Insufficient megakaryopoiesis and thrombopoiesis remain the main cause of persistent thrombocytopenia after hematopoietic stem and progenitor cell (HSPC) transplantation and after radiochemotherapy. Platelet transfusions are required to support patients with consequent increased risk of transfusion reactions, platelet alloimmunization, and transfusion-associated viral infections. Significant efforts are focused on identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow (BM) transplantation, chemotherapy, and to maximize the efficacy of in vitro human platelet production as an alternative method to increase the platelet supply for transfusion. Our ability to therapeutically optimize platelet recovery and in vitro platelet production is hindered by our paucity of knowledge of the molecular determinants that govern thrombopoiesis. Megakaryocytes (MKs) reside in the BM and maintain the continuous production of billions of circulating platelets in order to prevent bleeding. Recent work shows that MKs also maintain a functionally specific BM niche to support MK-biased HSPCs. Preliminary data suggest that: 1) MK-biased HSPCs express unusually high levels of α2,6-sialic acid (α2,6-Sia) which is unparalleled by cell intrinsic St6gal1 mRNA expression; 2) systemic ST6GAL1 deficiency promotes a myeloid skewed hematopoietic development profile, suggesting that α2,6-sialylation is a checkpoint of a particular HSPC population (Project 2); 3) B4galt1 deletion in MKs renders β1 integrin hyperactive and unexpectedly also regulates heparin sulfate proteoglycan (HS PG) and heparinase expression in MKs thereby severely impairing thrombopoiesis at steady state and following myeloablative injury; 4) Heparinase and glycosaminoglycan (GAG) mimetics improved thrombopoiesis in wild type and B4galt1-/- MKs, suggesting a role for GAGs in thrombopoiesis. The overarching hypothesis of this program is that “distinct cell-intrinsic and extrinsic glycan-mediated mechanisms regulate maintenance, differentiation, and function of hematopoietic cells”. Project 1 will test the specific hypothesis that glycosylation regulates MK-biased HSCs and thrombopoiesis in three aims: In Aim 1, a functionally defined MK-biased hematopoietic stem cell will be investigated together with Project 2, especially with respect to the heavily α2,6- sialylated cell surface despite the absence of St6gal1 expression necessary to generate this structure. We will establish the role of α2,6-Sia in MK-biased HSPCs. Based on the known requirement of the galactosyltransferase β4GalT1 in thrombopoiesis, Project 1 will also investigate the roles of β4GalT1, glycosaminoglycans (GAGs)/HS PG in thrombopoiesis at steady-state (Aim 2) and following myeloablative stress (Aim 3) using the novel combined shared “omics” together with Project 3 and standard approaches. A previously unknown role of β4GalT1 to regulate MK expression of HS PG will also be investigated. This P...