Loroch Stefan, Trabold Katharina, Gambaryan Stepan, Reiß Cora, Schwierczek Kathrin, Fleming Ingrid, Sickmann Albert, Behnisch Wolfgang, Zieger Barbara, Zahedi René P, Walter Ulrich, Jurk Kerstin
Kerstin Jurk, Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Langenbeckstr. 1, 55131 Mainz, Germany, Tel.: +49 6131 178278, Fax: +49 6131 176238, E-mail:
Thromb Haemost. 2017 Feb 28;117(3):556-569. doi: 10.1160/TH16-07-0515. Epub 2017 Jan 12.
Glanzmann thrombasthenia (GT) is one of the best characterised inherited platelet function disorders but global platelet proteome has not been determined in these patients. We investigated the proteome and function of platelets from two patients with type I GT, caused by different homozygous ITGA2b mutations, from family members and unrelated controls. The global proteome of highly purified washed platelets was quantified by liquid chromatography-mass spectrometry (LC-MS) and targeted MS-methods. Platelet function was analysed by flow cytometry, light transmission aggregometry and flow-based assays. Platelets from GT patients showed less than 5 % relative levels of the integrin subunit α and 5-9 % fibrinogen compared to controls. These patients demonstrated loss of αβ-dependent platelet function, but normal platelet granule secretion induced by physiological agonists. Platelets from heterozygous family members of a patient expressed 50-60 % of control α levels which were sufficient for normal αβ-dependent platelet function. Studying type I GT as model disease we established quantitative LC-MS to detect and clearly distinguish normal platelets, platelets from GT heterozygotes and platelets from GT patients. Diminished levels of factor XIIIB chain, plasminogen and carboxypeptidase 2B were identified in thrombasthenic platelets. Additionally, GT platelets showed up to 2.5-fold increased levels of FcγRIIA and laminin-α4 chain. Elevated levels of platelet FcγRIIA was associated with increased CD63-surface expression after FcγRIIA-crosslinking in one GT-patient which might present a compensatory mechanism of platelet activation in GT. We demonstrate that quantitative LC-MS based proteomics is suitable to validate known but also to identify previously unknown protein level changes of dysfunctional platelets.
血小板无力症（GT）是特征最明确的遗传性血小板功能障碍之一，但尚未测定这些患者的全球血小板蛋白质组。我们研究了两名由不同纯合ITGA2b突变引起的I型GT患者、家庭成员和无关对照的血小板蛋白质组和功能。通过液相色谱-质谱联用（LC-MS）和靶向质谱方法对高度纯化的洗涤血小板的全球蛋白质组进行定量。通过流式细胞术、光透射聚集测定法和基于流式的测定法分析血小板功能。与对照组相比，GT患者的血小板中整合素亚基α和纤维蛋白原的相对水平低于5%。这些患者表现出αβ依赖性血小板功能丧失，但生理激动剂诱导的血小板颗粒分泌正常。患者杂合家庭成员的血小板表达的α水平为对照水平的50-60%，足以实现正常的αβ依赖性血小板功能。以I型GT作为模型疾病进行研究，我们建立了定量LC-MS来检测并明确区分正常血小板、GT杂合子血小板和GT患者的血小板。在血小板无力症血小板中鉴定出因子XIIIB链、纤溶酶原和羧肽酶2B水平降低。此外，GT血小板的FcγRIIA和层粘连蛋白-α4链水平升高高达2.5倍。在一名GT患者中，FcγRIIA交联后血小板FcγRIIA水平升高与CD63表面表达增加相关，这可能是GT中血小板激活的一种补偿机制。我们证明基于定量LC-MS的蛋白质组学适用于验证已知的，也适用于识别功能失调血小板以前未知的蛋白质水平变化。
