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

Reticulated platelets (RP) are the youngest circulating platelets in peripheral blood. An increased amount of this platelet subpopulation is associated with cardiovascular events and mortality. It is unknown so far to what extent intrinsic properties of RP contribute to their hyperreactive features. This study is the first to provide a multifactorial explanatory approach based on ultrastructural, transcriptional and functional analysis of RP compared to non-RP.1,089 micrographs were analyzed for platelet size and amount of intracellular components (α-granules, dense granules, open canalicular system (OCS)-openings, mitochondria) and surrogates for platelet activation (shape, pseudopodia formation). Long and small RNA-seq of platelet populations sorted by flow cytometry were performed and differential gene expression analysis was accomplished. A functional analysis of P-selectin – an upregulated gene in RP – was performed in healthy subjects and patients on P2Y12-inhibitor therapy.The platelet micrographs exposed distinct ultrastructural differences in RP versus non-RP. Platelet cross sections were 1.9-fold higher 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 and 78 downregulated genes in RP (including P-selectin) which are predominantly associated to platelet shape change, aggregation and activation. Small RNA-seq did not reveal any differentially expressed genes. Functional analysis displayed a higher P-selectin expression as compared to non-RP upon stimulation with ADP or TRAP.Our results demonstrate that altered intrinsic properties contribute to the hyperreactivity of RP. These properties and an increased amount of RP account for the association with cardiovascular risk.