Sex-dependent molecular signatures of reticulated platelets

Introduction Reticulated platelets (RPs) exhibit enhanced reactivity and prothrombotic features compared with mature platelets (MPs) and are promising therapeutic targets in cardiovascular disease. Sex affects platelet function and thrombotic risk, yet sex-related molecular differences across platelet heterogeneity remain poorly defined. Aim To characterize sex-related transcriptional and proteomic profiles in RPs versus MPs with a multi-omics approach. Methods RPs and MPs were isolated from peripheral blood of patients with coronary artery disease (14 males, 5 females) using RNA-based staining and flow cytometric sorting. Total and small RNA sequencing were performed and comparisons between sorted RPs and MPs was conducted in a paired fashion within each individual. We performed sex-stratified differential expression and gene set enrichment analysis. MicroRNA and circular RNA profiles were integrated to explore potential post-transcriptional regulatory patterns. Mass cytometry of platelet-rich plasma was used to investigate proteomic profiles. Results Androgen receptor (AR, log2FC=1,06, padj=1,04e-09) and estrogen-related receptor alpha (ESRRA, log2FC=1,31, padj = 0,00018) transcripts were differentially upregulated in RPs compared with MPs (Figure 1A), whereas estrogen receptor beta showed no differential expression. Sex-stratified differential expression analysis demonstrated a moderate concordance of RP-MP differences across males and females (Pearson r=0.62), alongside a subset of genes exhibiting sex-specific transcriptional regulation (Figure 1B). Pathway-level gene set enrichment analyses showed that male RPs exhibited distinct lipid-inflammatory transcriptional programs with enrichment of cholesterol homeostasis (Figure 1C) and inflammatory response (Figure 1D) categories, suggesting that sex modifies RPs transcriptomic signatures. Mass cytometry confirmed the prothrombotic expression of RPs compared to MPs; however, no sex-based differences were detected at the proteomic level (Figure 1E). Conclusion This explorative analysis highlights biological sex as one component of platelet heterogeneity and provides a hypothesis-generating framework for future investigations into personalized antithrombotic strategies.