RNA-Seq of sorted non-RP and RP from healthy donors, CCS- and STEMI-patients

Background: Reticulated platelets (RP) are the youngest circulating platelets in blood. An increased amount of this platelet 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 to provide a multifactorial approach based on ultrastructural, transcriptional and functional analysis of RP compared to non-RP sorted by flow cytometry.Methods: Employing transmission electron microscopy, 1,089 micrographs were analyzed for platelet size, amounts of intracellular structures (α-granules, dense granules, open canalicular system (OCS)-openings, mitochondria) and surrogates for activation (shape, pseudopodia-formation). Long and small RNA-seq were performed and differential gene expression analysis was accomplished. Functional analysis of P-selectin – an upregulated mRNA in RP – was performed in healthy subjects and patients on P2Y12-inhibitor therapy.Results: Electron micrographs uncovered distinct ultrastructural differences in RP versus non-RP. Cross sections were 1.9-fold larger in RP (p<0.0001). Amounts of α-granules, dense granules, OCS-openings, and mitochondria were increased in RP which persisted after adjustment for platelet size. Long RNA-seq showed 131 upregulated (including P-selectin) and 78 downregulated mRNAs in RP which are predominantly associated to platelet shape change, aggregation and activation. Small RNA-seq did not reveal any differentially expressed genes. Functional analysis displayed higher P-selectin expression as compared to non-RP upon stimulation with ADP or TRAP.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.