A Journey Upstream: Fluctuating Platelet-Specific Genes in Cell Free Plasma as Proof-of-Concept for Using RNA Sequencing to Improve Understanding of Post-Injury Platelet Biology.

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 post-injury 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 to minimally-injured trauma controls (t-controls), filtered by healthy control (h-control) datasets.
High-read depth RNA sequencing was applied to CFP from ten 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 dataset 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 >1.5 or <.67 fold ex-vivo non-activated platelet-specific RNA transcripts.
42 differentially spliced activated platelet-specific RNA transcripts in 34 genes were altered in CFP from fTBI patients (both upregulated and downregulated).
We have discovered differentially-expressed activated platelet-specific spliced RNA transcripts present in CFP from isolated severe fatal TBI patients that are up- or downregulated compared to 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 post-injury platelet biology and treatment targets for aberrant platelet behavior.
VDiagnostic tests.