San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), Division of Regenerative Medicine, Stem Cells and Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.
Proteomic and Metabolomic Laboratory, Institute of Biomedical Technologies, National Research Council (ITB-CNR), Segrate, Italy.
J Allergy Clin Immunol. 2019 Sep;144(3):825-838. doi: 10.1016/j.jaci.2019.03.012. Epub 2019 Mar 27.
Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and life-threatening bleeding. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet counts remain low in GT-treated patients.
We carefully investigated platelet defects in terms of phenotype and function in untreated patients with WAS and assessed the effect of GT treatment on platelet dysfunction.
We analyzed a cohort of 20 patients with WAS/XLT, 15 of them receiving GT. Platelet phenotype and function were analyzed by using electron microscopy, flow cytometry, and an aggregation assay. Platelet protein composition was assessed before and after GT by means of proteomic profile analysis.
We show that platelets from untreated patients with WAS have reduced size, abnormal ultrastructure, and a hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores platelet size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein expression and length of follow-up.
Our study highlights the coexistence of morphologic and multiple functional defects in platelets lacking WAS protein and demonstrates that GT normalizes the platelet proteomic profile with consequent restoration of platelet ultrastructure and phenotype, which might explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in patients with XLT only presenting with microthrombocytopenia.
血小板减少症是所有经典 Wiskott-Aldrich 综合征（WAS）和 X 连锁血小板减少症（XLT）患者的严重问题，因为它会导致严重且危及生命的出血。针对 WAS 的慢病毒基因治疗（GT）在免疫重建方面显示出了有前景的结果。然而，尽管出血事件的严重程度和频率有所降低，GT 治疗的患者血小板计数仍然较低。
我们仔细研究了未经治疗的 WAS 患者的血小板缺陷表型和功能，并评估了 GT 治疗对血小板功能障碍的影响。
我们分析了 20 名 WAS/XLT 患者的队列，其中 15 名接受了 GT。通过电子显微镜、流式细胞术和聚集测定分析血小板表型和功能。通过蛋白质组谱分析评估 GT 前后血小板蛋白组成。
我们表明，未经治疗的 WAS 患者的血小板体积减小、超微结构异常，在稳态下表现出高激活表型，而对激动剂的激活和聚集反应降低。GT 在治疗后早期恢复血小板大小和功能，并与 WAS 蛋白表达和随访时间成正比地降低高激活表型的比例。
我们的研究强调了缺乏 WAS 蛋白的血小板存在形态和多种功能缺陷的共存，并证明 GT 使血小板蛋白质组谱正常化，随之恢复血小板超微结构和表型，这可能解释了 GT 后出血事件减少的现象。这些结果对于仅表现为微血小板减少症的 XLT 患者的未来临床试验也具有重要意义。
