A new trauma frontier: Exploratory pilot study of platelet transcriptomics in trauma patients.

BACKGROUND: The earliest measurable changes to postinjury platelet biology may be in the platelet transcriptome, as platelets are known to carry messenger ribonucleic acids (RNAs), and there is evidence in other inflammatory and infectious disease states of differential and alternative platelet RNA splicing in response to changing physiology. Thus, the aim of this exploratory pilot study was to examine the platelet transcriptome and platelet RNA splicing signatures in trauma patients compared with healthy donors. METHODS: Preresuscitation platelets purified from trauma patients (n = 9) and healthy donors (n = 5) were assayed using deep RNA sequencing. Differential gene expression analysis, weighted gene coexpression network analysis, and differential alternative splicing analyses were performed. In parallel samples, platelet function was measured with platelet aggregometry, and clot formation was measured with thromboelastography. RESULTS: Differential gene expression analysis identified 49 platelet RNAs to have differing abundance between trauma patients and healthy donors. Weighted gene coexpression network analysis identified coexpressed platelet RNAs that correlated with platelet aggregation. Differential alternative splicing analyses revealed 1,188 splicing events across 462 platelet RNAs that were highly statistically significant (false discovery rate <0.001) in trauma patients compared with healthy donors. Unsupervised principal component analysis of these platelet RNA splicing signatures segregated trauma patients in two main clusters separate from healthy controls. CONCLUSION: Our findings provide evidence of finetuning of the platelet transcriptome through differential alternative splicing of platelet RNA in trauma patients and that this finetuning may have relevance to downstream platelet signaling. Additional investigations of the trauma platelet transcriptome should be pursued to improve our understanding of the platelet functional responses to trauma on a molecular level.

A journey upstream: Fluctuating platelet-specific genes in cell-free plasma as proof-of-concept for using ribonucleic acid sequencing to improve understanding of postinjury platelet biology.

BACKGROUND: The mechanisms of aberrant circulating platelet behavior following injury remain unclear. Platelets retain megakaryocyte immature ribonucleic acid (RNA) splicing and protein synthesis machinery to alter their functions based on physiologic signals. We sought to identify fluctuating platelet-specific RNA transcripts in cell-free plasma (CFP) from traumatic brain injury (TBI) patients as proof-of-concept for using RNA sequencing to improve our understanding of postinjury platelet behavior. We hypothesized that we could identify differential expression of activated platelet-specific spliced RNA transcripts from CFP of patients with isolated severe fatal TBI (fTBI) compared with minimally injured trauma controls (t-controls), filtered by healthy control (h-control) data sets. METHODS: High-read depth RNA sequencing was applied to CFP from 10 patients with fTBI (Abbreviated Injury Scale [AIS] for head ≥3, AIS for all other categories <3, and expired) and five t-controls (Injury Severity Score ≤1, and survived). A publicly available CFP RNA sequencing data set from 23 h-controls was used to determine the relative steady state of splice-form RNA transcripts discoverable in CFP. Activated platelet-specific spliced RNA transcripts were derived from studies of ex vivo platelet activation and identified by splice junction presence greater than 1.5-fold or less than 0.67-fold ex vivo nonactivated platelet-specific RNA transcripts. RESULTS: Forty-two differentially spliced activated platelet-specific RNA transcripts in 34 genes were altered in CFP from fTBI patients (both upregulated and downregulated). CONCLUSION: We have discovered differentially expressed activated platelet-specific spliced RNA transcripts present in CFP from isolated severe fTBI patients that are upregulated or downregulated compared with minimally injured trauma controls. This proof-of-concept suggests that a pool of immature platelet RNAs undergo splicing events after injury for presumed modulation of platelet protein products involved in platelet function. This validates our exploration of injury-induced platelet RNA transcript modulation as an upstream "liquid biopsy" to identify novel postinjury platelet biology and treatment targets for aberrant platelet behavior. LEVEL OF EVIDENCE: Diagnostic tests, level V.