Canault Matthias, Ghalloussi Dorsaf, Grosdidier Charlotte, Guinier Marie, Perret Claire, Chelghoum Nadjim, Germain Marine, Raslova Hana, Peiretti Franck, Morange Pierre E, Saut Noemie, Pillois Xavier, Nurden Alan T, Cambien François, Pierres Anne, van den Berg Timo K, Kuijpers Taco W, Alessi Marie-Christine, Tregouet David-Alexandre
Institut National de la Santé et de la Recherche Médicale (Inserm), UMR_S 1062, 13005 Marseille, France Inra, UMR_INRA 1260, 13005 Marseille, France Aix Marseille Université, 13005 Marseille, France.
Post-Genomic Platform of Pitié-Salpêtrière (P3S), Pierre and Marie Curie University, F-75013 Paris, France.
J Exp Med. 2014 Jun 30;211(7):1349-62. doi: 10.1084/jem.20130477. Epub 2014 Jun 23.
The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1 and to perform proper αIIbβ3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet's ability to form thrombi under flow and spread normally as a consequence of reduced Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytes corrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis.
对三名患有严重出血的兄弟姐妹所患遗传性血小板疾病的性质进行了研究。通过全外显子组测序，我们在编码钙和二酰基甘油调节鸟嘌呤交换因子-1（CalDAG-GEFI）的RAS鸟苷释放蛋白-2（RASGRP2）基因中鉴定出致病突变（cG742T）。携带该突变的个体的血小板激活Rap1以及进行适当的αIIbβ3整合素外向内信号传导的能力降低。在HEK293T细胞中表达CalDAG-GEFI突变体可消除刺激后的Rap1激活。然而，蛋白激酶C和ADP依赖性途径在缺乏功能性CalDAG-GEFI的情况下允许残余的血小板激活。由于Rac1 GTP结合减少，该突变损害了血小板在流动状态下形成血栓和正常铺展的能力。功能缺陷仅限于血小板和巨核细胞，白细胞无改变。这与由Kindlin-3缺失引起的III型白细胞黏附缺陷的表型形成对比。杂合子不发生出血且血小板聚集正常；然而，他们的血小板在流动状态下不能正常黏附以及铺展，这与纯合子血小板类似。对培养的患者巨核细胞进行的挽救实验在用野生型RASGRP2转染后纠正了功能缺陷。值得注意的是，单个正常等位基因的存在足以预防出血，这使得CalDAG-GEFI成为预防血栓形成的一个新的且可能安全的治疗靶点。
