PROJECT SUMMARY Platelet studies have had a profound impact on our understanding of adhesion receptor function as exemplified by the adhesion and signaling roles of integrin αIIbβ3 in hemostasis and thrombosis. Less is known about whether and how αIIbβ3 signaling regulates platelets in their roles as sentinels and effectors of immunity and inflammation. In this regard, we have discovered that αIIbβ3 interacts through its αIIb cytoplasmic tail with SHARPIN, an obligate component of the linear ubiquitin chain assembly complex (LUBAC). This complex is the only known enzyme responsible for M1 linear ubiquitination of key proteins involved in immune and inflammatory signaling through the NF-κB pathway in leukocytes. Platelets express many proteins of the canonical NF-κB pathway. Therefore, we will now employ complementary approaches, including studies of peripheral blood platelets, megakaryocytes and platelets derived from human induced pluripotent stem cells, and gene-targeted mice to test the following central hypothesis: Through dynamic and mutually exclusive interactions with αIIbβ3 and LUBAC, SHARPIN regulates platelet function in inflammation and immunity as well as in hemostasis and thrombosis. Aim 1 will employ biochemical and advanced imaging techniques to test whether physical and functional linkages exist between αIIbβ3, LUBAC and the NF-κB pathway in human and mouse platelets. Attention will be focused on whether SHARPIN functions to maintain αIIbβ3 in a low-affinity state in resting platelets, but dissociates from αIIb and assembles the LUBAC complex to facilitate αIIbβ3 and NF-κB activation in response to platelet agonists generated during hemostasis and inflammation. Aim 2 will introduce genetic modifications into human megakaryocytes and platelets derived from induced pluripotent stem cells to model the effects of SHARPIN knockdown or knockout on αIIbβ3 and LUBAC functions in the appropriate primary cells. Using this system, optogenetic techniques new to the platelet field will be used to determine the functional consequences of conditional SHARPIN interactions with αIIbβ3 or with its two LUBAC protein partners. Aim 3 will address whether SHARPIN is required for αIIbβ3 and platelet functions in response to inflammatory stimuli and vascular injury in vivo. To accomplish this, platelet-specific SHARPIN knockout mice will be generated using the Pf4-Cre system and studied in several mouse models of platelet-dependent inflammation, hemostasis and thrombosis. Altogether, these studies will provide new insights into the role of SHARPIN in platelet pathobiology, with mechanistic and potential therapeutic implications beyond hemostasis and for platelets in inflammation and immunity.