PROJECT SUMMARY Megakaryocytes (MKs) are specialized blood cells that produce all of the platelets found in the human body. They reside primarily in the bone marrow, where they undergo endomitosis, synthesis of granules, membrane invagination, and finally proplatelet formation (PPF), a process characterized by dramatic changes to the cytoskeleton. Impaired platelet production leads to thrombocytopenia (low platelet count), which can cause life- threatening bleeding complications. Treating thrombocytopenia requires millions of platelet transfusions annually in the US alone. The high demand for platelet concentrates presents a significant problem in transfusion medicine, as platelets have a short shelf-life so must be supplied frequently by volunteer donors. Multifaceted approaches are under investigation to address this problem by producing platelets in vitro, but the low efficiency of PPF and the limited quality of the platelets produced remain major obstacles. The overarching goal of this proposal is to elucidate the mechanism of PPF, and thereby develop strategies for carefully timed perturbations of Rap and/or Rho GTPase activity as a means to significantly improve PPF efficiency and the quality of the platelet product. Rho-family GTPases are master regulators of the cytoskeleton that control morphodynamics through localized, precisely timed activation events. It has recently become clear that they are important regulators of PPF, but very little is known about their spatio-temporal dynamics or coordination. In addition, we here present the first direct proof that PPF also requires signaling by the Ras-family GTPases Rap1A and Rap1B; mice deficient in both Rap1 isoforms in MKs show significant thrombocytopenia and a near complete loss of PPF in vitro. To elucidate how GTPases orchestrate the complex morphological changes of PPF, we have assembled an interdisciplinary team of investigators with expert knowledge in platelet/megakaryocyte biology, small GTPase signaling, and the design of molecules to visualize and photo- manipulate GTPase activity in living cells. We will define the contribution of Rap1 isoforms and their regulators to MK development and PPF (Aim 1), establish GTPase “activity signatures” and network connections critical to PPF (Aims 2 & 3), and establish proof-of-principle that precisely targeted perturbation of GTPase activity is a viable strategy to optimize in vitro platelet production (Aim 3). Successful completion of these studies will provide critical novel insights into the cell biology of PPF and may have important implications for improving transfusion medicine.