Ultrastructural, transcriptional, and functional differences between human reticulated and non-reticulated platelets

Background Reticulated platelets (RP) are the youngest circulating platelets in blood. An increased amount of this subpopulation is associated with higher cardiovascular risk and mortality. Objectives It is unknown to what extent intrinsic properties of RP contribute to their hyperreactive features. This study is the first providing a multifactorial approach based on ultrastructural, transcriptional, and functional analysis of RP compared to non-RP sorted by flow cytometry. Methods Reticulated platelets and non-RP were sorted after platelet staining with SYTO 13. Employing transmission electron microscopy, 1089 micrographs were analyzed for platelet size, amounts of intracellular structures, and anatomical surrogates indicating activation. Long and small RNA-sequencing (RNA-seq) were performed for analyzing differential gene expression. Functional analysis of P-selectin-an upregulated mRNA in RP-was performed in healthy subjects and patients on P2Y(12)-inhibitors. Results Electron micrographs uncovered distinct ultrastructural differences in RP versus non-RP. Cross sections were 1.9-fold larger in RP (P < .0001). Amounts of alpha-granules, dense granules, open canalicular system-openings, and mitochondria were increased in RP, which persisted after adjustment for platelet size. Long RNA-seq showed 1212 upregulated transcripts that are predominantly associated to platelet shape change, aggregation, and activation; 1264 mRNAs were downregulated in RP. Small RNA-seq did not reveal any differentially expressed transcripts. Functional analysis displayed higher P-selectin expression as compared to non-RP upon ADP- or TRAP-stimulation. Conclusions Our results demonstrate that altered intrinsic structural and molecular properties contribute to the hyperreactivity of RP. These properties and an increased amount of RP may account for the association with cardiovascular risk.