Differential Expression Analysis by RNA-Seq Reveals Perturbations in the Platelet mRNA Transcriptome Triggered by Pathogen Reduction Systems.

Abdimajid Osman,Walter E. Hitzler,Patrick Provost,Adam Ameur,Michael Schubert

doi:10.1371/journal.pone.0133070

Abstract

Platelet concentrates (PCs) are prepared at blood banks for transfusion to patients in certain clinical conditions associated with a low platelet count. To prevent transfusion-transmitted infections via PCs, different pathogen reduction (PR) systems have been developed that inactivate the nucleic acids of contaminating pathogens by chemical cross-linking, a mechanism that may also affect platelets’ nucleic acids. We previously reported that treatment of stored platelets with the PR system Intercept significantly reduced the level of half of the microRNAs that were monitored, induced platelet activation and compromised the platelet response to physiological agonists. Using genome-wide differential expression (DE) RNA sequencing (RNA-Seq), we now report that Intercept markedly perturbs the mRNA transcriptome of human platelets and alters the expression level of >800 mRNAs (P<0.05) compared to other PR systems and control platelets. Of these, 400 genes were deregulated with DE corresponding to fold changes (FC) ≥2. At the p-value < 0.001, as many as 147 genes were deregulated by ≥ 2-fold in Intercept-treated platelets, compared to none in the other groups. Finally, integrated analysis combining expression data for microRNA (miRNA) and mRNA, and involving prediction of miRNA-mRNA interactions, disclosed several positive and inverse correlations between miRNAs and mRNAs in stored platelets. In conclusion, this study demonstrates that Intercept markedly deregulates the platelet mRNA transcriptome, concomitant with reduced levels of mRNA-regulatory miRNAs. These findings should enlighten authorities worldwide when considering the implementation of PR systems, that target nucleic acids and are not specific to pathogens, for the management of blood products.

Highlights

Circulating blood platelets play a central role in diverse physiological processes including hemostasis, fibrinolysis, blood vessel repair and inflammation
This Figure shows the number of genes that were above the threshold 1/10,000 of βactin expression and differentially expressed at specific levels of statistical significance. In these messenger RNA (mRNA) differential expression (DE) analysis, we found that none of the > 5,300 platelet transcripts detected was differentially expressed in the irradiated platelets, compared to control platelets, indicating that gamma-irradiation has no impact on the platelet mRNA
McCloskey et al [23] recently reported that Kv1.3 forms the voltage-gated K+ channel of platelets and megakaryocytes, is responsible for the major K+ conductance and resting potential of platelets, and influences the number of circulating platelets. These results indicate that the Intercept system, which is either used or considered to be implemented for pathogen reduction of blood components by blood banks, markedly alters the platelet mRNA transcriptome, which may negatively impact the platelets’ response and function involving de novo mRNA translation into bioactive effector proteins

Summary

Introduction

Circulating blood platelets play a central role in diverse physiological processes including hemostasis, fibrinolysis, blood vessel repair and inflammation. Derived from their bone marrow precursor megakaryocytes, platelets are anucleate and are deprived of genomic DNA, so they are incapable of achieving de novo genomic DNA transcription. A platelet count lower than 100 × 109/L is internationally recognized as the threshold for diagnosis of thrombocytopenia and is associated with increased risk of bleeding [6] This clinical condition can be treated by transfusion of platelet concentrates (PCs) prepared from the blood of healthy donors by blood banks, where they are stored at ambient temperature for several days prior to transfusion. We proposed that Intercept-treatment might activate platelets and induce the release of nucleic acids from platelets, and impoverish platelets in microRNAs

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Discussion

Conclusion