Sex- and age-specific transcriptomic changes drive the prothrombotic potential of megakaryocytes

Background Platelet (Plt) reactivity increases with age and contributes to cardiovascular (CV) and thromboembolic disease (TE). Despite the known involvement of genetic and acquired risk factors, the mechanisms underlying age-related platelet dysfunction remain unclear. Here, we studied the age-specific pro-thrombotic and pro-inflammatory characteristics of megakaryocytes (Mk) in an aging mouse model, with specific focus on sex differences. Methods Single-cell RNAseq was conducted on bone marrow (BM) samples obtained from femora and tibiae of young (3 months) and aged (>24 months) male and female mice, as well as from young and middle-aged human donors. Sorting was employed to enrich Mk from the BM. Cells were processed and sequenced using the 10x Genomics single platform. Mk cells were defined transcriptionally by the expression of Pf4, Vwf, Gp6, Gp1ba, Itgb3, and Mpl genes. By functional pathway analyses, based on Gene Ontology (GO), we compared transcriptional signatures between aged and young mice using Seurat workflow and RStudio (v.2023.12.0). Results In mice samples, we identified five distinct Mk clusters (Mk1 to Mk5) (Fig. 1A), with Mk3 and Mk4 notably increased in aged mice, consistently in both males and females (Fig. 1B). Pathway enrichment analyses revealed that Mk3 was enriched for procoagulant/pro-aggregatory genes, while Mk4 showed enrichment for pro-immune-inflammatory genes (Fig. 1C). Additionally, differential gene expression (DGE) analysis found that aged Mk populations exhibited higher expression of platelet-related pathways, including integrin signaling, aggregation, and platelet-leukocyte interactions. Further examination of gene expression changes revealed sex-specific differences in key platelet-related receptors. In females, Itgb1 (integrin β1) and Itga2b (integrin αIIbβ3) were reduced to 0.54-fold and 0.57-fold, respectively. In males, Itga2b expression increased to 1.39-fold, while Par1/F2r (thrombin receptor) was reduced to 0.7-fold. Notably, aged females exhibited a strong reduction in the ADP receptor P2ry12 (0.25-fold), potentially limiting platelet responsiveness. However, this was accompanied by a significant increase in the serotonin receptor Htr2a (8.75-fold), suggesting a compensatory mechanism. Findings in human donors showed only middle-aged females has a robust PF4 upregulation and a trend toward increased expression of collagen receptors (GP9, GP6, ITGA2), vWF receptor (GP1BA), P2RY12, and HTR2A, indicating age-dependent shifts in platelet function. Conclusions Aging is associated with the upregulation of immune-related and pro-thrombotic Mk subsets in BM, in both male and female. Interestingly, aged female mice exhibited a shift from integrin-related signaling to increased expression of the serotonin receptor gene, potentially as a compensatory response to the observed reduction in ADP receptor expression.