Megakaryocyte Mechanosensing Toward Platelet Biogenesis

摘要 严格控制血小板计数，以防止相关的血栓或出血并发症 分别伴有血小板增多症或血小板减少症。虽然我们在认识上取得了很大进展 血小板原形成(PPF)，关于骨骼通过什么机制仍知有限 骨髓(BM)细胞外基质(ECM)调节血小板生成。事实上，BM包括一个丰富的ECM， 可能会生成机械约束。然而，骨髓机制对巨核细胞的影响 性质和血小板的形成一直没有得到充分的研究。在这里，我们提出了一种综合的方法来 研究与MK机械生物受体在控制MK中的作用相关的新概念 黏附于细胞外基质和血小板的产生。我们的最终目标是了解具体的MK 机械传感器感应BM基质以影响细胞骨架、MK属性，重要的是， 血小板水平。在我们新发现的基础上，目标1探索了新的范式和假设 MK阳离子通道优先与不同的BM基质蛋白反应，并反向影响MK 细胞骨架和血小板水平。实验将集中在阳离子通道机械传感器的Piezo系列上， 与瞬时受体电位相比，阳离子通道亚家族V成员4机械传感器。在……里面 最近的研究发现，这些机械传感器对不同的基质蛋白有不同的偏好 对PPF产生相反的影响。调查将使用药理学方法以及新的 在基线和对挑战的反应中产生基因敲除小鼠，如骨髓抑制或 血小板减少症。在使用人类原代MK的初步研究的鼓舞下，继续研究将 证实了老鼠的发现。目标2描述了MK和MK之间新的联系的调节机制 机械感受器、整合素受体激活、细胞骨架变化和MK机械敏感转录 各种因素。这一建议意义重大，因为需要确定新的和替代的血栓生成途径 以及调节血小板计数的药物。除了概念创新，在技术层面上，我们还将 分析我们开发的新的小鼠模型，删除了MK中的特定机械传感器，并将应用 最先进的成像和测量，在流动下跟踪细胞过程。建议的研究包括 预计将对MK选择性ECM传感在控制MK细胞骨架中的作用产生新的见解， 粘附性和血小板生成，有很大的潜力影响我们调节血小板水平的能力。

ABSTRACT Platelet counts are tightly regulated in order to prevent thrombotic or hemorrhagic complications associated with thrombocytosis or thrombocytopenia, respectively. While strides have been made in our understanding of proplatelet formation (PPF), there is still limited knowledge regarding the mechanisms through which the bone marrow (BM) extracellular matrix (ECM) regulates platelet production. Indeed, the BM includes a rich ECM with potential to generate mechanical constraints. Yet, the impact of BM mechanics on megakaryocyte (MK) properties and platelet formation has been understudied. Here, we propose an integrative approach to investigate emerging concepts related to the role of MK mechanobiological receptors in controlling MK adhesion to the ECM and platelet production. Our ultimate goal is to understand how specific MK mechanosensors sense the BM matrix to affect the cellular cytoskeleton, MK properties and, importantly, platelet level. Building upon our novel findings, Aim 1 explores the new paradigm and hypothesis that distinct MK cation channels preferentially respond to different BM matrix proteins and inversely impact the MK cytoskeleton and platelet levels. Experiments will focus on the Piezo family of cation channel mechanosensors, as compared to the Transient Receptor Potential cation channel subfamily V member 4 mechanosensor. In recent studies, we found these mechanosensors to have distinct preferences for different matrix proteins and opposing effects on PPF. Investigations will be carried out using pharmacological approaches as well as newly generated knockout mice at baseline and in response to challenges, such as myelosuppression or thrombocytopenia. Encouraged by preliminary studies using human primary MKs, continued studies will confirm murine findings. Aim 2 delineates mechanisms mediating novel connections between MK mechanosensors, integrin receptors activation, cytoskeletal changes, and MK mechano-sensitive transcription factors. This proposal is significant as there is need to identify new and alternative thrombopoietic pathways and agents that modulate platelet counts. In addition to conceptual innovation, at the technical level we will analyze new mouse models we developed with deletion of specific mechanosensors in MKs, and will apply state-of-the-art imaging and measurements under flow to follow cellular processes. Proposed studies are expected to yield new insights on the role of selective ECM sensing by MKs in controlling the MK cytoskeleton, adhesion and platelet production, with significant potential to impact our ability to modulate platelet levels.