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

Abstract

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.

Trauma-induced coagulopathy

“…Efforts for deeper molecular phenotyping 89,[127][128][129][130] have uncovered multiple molecular phenotypes of platelet dysfunction characteristic of TIC, both adaptive and maladaptive in nature (fig. 4).…”

“…Further, platelet-mediated Toll-like receptor 4 (TLR4) signalling, attachment of histone H4 to platelets, platelet ballooning (a shape change that has procoagulant effects), recruitment of monocytes by platelet-derived high mobility group protein B1 (HMGB1), and neutrophil extracellular trap formation 89,132,133 are all pro-inflammatory mechanisms identified in association with early failures in platelet haemostasis and later hypercoagulability. Whether the diverse qualitative changes in platelet behaviour characteristic of TIC are favourable remains unclear 80 , and investigations beyond platelet biomarkers including microfluidics, cell culture, mitochondrial respiration, ultrastructure microscopy and genomic methods, are necessary to uncover platelet targets for alternative TIC therapies beyond human-donated blood products 89,99,127,134,135 .…”

The intersection of coagulation activation and inflammation after injury: What you need to know

“…31,32 It remains incompletely understood whether described platelet alterations are maladaptive biology or evidence of adaptive functions in the setting of hypoperfusion and hemorrhage. 33,34 Furthermore, evidence highlights that platelets play a crucial role in vascular biology beyond hemostasis. 35 The activation of platelets and complex interplay of receptors and signaling molecules mediates thromboinflammation through the vWF axis, 36 platelet shape and receptor conformation changes, 37 secretion of granule contents, 25,30 and exposure of procoagulant surfaces.…”

“…In the setting of injury and shock, platelet hemostatic functions are altered and have been the subject of much research related to TIC 31,32 . It remains incompletely understood whether described platelet alterations are maladaptive biology or evidence of adaptive functions in the setting of hypoperfusion and hemorrhage 33,34 . Furthermore, evidence highlights that platelets play a crucial role in vascular biology beyond hemostasis 35 .…”

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

“…[17][18][19][20][21][22][23][24][25][26] Therefore, transcriptomics is emerging as an attractive means of studying the platelet response to disease. 25,[27][28][29][30] Recently, we identified numerous RNAs potentially involved in platelet function in the plasma of traumatic brain injury patients 31 and hypothesized that platelets themselves were the most likely source of this. However, it remains unknown if the physiology of trauma alters the platelet transcriptome or if differential alternative splicing of platelet RNA could be responsible for the described changes in platelet function after injury.…”