PROPOSAL SUMMARY/ABSTRACT Venous thromboembolism (VTE) is a common disease with an incidence of 300,000-600,000 events in the United States annually. Current therapies include anticoagulation and pharmacomechanical thrombolysis, both of which are associated with bleeding risks. Preliminary studies from our group suggest that RIPK3, a regulator of necroptosis, contributes to venous thrombosis (VT). In the inferior vena cava (IVC) ligation model of VT, Ripk3-/- mice formed smaller thrombi compared to wild type (WT) mice, and treatment with GSK’074/C9, a RIPK1/RIPK3 inhibitor, decreased thrombus size. Furthermore, thrombin generation was decreased in plasma from Ripk3-/- mice, and supplementation of WT and Ripk3-/- deficient plasma with recombinant RIPK3 increased and restored thrombin generation in a dose dependent fashion respectively. Publications have shown that neutrophils and NETosis, a process by which neutrophils unravel and extrude anti-microbial and pro- thrombotic DNA NETs into the extracellular space, play a pivotal role in venous thrombogenesis. Mice deficient in NETosis (Pad4-/ mice-) form thrombi at a reduced rate compared to WT mice. A potential link has recently been identified between the RIPK1/RIPK3/MLKL signaling pathway, PAD4 activation, and NETosis. The goal of this proposal is to investigate the hypothesis that RIPK3 promotes thrombosis through a NETosis dependent pathway, and that RIPK3 within neutrophils contributes meaningfully to local and circulating levels of RIPK3 in venous thrombosis. We will investigate this hypothesis using WT, Ripk3-/-, and GSK’074 treated mice and the IVC stenosis model of VT. In aim 1 we will determine if RIPK3 promotes thrombosis in the IVC stenosis model of VT, determine RIPK3 localization, and evaluate if RIPK3 upregulates NETosis. Moreover, we will investigate the importance of RIPK3 within neutrophils during VT. WT and Ripk3-/- mice will undergo IVC stenosis or sham surgery. At euthanasia, blood will be subjected to thrombin generation assay, RIPK3 and citrullinated H3 ELISA, and analyzed for circulating DNA. Thrombus weight/length will be measured. Thrombus/Vein wall samples will be analyzed by WB and IF to determine RIPK3 localization and NET burden. Separately, WT and Ripk3-/- mice will be treated with WT or Ripk3-/- deficient neutrophils to investigate whether or not Ripk3+/+ neutrophil infusion can restore thrombosis in Ripk3-/- deficient mice. In aim 2, we will study the effects of GSK’074 treatment on thrombus size and NETosis burden. This research strategy addresses the proposed hypothesis and will be carried out with the support and mentorship of Dr. Bo Liu, who is a well-funded and well-published researcher in the field of blood vessel biology. Her experience combined with the research environment at UW-Madison will provide an outstanding foundation for professional growth for a future surgeon-scientist in vascular biology.