Apoptosis is a critical process for the maintenance of cell populations, and involves mitochondrial depolarization, the sequential cleavage of caspase-9 and -3, followed by the externalization of phosphatidylserine (PS) on the plasma membrane. The actin cytoskeleton and its accessory proteins are known regulators of apoptotic signaling in nucleated cells but their roles in platelet apoptosis are undefined. Filamin A (FLNA) is a ubiquitously expressed actin-crosslinking protein that also serves as an intracellular signaling scaffold. Here we used platelets from mice with a platelet-specific FLNA deficiency (Flnafl/Y, Pf4-cre/+, termed platelet-specific knockout) to test the role of FLNA in platelet apoptosis. Treatment with the BH3-mimetic drug ABT-737 induced caspase-3 cleavage and PS exposure in platelets from floxed mice (Flnafl/Y, termed control) but these effects were essentially abrogated in FLNA-null platelets (platelet-specific knockout). Protein kinase C (PKC), a known FLNA ligand, was also activated by ABT-737, and PKC's phosphorylation of its downstream substrates was attenuated in FLNA-null platelets. The PKC inhibitor bisindolylmaleimide (BIM) also reduced caspase-3 cleavage, thus essentially phenocopying the FLNA-null platelets. Notably, the caspase-3 cleavage defect in FLNA-null platelets was rescued by the PKC-activating phorbol ester PMA, suggesting that FLNA and PKC share a common pathway in regulating platelet apoptosis. Mitochondrial depolarization and caspase-9 cleavage were unaffected by BIM treatment, suggesting that PKC specifically controls the downstream caspase-3 point of the pro-apoptotic signaling pathway. These data point to a novel role for FLNA in the regulation of platelet apoptosis, thus providing an improved understanding of how circulating platelet counts are maintained.

中文翻译：

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细丝蛋白 A 以蛋白激酶 C (PKC) 依赖性方式调节血小板中的 caspase-3 裂解

细胞凋亡是维持细胞群的关键过程，涉及线粒体去极化、caspase-9 和 -3 的连续裂解，然后是质膜上磷脂酰丝氨酸 (PS) 的外化。肌动蛋白细胞骨架及其辅助蛋白是有核细胞中凋亡信号的已知调节剂，但它们在血小板凋亡中的作用尚不明确。细丝蛋白 A (FLNA) 是一种普遍表达的肌动蛋白交联蛋白，也可作为细胞内信号支架。在这里，我们使用来自具有血小板特异性 FLNA 缺陷（Flnafl/Y、Pf4-cre/+，称为血小板特异性敲除）的小鼠的血小板来测试 FLNA 在血小板凋亡中的作用。用 BH3 模拟药物 ABT-737 治疗诱导 floxed 小鼠血小板中的 caspase-3 裂解和 PS 暴露 (Flnafl/Y, 称为对照），但这些影响在 FLNA 无效血小板（血小板特异性敲除）中基本上被消除。蛋白激酶 C (PKC) 是一种已知的 FLNA 配体，它也被 ABT-737 激活，并且 PKC 对其下游底物的磷酸化在 FLNA 缺失的血小板中减弱。PKC 抑制剂双吲哚马来酰亚胺 (BIM) 也减少了 caspase-3 的切割，因此基本上表型复制了 FLNA 缺失的血小板。值得注意的是，激活 PKC 的佛波醇酯 PMA 挽救了 FLNA 无效血小板中的 caspase-3 切割缺陷，这表明 FLNA 和 PKC 在调节血小板凋亡方面具有共同的途径。线粒体去极化和 caspase-9 切割不受 BIM 处理的影响，表明 PKC 特异性控制促凋亡信号通路的下游 caspase-3 点。