Nascent preplatelets and B4GALT1 glycosylation contribute to 5-FU-induced bone marrow recovery.

Megakaryocytes (MKs) are essential for hemostasis, vascular integrity, and hematopoietic stem and progenitor cell (HSPC) support, but their role in bone marrow (BM) recovery has remained underexplored. Here, using a 5-fluorouracil (5-FU) injury model, we identify a transient intramedullary space enriched in extracellular matrix (ECM) proteins such as perlecan, von Willebrand factor, and heparanase, as well as functionally responsive GPIbα+ platelet particles (termed preplatelets). During 5-FU-induced injury, this compartment undergoes dynamic changes, and its resolution depends on the presence of functional preplatelets and ECM components. Consistent with this, platelet depletion further delays restoration of the intramedullary space despite preserved MK numbers, supporting a critical local role for nascent platelets during 5-FU-induced injury. Furthermore, mice lacking β-1,4-galactosyltransferase 1 (B4GALT1) develop persistent thrombocytopenia, exhibit mislocalized and morphologically abnormal MKs, and display expansion of MK-biased HSPCs after 5-FU injury, collectively leading to delayed hematopoietic recovery and expansion of the BM intramedullary space. Single-cell transcriptomic analysis of B4GALT1-/- MK-biased HSPCs at steady state further revealed disruption of adhesion, cytoskeletal, and Notch1-associated programs required for proplatelet formation and MK interactions with ECM components. Our findings reveal a previously unrecognized role for locally retained platelet intermediates and identify B4GALT1-dependent glycosylation as a key regulator of megakaryocyte integrity, platelet production, and hematopoietic recovery after 5-FU-induced myeloablation.