The pro-thrombotic transcriptomic signature of reticulated platelets in patients with chronic coronary syndrome

Dario Bongiovanni (Augsburg)1, K. Kirmes (München)2, M. Klug (München)2, L. Raka (Freising)3, O. Tsoy (Hamburg)4, Z. Louadi (Hamburg)5, C. Peano (Rozzano (Milan))6, C. Bloxham (München)2, J. Han (München)2, K.-L. Laugwitz (München)2, G. Condorelli (Freising)7, M. List (Freising)3, P. Raake (Augsburg)1, M. I. Bernlochner (München)2

1Universitätsklinikum Augsburg I. Medizinische Klinik Augsburg, Deutschland; 2Klinikum rechts der Isar der Technischen Universität München Klinik und Poliklinik für Innere Medizin I München, Deutschland; 3Chair of Experimental Bioinformatics Technical University of Munich Freising, Deutschland; 4Universität Hamburg Computational Systems Biology Hamburg, Deutschland; 5Chair of Computational Systems Biology University of Hamburg Hamburg, Deutschland; 6University of Milan Humanitas Clinical and Research Center Rozzano (Milan), Italien; 7Chair of Experimental Bioinformatics Freising, Deutschland


RPs are young, hyperreactive and prothrombotic, and RNA-rich platelets. They are predictors of an insufficient response to antiplatelet therapy after myocardial infarction and are suggested to play a key role in chronic coronary syndrome (CCS) patients with a high on-treatment platelet reactivity. Moreover, RPs are promising novel biomarkers for predicting adverse cardiovascular events in different pathological settings. 
We aimed to compare, for the first time, the transcriptomic profiles of RPs and MPs in CCS patients.
Using fluorescent activated cell sorting (FACS), we isolated RPs and MPs based on their RNA content from the peripheral blood of 19 CCS patients. After sorting, RNA was extracted and quality, concentration, and integrity were assessed with the Tapestation 4200 platform (Agilent). TotalRNA 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 and a cut-off of p <0.005 and a log2fc >1 was applied. Alternative splicing event detection and analysis was performed with MAJIQ. We performed circular RNA (circRNA) analysis using CIRCexplorer. 
With total RNA-sequencing, we detected 1589 genes differentially expressed with 1100 transcripts upregulated in RPs, while 489 were enriched in MPs (Figure 1 A and B). Of interest, the transcripts for the collagen receptor GP6 (log2FC 1.12, p=6.89x10-41), thrombin receptor PAR4 (F2RL3, log2FC 1.1, p=3.54*10-21) and Von Willebrand Factor (log2FC 1.2, p value 1.26*10-38) were significantly enriched in RPs. These results are in line with our previous finding and support the hypothesis that RPs in cardiovascular disease are hyperreactive and prothrombotic due to their different RNA content, compared to other platelets. In contrast to these prothrombotic transcripts, we found an enrichment of transcripts coding for genes involved in RNA processing such as the RNA regulator and splicing factor LUC7L3 (log2FC -1.01, p value 6.65*10-22) in MPs. We detected several splicing events differentially regulated: an alternative splicing on the collagen receptor transcript GP6 is upregulated in RPs (Figure 1E); near the transcript of the G-protein GNAQ, one alternative splicing event was found to be upregulated in RPs. Additionally, we detected differentially regulated miRNAs in RPs compared to MPs (Figure 1F&G). Nevertheless, several miRNAs that have not been described before were found enriched in RPs (Figure 1F). 
This study represents the first transcriptomic profiling of RPs and MPs in patients with CCS and provides for the first time a biological explanation of RPs’ hyperreactivity by reporting a differential enrichment of transcripts involved in platelet activation. The clear upregulation of prothrombotic signaling in RPs could provide and explanation to their hyperactivity and their correlation with adverse cardiovascular events in different pathological settings. Altogether, these findings shed light on a new therapeutic niche in CCS patients. However, the detrimental role of RPs in patients with coronary artery disease requires further investigation.

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