The pro-thrombotic transcriptomic signature of reticulated platelets in patients with chronic coronary syndrome
Authors: Bongiovanni D , Klug M , Mueller M , Santovito D , Weber C , Peano C , Laugwitz K.L , Bernlochner I 
Published: November 2020

Abstract

Introduction
Reticulated or immature platelets (RPs) are hyper-reactive young platelets that are larger and contain significantly more RNA compared to mature platelets (MPs). High levels of RPs in peripheral blood are predictors of an insufficient response to dual antiplatelet therapy and of adverse cardiovascular events in cardiovascular patients. Recently, we reported for the first time an enrichment of prothrombotic transcripts in RPs transcriptome of healthy donors. However, the biology of RPs in patients with coronary artery disease has not been investigated yet.

Purpose
We aimed to compare for the first time the transcriptomic profiles of RPs and MPs in patients with chronic coronary syndrome (CCS).

Methods
RPs and MPs from peripheral blood of CCS patients were isolated using fluorescent activated cell sorting (FACS) based on their RNA-content. After sorting, RNA was extracted and quality, concentration and integrity were assessed with the Tapestation 4200 platform (Agilent). Total-RNA libraries were prepared, multiplexed and sequenced on a NextSeq 500 Illumina platform obtaining 80 to 100 million paired-end reads per sample. RNA-sequencing analysis was performed with R and DESeq2.

Results
Total-RNA-sequencing detected 538 genes differentially expressed (300 downregulated, 238 upregulated) in RPs compared to MPs in CCS patients (Figure 1A). In particular, transcripts for the collagen receptor GP6 (FC 1.89, p=4.7x10–23), thrombin receptor PAR4 (F2RL3, FC 1.97, p=3.5x10–11), the ATP receptor P2RX1 (FC 1.94, p=3.1x10–15) and the ADP receptor P2RY1 (FC 1.82, p=3.15x10–10) were significantly enriched in RPs, whereas RNA regulators as the RISC-component TNRC6A (FC 0.5, p=7.98x10–13) and the splicing factor LUC7L3 (log2FC 0.55, p=1.76x10–11) were downregulated in RPs. Gene ontology analysis revealed an enrichment of relevant biological categories in RPs including “platelet activation” (fold enrichment = 10.5, p=1.8x10–8) and “blood coagulation” (fold enrichment = 4.4, p=2.4x10–3). Splicing analysis detected several differential splicing events. Of note, we detected an alternative splicing on GP6 transcript present only in RPs and absent in MPs (p=0.03, Figure 1B) At last, backsplicing analysis detected an enrichment of circular-RNAs in MPs.

Conclusions
This study represents the first deep transcriptomic profiling of RPs and MPs in patients with CCS and reports for the first time a differential enrichment of transcripts involved in platelet activation. Moreover, we could detect for the first time alternative splicing events in RPs and an enrichment of circular-RNAs in MPs. The clear upregulation of prothrombotic signaling in RPs (schematic overview Figure 1C) could explain, at least in part, their hyper-activity and their correlation with cardiovascular events in different pathological settings at it may offer a new therapeutic niche in patients with CCS.
Figure 1

Funding Acknowledgement
Type of funding source: Foundation. Main funding source(s): German society of cardiology (DGK)