Background Reticulated platelets (RP), also termed immature platelets, represent the youngest platelets within the platelet pool. They are characterized by distinct features such as an increased content of ribonucleic acids (RNA), greater volume, more dense granules, higher levels of surface activation markers and higher platelet reactivity. To date, several studies have shown an association of higher RP levels and cardiovascular events including mortality in patients with coronary artery disease.

Objective The current study sought to investigate by qualitative assessment of the RNA content whether the platelet transcriptome may contribute to specific properties of RNA-rich versus RNA-low platelets.  

Methods Citrate-anticoagulated blood samples were obtained from three healthy donors on two different days. Staining of washed platelets with SYTO® 13, a highly specific nucleic acid binding fluorescent dye showed that all platelets contain RNA, albeit to a substantially different extent. At the borders of the continuum, two gates representing the 20% of platelets with highest staining intensity (RNA-rich platelets) and the 20% of platelets with lowest staining intensity (RNA-low platelets) were used to sort platelets based on their RNA amount. Given the non-linear time-dependent RNA decay and the platelet lifespan of 8 to 10 days, RNA-rich platelets contain the youngest platelets, whereas RNA-low platelets constitute a population at the end of the platelet lifespan. After RNA extraction, RNA yield was measured by Bioanalyzer®. After preparation of libraries for long and small RNAs, Illumina Next Generation Sequencing was performed. Sequenced reads were analyzed by use of the local Galaxy platform. Differential gene expression and gene ontologies were assessed with DESeq2 and ClueGO, respectively.

Results RNA quantity measurement resulted in a median RNA amount of 0.52 [0.33 – 0.68] fg per platelet in RNA-low platelets and 1.04 [0.67 – 1.15] fg per platelet in RNA-rich platelets.   
Long RNA sequencing revealed 131 significantly enriched genes in RNA-rich platelets (adjusted p < 0.05 FDR by Benjamini-Hochberg correction) and 78 enriched genes in RNA-low platelets. Gene ontology analysis yielded 36 GO-terms for genes with increased expression in RNA-rich platelets (Fig.1) – particularly genes which are responsible for platelet reactivity, shape change, clotting and cell-cell-interactions (such as DMTN, SELP, ITGA2B, ITGB3). Only 1 GO-term -‘vesicle docking for exocytosis’- was found for genes enriched in RNA-low platelets.   
Small RNA sequencing of the examined platelet populations showed no significant up- or downregulation in miRNAs or YRNAs.

Conclusion RNA-sequencing of RNA-low and RNA-rich platelets indicated notable changes in transcripts of protein-coding genes. Especially genes and gene clusters involved in platelet reactivity are enriched in RNA-rich platelets. Thus, the increased reactivity of RNA-rich platelets is reflected in their transcriptome.  Further studies are needed to clarify whether these characteristics of RNA-rich platelets offer a window to potentially modify the properties of reticulated platelets.

Fig. 1: GO-terms of enriched genes in RNA-rich platelets.