Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada.
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
PLoS One. 2022 Nov 15;17(11):e0276584. doi: 10.1371/journal.pone.0276584. eCollection 2022.
Homeostasis between platelet production and clearance is essential for human health. A critical facet of the balance that facilitates platelet clearance from the circulation is apoptosis (programmed cell death). The precise cellular mechanisms that underpin platelet apoptosis are not defined. In nucleated cells, reorganization of the actin cytoskeleton is known to regulate platelet apoptosis. However, the role of the actin cytoskeleton in regulating apoptosis in platelets has not been extensively studied as they are anucleate and exhibit a distinctive physiology. Here, apoptosis was induced in washed human platelets using ABT-737, a BH3-mimetic drug. Mitochondrial depolarization was measured using the ratiometric dye JC-1; surface phosphatidylserine (PS) exposure was measured by annexin V binding; caspase-3 activation was measured by Western blotting. All three apoptotic markers were unaffected by the presence of either the actin depolymerizing drug cytochalasin D or the actin polymerizing drug jasplakinolide. Moreover, platelets were isolated from wild-type (WT) mice and mice deficient in gelsolin (Gsn), an actin-binding protein that is essential for normal cytoskeletal remodeling. In response to ABT-737, gelsolin-null (Gsn-/-) platelets initially showed accelerated PS exposure relative to WT platelets, however, both WT and Gsn-/- platelets exhibited similar levels of mitochondrial depolarization and caspase-3 activation in response to ABT-737. We conclude that ABT-737 induces established markers of platelet apoptosis in an actin-independent manner.
血小板生成与清除之间的平衡对人类健康至关重要。促进血小板从循环中清除的平衡的一个关键方面是细胞凋亡（程序性细胞死亡）。支持血小板凋亡的精确细胞机制尚未确定。在有核细胞中，已知细胞骨架肌动蛋白的重排调节血小板凋亡。然而，由于血小板无核且表现出独特的生理学特性，因此尚未广泛研究细胞骨架肌动蛋白在调节血小板凋亡中的作用。在这里，使用 BH3 模拟药物 ABT-737 诱导洗涤后的人血小板发生凋亡。使用比率染料 JC-1 测量线粒体去极化；通过 annexin V 结合测量表面磷脂酰丝氨酸 (PS) 暴露；通过 Western blot 测量半胱天冬酶-3 的激活。所有三种凋亡标志物均不受肌动蛋白解聚药物细胞松弛素 D 或肌动蛋白聚合药物 jasplakinolide 的存在影响。此外，从野生型 (WT) 小鼠和缺乏肌动蛋白结合蛋白凝胶蛋白 (Gsn) 的小鼠中分离血小板，Gsn 对于正常细胞骨架重塑至关重要。在用 ABT-737 处理后，与 WT 血小板相比，gelsolin 缺陷型 (Gsn-/-) 血小板最初表现出加速的 PS 暴露，但 WT 和 Gsn-/-血小板在对 ABT-737 的反应中均表现出相似水平的线粒体去极化和半胱天冬酶-3 激活。我们得出结论，ABT-737 以肌动蛋白非依赖性方式诱导血小板凋亡的既定标志物。
