Platelets are critical in maintaining hemostasis and platelet counts are tightly regulated in healthy individuals. In several pathologies, the disruption of thrombopoiesis leads to abnormal platelet counts which significantly impact clotting and/or bleeding risks. Thrombopoietin (TPO) signaling through the myeloproliferative leukemia virus (MPL) receptor is the only known driver of megakaryocyte (MK) differentiation and maturation from hematopoietic stem cells (HSCs). However, therapies targeting the TPO/MPL axis do not work for all patients, leading to an unmet need for identifying new targets to modulate platelet counts. We have found that membrane polyunsaturated fatty acids (PUFAs) in MK progenitors regulate MK development and platelet production, representing a novel target for modulating megakaryopoiesis and platelet count. Our main hypothesis is that MKs and their progenitor cells utilize the CD36 receptor to take up PUFAs from exogenous sources, and accumulation of membrane PUFAs is essential for maintaining normal MK development and platelet count. This hypothesis will be tested in three specific aims using novel techniques such as click chemistry, cellular barcoding and phospho-flow cytometry, and the newly published Fatty Acid Library for Comprehensive ONtologies (FALCON) platform paired with human and murine in vitro models, in vivo murine models, and human iPSC-derived organoids. Aim 1 will determine the cell-type-specific enzyme requirement for PUFA accumulation during megakaryopoiesis. We will use in vivo and in vitro models to determine which enzymes play a role in PUFA accumulation in MKs and whether they are viable targets to manipulate MK development. Aim 2 will reveal if the CD36 receptor preferentially takes up PUFAs at the expense of saturated fatty acids. We will use CD36-/- mice and a novel in vivo click-chemistry technique to provide the first in vivo evidence of selective PUFA uptake. We will determine if CD36 within MKs and MK progenitors preferentially promotes the internalization of fatty acids with a specific saturation status and whether this process is impacted by dietary fatty acid composition. Aim 3 will examine the mechanisms by which PUFA uptake influences megakaryopoiesis. This inquiry will be addressed using 1) a targeted approach focusing on MPL signaling via interaction with membrane lipids and 2) a hypothesis-generating approach using the FALCON platform to generate new leads into potential TPO-independent MK differentiation pathways impacted by fatty acids. Successful completion of the proposed experiments will extend the discovery of the role of membrane PUFAs in MK development and platelet production to reveal a detailed and actionable roadmap of the mechanisms by which both human and murine MKs and progenitors accumulate and utilize PUFAs to drive platelet production, potentially leading to therapeutic and dietary interventions to modulate platelet production for patients with diverse thrombotic and pl...