Age- and sex-specific transcriptomic changes drive the prothrombotic potential of megakaryocytes

Aging is associated with enhanced platelet activation and inflammatory responses, contributing to an increased risk of thrombotic and cardiovascular events. However, how megakaryocytes (Mk) change with age or between sexes is still unclear.We performed single-cell RNA sequencing to profile bone marrow from young (3-month-old) and old (> 24-month-old) C57BL/6 mice, as well as from young and middle-aged human donors. In parallel, platelet activation was assessed by CD62P expression using flow cytometry in isolated and washed mouse platelets from young and old donors. Single-cell clustering in mouse bone marrow revealed five distinct Mk subpopulations. The age-dependent shift was more pronounced for subpopulations associated with platelet activation (mMk3) and immune/inflammatory responses (mMk4). mMk3 cells were more abundant in females, whereas mMk4 predominated in males. An increased platelet activation phenotype in old mice was confirmed in both sexes by CD62P expression analyses. Differential gene expression analyses showed that aging alters gene expression patterns related to platelet activation and aggregation via receptor signaling, granule formation, and inflammation. Aged males exhibited enrichment in genes linked to mitochondrial function, oxidative stress, lipid metabolism and membrane dynamics, whereas aged females showed upregulation of serotonin receptor genes. Human transcriptomic data support our murine findings, exhibiting increased expression of genes involved in mitochondrial electron transport and immune signaling in middle-aged individuals. Our study reveals a complex, sex-specific remodeling of Mk subpopulations in the aging organisms, characterized by distinct pro-thrombotic and pro-inflammatory transcriptional profiles. These observations suggest potential targets for developing age- and sex-tailored antithrombotic therapies.