Proteogenomics of ex vivo-activated platelets.

Platelets are major players in the process of intravascular thrombus formation. Current therapeutic strategies still fail to prevent thrombotic coronary events in a substantial number of patients, indicating that the complex mechanisms modulating platelet response during activation are not fully elucidated. The evidence that platelets are capable of de novo protein synthesis has raised the issue of whether and how these resident mRNAs are regulated in circulating platelets. Among the several mechanisms potentially involved, mRNA splicing may be relevant. Purified platelet-rich plasmas from healthy volunteers were collected and in vitro activated with collagen or Thrombin Receptor Activating Peptide (TRAP). Transcriptome profiling by RNA-Seq and in silico intron representation analysis were applied to search for the presence of pre-mRNA molecules and splicing events affected by platelet activation. HiRIEF LC-MS allowed platelet proteome characterization at deep coverage to investigate a possible correlation between splicing events and protein levels. By comparing computational and wet-lab analyses it was possible to identify a set of transcripts influenced at both intron and protein level.

血小板是血管内血栓形成过程中的核心参与者。当前的治疗策略仍无法在大量患者中预防血栓性冠状动脉事件，这表明调控血小板活化过程中应答反应的复杂机制尚未完全阐明。已有研究证实血小板具备蛋白质从头合成（de novo protein synthesis）的能力，这引发了一个关键科学问题：循环血小板内的驻留mRNA是否受到调控、以及如何被调控。在诸多潜在参与的调控机制中，mRNA剪接可能发挥相关作用。本研究收集了健康志愿者的纯化富血小板血浆，并采用胶原蛋白或凝血酶受体激活肽（Thrombin Receptor Activating Peptide，TRAP）进行体外活化处理。通过RNA测序（RNA-Seq）开展转录组谱分析，并结合硅基内含子表征分析，以探究血小板活化过程中前mRNA（pre-mRNA）分子的存在情况，以及受活化影响的剪接事件。采用HiRIEF LC-MS技术对血小板蛋白质组进行深度覆盖表征，以探究剪接事件与蛋白质表达水平之间的潜在关联。通过对比计算机分析与实验室湿实验分析结果，最终鉴定得到一批同时在剪接内含子层面与蛋白质层面受到调控的转录本。