2181The prothrombotic transcriptome of reticulated platelets

Introduction Reticulated platelets (RPs) are young, hyper-reactive platelets that are larger and contain significantly more RNA compared to older mature platelets. High levels of RPs in peripheral blood are predictors of an insufficient response to dual antiplatelet therapy in cardiovascular patients and of adverse cardiovascular events also in non-cardiac patients. However, the mechanisms underlying these correlations remains widely unknown and the biology of RPs has not been investigated yet. Purpose We aimed to compare for the first time the transcriptomic profiles of RPs and mature platelets (MPs). Methods RPs and MPs from peripheral blood of healthy donors were identified and isolated using FACS/Sorting based on their RNA-content. Immediately after sorting, RNA was extracted and quality, concentration and integrity was assessed with the Tapestation 4200 platform (Agilent). Total- and small-RNA libraries were prepared, multiplexed and sequenced on a NextSeq 500 Illumina platform Results Total-RNA-sequencing revealed 1744 differentially expressed genes (670 downregulated 1074 upregulated) in RPs compared to MPs (Figure 1A, B). In particular, transcripts for the collagen receptor GP6, thromboxane receptor A2 (TBXA2R), thrombin receptor PAR4 (F2RL3) and ATP receptor P2RX1 were significantly enriched in RPs, whereas several RNA regulators as the ribonuclease PARN, the RISC-component TNRC6A and the splicing factor LUC7L3 were downregulated in RPs. Gene ontology analysis revealed an enrichment of relevant biological categories in RPs including platelet activation and blood coagulation (Figure 1C). Gene Set Enrichment Analysis showed an enrichment of several activation pathways like thrombin, thromboxane and GPIIb/IIIa signaling in RPs. Small-RNA-sequencing reported 9 miRNAs significantly downregulated in RPs with targets involved in platelet reactivity. Figure 1 Conclusions This study represents the first comparative transcriptome analysis of RPs and MPs and reports for the first time a differential enrichment of transcripts involved in platelet activation. The clear upregulation of prothrombotic signaling in RPs could explain, at least in part, their hyper-activity and their correlation with cardiovascular events in different pathological settings (trancripts enriched in RPs: Figure 1D). Acknowledgement/Funding German Society of Cardiology (DGK Nr.102018) ESC First conctact initiative grant 2018