Department of Pharmacology, University of Cambridge , Cambridge, UK.
Platelets. 2021 Jan 2;32(1):105-112. doi: 10.1080/09537104.2020.1724276. Epub 2020 Feb 21.
Platelet lifespan is regulated by intrinsic apoptosis. Platelet apoptosis can be triggered by BH3 mimetics that inhibit the pro-survival Bcl-2 family protein, Bcl-xL. Here, we investigated several small molecules that are reported to act as BH3 mimetics and compared their effects to the well-established BH3 mimetic, ABT-737. Platelet phosphatidylserine (PS) exposure was determined by flow cytometry. Changes in cytosolic Ca signaling were detected using Cal-520. Plasma membrane integrity was determined by calcein leakage. The roles of caspases and calpain in these processes were determined using Q-VD-OPh and calpeptin, respectively. As previously reported, ABT-737 triggered PS exposure in a caspase-dependent manner and calcein loss in a caspase and calpain-dependent manner. In contrast, AT-101 and sabutoclax triggered PS exposure independently of caspases. HA14-1 also triggered PS exposure in a caspase-independent but calpain-dependent manner. There were also significant differences in the pattern and protease-dependency of cytosolic Ca signaling in response to these drugs compared to ABT-737. Since there are clear differences between the action of ABT-737 and the other putative BH3 mimetics investigated here, AT-101, HA14-1 and sabutoclax cannot be considered as acting as BH3 mimetics in platelets. Furthermore, the platelet death caused by these drugs is likely to be distinct from apoptosis.
血小板寿命受内在凋亡调控。血小板凋亡可被 BH3 模拟物触发，该模拟物抑制生存 Bcl-2 家族蛋白 Bcl-xL。在此，我们研究了几种被报道为 BH3 模拟物的小分子，并将其作用与公认的 BH3 模拟物 ABT-737 进行了比较。血小板磷脂酰丝氨酸（PS）暴露通过流式细胞术测定。使用 Cal-520 检测细胞溶质 Ca 信号变化。通过钙黄绿素渗漏测定质膜完整性。使用 Q-VD-OPh 和 calpeptin 分别确定了这些过程中半胱天冬酶和钙蛋白酶的作用。如先前报道，ABT-737 以半胱天冬酶依赖性方式触发 PS 暴露，并以半胱天冬酶和钙蛋白酶依赖性方式触发钙黄绿素损失。相比之下，AT-101 和 sabutoclax 以不依赖半胱天冬酶的方式触发 PS 暴露。HA14-1 也以不依赖半胱天冬酶但依赖钙蛋白酶的方式触发 PS 暴露。与 ABT-737 相比，这些药物对细胞溶质 Ca 信号的作用模式和蛋白酶依赖性也存在显著差异。由于 ABT-737 和我们在此研究的其他假定 BH3 模拟物之间的作用存在明显差异，因此 AT-101、HA14-1 和 sabutoclax 不能被认为是血小板中的 BH3 模拟物。此外，这些药物引起的血小板死亡可能与凋亡不同。
