Abstract
Reticulated platelets are newly formed, RNA-rich platelets with heightened reactivity. Although elevated levels are observed after myocardial ischemia/reperfusion injury, their functional contributions to postischemic pathology remains poorly defined. We aimed to determine whether reticulated platelets actively contribute to inflammation and repair following myocardial ischemia and reperfusion, rather than serving solely as biomarkers of platelet turnover. We generated Pf4-Cre:RiboTag mice, in which hemagglutinin-tagged ribosomal proteins are selectively expressed in megakaryocytes and platelets. Using hemagglutinin-based flow cytometry, we identified reticulated platelets without relying on nucleic acid dyes. Surface marker expression and agonist responsiveness were evaluated ex vivo. Bulk RNA sequencing was performed on sorted reticulated and non-reticulated platelets 48 hours after ischemia/reperfusion injury. Hemagglutinin-based detection revealed a time-dependent increase in circulating reticulated platelets after myocardial ischemia/reperfusion, confirmed by conventional dye-based methods. These platelets exhibited higher baseline expression of glycoprotein Ibα and greater agonist-induced activation of glycoprotein IIb/IIIa and P-selectin. Transcriptomic profiling demonstrated enrichment of genes associated with platelet activation, cytoskeletal reorganization, and wound healing. Ligand-receptor analysis suggested interactions between reticulated platelets and cardiac endothelial cells, fibroblasts, and macrophages. In conclusion, reticulated platelets constitute a transcriptionally distinct, hyperreactive platelet subset that may modulate post-ischemia/reperfusion inflammation and tissue remodeling. This genetic model provides a platform for mechanistic studies and may inform therapeutic strategies targeting platelet-mediated responses in cardiovascular disease.