From the Department of Surgery (L.Z.K., A.T.F., Z.A.M., B.N.-G., R.A.C.), Department of Anesthesiology and Perioperative Care (C.M.V.B., N.T.M., A.P., P.A.K., R.J.B.), Zuckerberg San Francisco General Hospital and the University of California, San Francisco, San Francisco, California; and Department of Surgery (M.J.C.), Denver Health Medical Center and the University of Colorado, Denver, Colorado.
J Trauma Acute Care Surg. 2020 Jun;88(6):742-751. doi: 10.1097/TA.0000000000002681.
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.
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.
Forty-two 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 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.
Diagnostic tests, level V.
受伤后循环血小板行为异常的机制仍不清楚。血小板保留巨核细胞不成熟的 RNA（RNA）剪接和蛋白质合成机制，根据生理信号改变其功能。我们试图鉴定创伤性脑损伤（TBI）患者无细胞血浆（CFP）中波动的血小板特异性 RNA 转录本，作为使用 RNA 测序来提高我们对损伤后血小板行为的理解的概念验证。我们假设，与最小受伤的创伤对照（t-controls）相比，我们可以从孤立性严重致命性 TBI（fTBI）患者的 CFP 中鉴定出差异表达的激活血小板特异性剪接 RNA 转录本，这些患者通过健康对照（h-controls）数据集进行过滤。
高读深度 RNA 测序应用于 10 例 fTBI 患者（头部 AIS≥3，所有其他类别 AIS<3，死亡）和 5 例 t-controls（损伤严重程度评分≤1，存活）的 CFP。使用来自 23 例 h-controls 的公开可用 CFP RNA 测序数据集来确定可在 CFP 中发现的剪接形式 RNA 转录本的相对稳定状态。激活的血小板特异性剪接 RNA 转录本源自体外血小板激活研究，并通过剪接接头存在大于 1.5 倍或小于 0.67 倍的体外非激活血小板特异性 RNA 转录本来鉴定。
fTBI 患者 CFP 中 34 个基因的 42 个差异剪接的激活血小板特异性 RNA 转录本发生改变（上调和下调）。
我们发现，与最小受伤的创伤对照相比，孤立性严重 fTBI 患者的 CFP 中存在差异表达的激活血小板特异性剪接 RNA 转录本，这些转录本上调或下调。这一概念验证表明，损伤后，一组不成熟的血小板 RNA 经历剪接事件，推测是为了调节血小板蛋白产物参与血小板功能。这验证了我们对损伤诱导的血小板 RNA 转录本调节的探索，作为一种识别异常血小板行为的新的损伤后血小板生物学和治疗靶点的上游“液体活检”。
诊断试验，等级 V。
