Garcia Cedric, Dejean Sebastien, Savy Nicolas, Bordet Jean-Claude, Series Jennifer, Cadot Sarah, Ribes Agnès, Voisin Sophie, Rugeri Lucia, Payrastre Bernard, Sié Pierre
CHU de Toulouse, Laboratoire d'Hématologie, Toulouse, France.
Institut des Maladies Métaboliques et Cardiovasculaires INSERM U1048, Université de Toulouse, Toulouse, France.
Res Pract Thromb Haemost. 2023 May 16;7(4):100180. doi: 10.1016/j.rpth.2023.100180. eCollection 2023 May.
Availability of multichannel cytometers and specific commercial antibodies makes flow cytometry a new option to simultaneously assess multiple intracellular platelet signaling pathways for clinical purposes, in small volume of blood or low platelet count.
To describe a multicolor flow cytometry with fluorescent barcoding technique for screening signaling pathways downstream membrane receptors of major platelet agonists (adenosine diphosphate, thrombin, thromboxane, and collagen).
By comparison with immunoblotting, we first selected the target phosphoproteins, AKT, P38MAPK, LIMK, and SPL76; the times of stimulation; and phosphoflow barcoding conditions. We then performed a clinical study on whole blood of patients without evidence of blood platelet disorder on standard biological screening, consulting for trivial or occasionally provoked bleeds without familial antecedent (bleeding of unknown origin, = 23) or type-1 von Willebrand disease ( = 9). In addition, we included a small group of patients with definite platelet disorders (Glanzmann thrombasthenia, δ-storage pool deficiency, and immune glycoprotein VI-related disease with granule secretion defect).
The range, kinetics, and distribution of fluorescence intensity were established for each agonist-target protein combination. Principal component analysis indicates a correlation in response to a target phosphoprotein (AKT and P38MAPK) to different agonists but no correlation in the response of different target phosphoproteins to the same agonist. The heterogeneity of individual responses in the whole population displayed was analyzed using clustering algorithm. Patients with platelet storage pool deficiency were positioned as lowest responders on the heatmap.
In complement of functional tests, this study introduces a new approach for rapid platelet signaling profiling in clinical practice.
多通道细胞仪和特定商业抗体的可用性使流式细胞术成为一种新的选择，可用于在少量血液或低血小板计数的情况下，出于临床目的同时评估多种细胞内血小板信号通路。
描述一种采用荧光条形码技术的多色流式细胞术，用于筛选主要血小板激动剂（二磷酸腺苷、凝血酶、血栓素和胶原蛋白）膜受体下游的信号通路。
通过与免疫印迹法比较，我们首先选择了靶磷酸化蛋白、AKT、P38MAPK、LIMK和SPL76；刺激时间；以及磷酸化流式细胞术条形码条件。然后，我们对标准生物学筛查无血小板疾病证据、因轻微或偶尔发生的出血前来咨询且无家族病史（不明原因出血，n = 23）或1型血管性血友病（n = 9）的患者的全血进行了临床研究。此外，我们纳入了一小群患有明确血小板疾病（Glanzmann血小板无力症、δ-储存池缺乏症以及伴有颗粒分泌缺陷的免疫糖蛋白VI相关疾病）的患者。
确定了每种激动剂-靶蛋白组合的荧光强度范围、动力学和分布。主成分分析表明，针对不同激动剂的靶磷酸化蛋白（AKT和P38MAPK）的反应存在相关性，但不同靶磷酸化蛋白对同一激动剂的反应不存在相关性。使用聚类算法分析了所显示的整个人群中个体反应的异质性。血小板储存池缺乏症患者在热图上被定位为反应最低的人群。
作为功能测试的补充，本研究引入了一种在临床实践中快速进行血小板信号分析的新方法。
