there is a clear need to further understand the molecular basis of hemostasis and thrombosis in order develop safer and more effective therapies. In this regard, one recent therapeutic group of interest is tyrosine kinase inhibitors (TKIs). Tyrosine kinase inhibitors targeting Bruton’s tyrosine kinase (BTK), including ibrutinib, have traditionally been used with great success in treating hematological malignancies, such as chronic lymphocytic leukemia (CLL), and inflammatory conditions, such as rheumatoid arthritis. Due to the central role of BTK also seen in platelet activation, BTK inhibitors have recently been studied as a potential anti-platelet agent, demonstrating effects against atherosclerotic plaque-triggered thrombus formation; however, the mechanisms by which platelet BTK is activated and its functional effects remain largely ill-defined. To this end, our proposal aims to characterize in platelets the regulation of BTK (Aim 1) and functional effects of BTK (Aim 2) in atherosclerosis, to reduce thrombotic complications while minimally affecting hemostasis. To investigate the role of platelet BTK in atherosclerosis, 1) we will bring together for the first time physiological and phosphoproteomics methods to perform a combinatorial analysis delineating the regulatory signaling cascades that activate BTK in platelets; and 2) we will define the effects of BTK activation on platelet functional responses and investigate the interplay between platelets, endothelial cells, and leukocytes classically seen at the microenvironment of atherosclerotic plaque rupture. The potential translational relevance of our project will be the identification of safe and druggable molecular target and mechanisms within the platelet activation pathway. Our research may ultimately provide rationale for the development and use of classic BTK inhibitors as secondary anti-platelet and anti-thrombotic agents that could safely benefit patients who suffer from atherosclerosis and its complications.