National Medical Research Centеr of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev, 1 Samory Mashela St, Moscow 117198, Russia.
Center for Theoretical Problems of Physico-Сhemical Pharmacology, Russian Academy of Sciences, 30 Srednyaya Kalitnikovskaya str., Moscow 109029, Russia.
Int J Mol Sci. 2020 Apr 25;21(9):3035. doi: 10.3390/ijms21093035.
Immune thrombocytopenia (ITP) is an autoimmune condition primarily induced by the loss of immune tolerance to the platelet glycoproteins. Here we develop a novel flow cytometry approach to analyze integrin αβ functioning in ITP in comparison with Glanzmann thrombasthenia (GT) (negative control) and healthy pediatric donors (positive control). Continuous flow cytometry of Fura-Red-loaded platelets from whole hirudinated blood was used for the characterization of platelet responses to conventional activators. Calcium levels and fibrinogen binding were normalized to ionomycin-induced responses. Ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Platelets from all ITP patients had significantly higher cytosolic calcium concentration in the quiescent state compared to healthy donors (15 ± 5 nM vs. 8 ± 5 nM), but calcium increases in response to all activators were normal. Clustering analysis revealed two subpopulations of ITP patients: the subgroup with high fibrinogen binding (HFB), and the subgroup with low fibrinogen binding (LFB) (8% ± 5% for LFB vs. 16% ± 3% for healthy donors in response to ADP). GT platelets had calcium mobilization (81 ± 23 nM), fibrinogen binding (5.1% ± 0.3%) and thrombus growth comparable to the LFB subgroup. Computational modeling suggested phospholipase C-dependent platelet pre-activation for the HFB subgroup and lower levels of functional integrin molecules for the LFB group.
免疫性血小板减少症（ITP）是一种主要由血小板糖蛋白的免疫耐受丧失引起的自身免疫性疾病。在这里，我们开发了一种新的流式细胞术方法，用于分析 ITP 中整合素 αβ 的功能，与 Glanzmann 血小板无力症（GT）（阴性对照）和健康儿科供体（阳性对照）进行比较。使用全血中加载 Fura-Red 的血小板的连续流式细胞术来表征血小板对常规激活剂的反应。将钙水平和纤维蛋白原结合归一化为离子霉素诱导的反应。在平行板流动室中观察到胶原上的体外血栓形成。与健康供体相比（15±5 nM 比 8±5 nM），所有 ITP 患者的静止状态下的胞质钙浓度明显更高（15±5 nM 比 8±5 nM），但对所有激活剂的钙增加均正常。聚类分析显示 ITP 患者存在两个亚组：高纤维蛋白原结合（HFB）亚组和低纤维蛋白原结合（LFB）亚组（ADP 反应中 LFB 组为 8%±5%，健康供体为 16%±3%）。GT 血小板具有钙动员（81±23 nM）、纤维蛋白原结合（5.1%±0.3%）和与 LFB 亚组相当的血栓生长。计算模型表明 HFB 亚组存在依赖于 PLC 的血小板预激活，而 LFB 组的功能性整合素分子水平较低。
