BCR-ABL tyrosine kinase inhibitors (TKIs) are effective drugs in the treatment of patients with chronic myeloid leukemia. However, based on clinical studies, ponatinib was associated with the development of thrombotic complications. Since endothelial cells (ECs) regulate blood coagulation, their abnormal phenotype may play a role in the development of thrombotic events. We here aimed to investigate the effect of ponatinib on the procoagulant activity of cultured endothelial cells in vitro. Human coronary artery endothelial cells (HCAECs) were incubated with 50, 150, and 1000 nM of ponatinib. Subsequently, phosphatidylserine (PS) exposure and endothelial microvesicles (EMVs) were measured by flow cytometry. In addition, EC- and EMV-dependent thrombin generation was analyzed. To investigate pro-apoptotic effects of ponatinib, the level of Bax and Bcl-xL proteins were studied using Western blot and F3, THBD, and VCAM1 mRNAs were quantified by qPCR. Therapeutic concentrations of ponatinib significantly increased PS expression on ECs and the amount of EMVs which significantly shortened the time parameters of thrombin generation. In addition, these changes were associated with an increased ratio of Bax and Bcl-xL proteins in the presence of the decreased THBD mRNA level. Overall, ponatinib enhances the procoagulant activity of ECs via inducing apoptosis, which may contribute to thrombotic events.

中文翻译：

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帕纳替尼通过诱导细胞凋亡在人冠状动脉内皮细胞中诱导促凝血表型

BCR-ABL酪氨酸激酶抑制剂（TKI）是治疗慢性粒细胞白血病的有效药物。然而，根据临床研究，普纳替尼与血栓并发症的发生有关。由于内皮细胞（EC）调节血液凝固，因此它们的异常表型可能在血栓事件的发生中发挥作用。我们的目的是研究普纳替尼对体外培养的内皮细胞促凝血活性的影响。将人冠状动脉内皮细胞 (HCAEC) 与 50、150 和 1000 nM 的 ponatinib 一起孵育。随后，通过流式细胞术测量磷脂酰丝氨酸（PS）暴露和内皮微泡（EMV）。此外，还分析了 EC 和 EMV 依赖性凝血酶的生成。为了研究 ponatinib 的促凋亡作用，使用蛋白质印迹研究了 Bax 和 Bcl-xL 蛋白的水平，并通过 qPCR 定量了 F3、THBD 和 VCAM1 mRNA。 ponatinib 的治疗浓度显着增加了 EC 上的 PS 表达和 EMV 的数量，从而显着缩短了凝血酶生成的时间参数。此外，这些变化与 THBD mRNA 水平降低的情况下 Bax 和 Bcl-xL 蛋白比例增加有关。总体而言，ponatinib 通过诱导细胞凋亡增强 EC 的促凝血活性，这可能导致血栓事件。