Platelet function tests utilizing agonists or patient serum are generally performed to assess platelet activation ex vivo. However, inter-individual differences in platelet reactivity and donor requirements make it difficult to standardize these tests. Here, we established a megakaryoblastic cell line for the conventional assessment of platelet activation. We first compared intracellular signaling pathways using CD32 crosslinking in several megakaryoblastic cell lines, including CMK, UT-7/TPO, and MEG-01 cells. We confirmed that CD32 was abundantly expressed on the cell surface, and that intracellular calcium mobilization and tyrosine phosphorylation occurred after CD32 crosslinking. We next employed GCaMP6s, a highly sensitive calcium indicator, to facilitate the detection of calcium mobilization by transducing CMK and MEG-01 cells with a plasmid harboring GCaMP6s under the control of the human elongation factor-1α promoter. Cells that stably expressed GCaMP6s emitted enhanced green fluorescent protein fluorescence in response to intracellular calcium mobilization following agonist stimulation in the absence of pretreatment. In summary, we have established megakaryoblastic cell lines that mimic platelets by mobilizing intracellular calcium in response to several agonists. These cell lines can potentially be utilized in high-throughput screening assays for the discovery of new antiplatelet drugs or diagnosis of disorders caused by platelet-activating substances.

中文翻译：

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建立用于常规评估血小板钙信号的巨核细胞系。

通常进行利用激动剂或患者血清的血小板功能测试以评估离体血小板活化。然而，个体间血小板反应性和供体需求的差异使得标准化这些检测变得困难。在这里，我们建立了巨核细胞系，用于常规的血小板活化评估。我们首先比较了CD32交联在几种巨核细胞系中的细胞内信号传导途径，包括CMK，UT-7 / TPO和MEG-01细胞。我们确认CD32在细胞表面上大量表达，并且CD32交联后发生了细胞内钙动员和酪氨酸磷酸化。接下来，我们使用高灵敏度钙指示剂GCaMP6s，通过在人延伸因子-1α启动子的控制下，通过携带带有GCaMP6s的质粒转导CMK和MEG-01细胞来促进钙动员的检测。在没有预处理的情况下，激动剂刺激后，稳定表达GCaMP6s的细胞会发出增强的绿色荧光蛋白荧光，以响应细胞内钙动员。总之，我们已经建立了巨核细胞系，通过响应几种激动剂动员细胞内钙来模拟血小板。这些细胞系可潜在地用于高通量筛选测定中，以发现新的抗血小板药物或诊断由血小板活化物质引起的疾病。在没有预处理的情况下，激动剂刺激后，稳定表达GCaMP6s的细胞会发出增强的绿色荧光蛋白荧光，以响应细胞内钙动员。总之，我们已经建立了巨核细胞系，通过响应几种激动剂动员细胞内钙来模拟血小板。这些细胞系可潜在地用于高通量筛选测定中，以发现新的抗血小板药物或诊断由血小板活化物质引起的疾病。在没有预处理的情况下，激动剂刺激后，稳定表达GCaMP6s的细胞会发出增强的绿色荧光蛋白荧光，以响应细胞内钙动员。总之，我们已经建立了巨核细胞系，通过响应几种激动剂动员细胞内钙来模拟血小板。这些细胞系可潜在地用于高通量筛选测定中，以发现新的抗血小板药物或诊断由血小板活化物质引起的疾病。