MAPK-interacting kinase 1 (Mnk1) regulates platelet production, activation, and thrombosis

The MAPK-interacting kinase (Mnk) family includes Mnk1 and Mnk2, which are phosphorylated and activated in response to extracellular stimuli. Mnk1 contributes to cellular responses by regulating mRNA translation and mRNA translation influences platelet production and function. However, the role of Mnk1 in megakaryocytes and platelets has not previously been studied. The present study investigated Mnk1 in megakaryocytes and platelets using both pharmacological and genetic approaches. We demonstrate that Mnk1, but not Mnk2, is expressed and active in human and murine megakaryocytes and platelets. Stimulating human and murine megakaryocytes and platelets induced Mnk1 activation and phosphorylation of eIF4E, a downstream target of activated Mnk1 that triggers mRNA translation. Mnk1 inhibition or deletion significantly diminished protein synthesis in megakaryocytes, as measured by polysome profiling and [35S]-methionine incorporation assays. Depletion of Mnk1 also reduced megakaryocyte ploidy and proplatelet forming megakaryocytes in vitro, and resulted in thrombocytopenia, However, Mnk1 deletion did not affect the half-life of circulating platelets. Platelets from Mnk1 knockout mice exhibited reduced platelet aggregation, alpha granule secretion, and integrin aIIbb3 activation. Ribosomal footprint sequencing indicated that Mnk1 regulates the translation of PLA2G4A mRNA (which encodes cPLA2) in megakaryocytes. Consistent with this, Mnk1 ablation reduced cPLA2 activity and thromboxane generation in platelets and megakaryocytes. In vivo, Mnk1 ablation in platelets reduced mortality from pulmonary thromboembolism. These results provide previously unrecognized evidence that Mnk1 in platelets and megakaryocytes regulates mRNA translation, thrombopoiesis, platelet reactivity, and thrombosis.

丝裂原活化蛋白激酶相互作用激酶（MAPK-interacting kinase，Mnk）家族包含Mnk1与Mnk2，二者可响应细胞外刺激发生磷酸化并被激活。Mnk1通过调控mRNA翻译参与细胞应答，而mRNA翻译过程可影响血小板生成与功能。然而此前尚未有关于Mnk1在巨核细胞与血小板中作用的研究报道。本研究采用药理学与遗传学手段，对巨核细胞及血小板中的Mnk1展开了探究。我们证实，在人类与小鼠的巨核细胞及血小板中，仅Mnk1（而非Mnk2）存在表达与活性。刺激人类及小鼠的巨核细胞与血小板，可诱导Mnk1激活及其下游靶标真核翻译起始因子4E（eukaryotic translation initiation factor 4E，eIF4E）的磷酸化，而eIF4E可触发mRNA翻译过程。通过多聚核糖体谱分析与[35S]-甲硫氨酸掺入实验检测发现，抑制或敲除Mnk1可显著降低巨核细胞内的蛋白质合成速率。敲除Mnk1还可在体外减少巨核细胞的倍性以及前血小板形成巨核细胞的数量，并引发血小板减少症。不过，Mnk1敲除并不会影响循环血小板的半衰期。来自Mnk1敲除小鼠的血小板表现出血小板聚集、α颗粒分泌以及整合素αIIbβ3激活水平降低的现象。核糖体足迹测序结果表明，Mnk1可调控巨核细胞中编码胞质磷脂酶A2（cytosolic phospholipase A2，cPLA2）的PLA2G4A mRNA的翻译过程。与此一致的是，敲除Mnk1可降低血小板与巨核细胞中的cPLA2活性及血栓素生成量。在活体实验中，血小板内的Mnk1敲除可降低肺血栓栓塞症所致的小鼠死亡率。上述研究结果提供了此前未被发现的证据，表明巨核细胞与血小板中的Mnk1可调控mRNA翻译、血小板生成、血小板反应性以及血栓形成。