The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4^-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4^-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.

ABCC4 （一种 ATP 结合盒转运蛋白）在血小板形成（巨核细胞生成）过程中的作用尚不清楚。在这里，我们发现 ABCC4 在巨核细胞 (MK) 中高度表达。对公共基因组数据（ATAC-seq 和全基因组染色质相互作用，Hi-C）的挖掘表明，关键的巨核细胞生成转录因子 (TF) 与 ABCC4 调控元件相互作用，并可能解释了 MK 中 ABCC4 的高表达。重要的是，ABCC4 的这些基因组相互作用排名高于在巨核细胞生成中已知作用的基因，这表明 ABCC4 在巨核细胞生成中的作用。然后，我们证明 ABCC4 是最佳血小板形成所必需的，因为来自Abcc4 ^-/-小鼠的胎肝衍生 MK 的体外分化表现出受损的前血小板形成和多倍化，这是最佳巨核细胞生成所需的特征。同样，人类巨核细胞系 MEG-01 显示，急性 ABCC4 抑制可显着抑制巨核细胞生成的关键过程，并且这些效应与 cAMP 输出减少和巨核细胞生成负调节因子蛋白激酶 A (PKA) 从 ABCC4 解离增强有关。ABCC4 抑制后 PKA 活性随之增加，同时 GATA-1 表达显着降低，GATA-1 是最佳巨核细胞生成所需的 TF。此外，ABCC4 保护 MK 免受 6-巯基嘌呤 (6-MP) 的影响，因为Abcc4 ^-/-小鼠在 6-MP 治疗后显示 MK 显着减少。总的来说，我们的研究表明 ABCC4 不仅可以保护 MK，而且也是 MK 产生最大血小板所必需的，这表明调节 ABCC4 功能可能是调节血小板产生的潜在治疗策略。