Megakaryocyte Mechanosensing Toward Platelet Biogenesis

摘要 严格控制血小板计数，以防止血栓形成或出血性并发症 血小板增多症或血小板减少症。虽然我们对人类的理解已经有了长足的进步， 尽管有血小板前体形成（PPF），但关于骨形成机制的知识仍然有限。 骨髓（BM）细胞外基质（ECM）调节血小板产生。事实上，BM包括丰富的ECM， 可能产生机械约束。然而，骨髓力学对巨核细胞（MK）的影响 性质和血小板形成的研究不足。在这里，我们提出了一个综合的方法， 研究与MK机械生物学受体在控制MK中的作用相关的新概念 粘附于ECM和血小板产生。我们的最终目标是了解MK 机械传感器感测BM基质以影响细胞的细胞骨架、MK性质，重要的是， 血小板水平基于我们的新发现，目标1探索了新的范式和假设， MK阳离子通道优先响应不同的BM基质蛋白，并对MK产生相反的影响。 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压电家族， 与瞬时受体电位阳离子通道亚家族V成员4机械传感器相比。在 最近的研究，我们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的影响。研究将使用药理学方法以及新的 在基线和响应于挑战（例如骨髓抑制或 血小板减少症受使用人原代MK的初步研究的鼓舞，继续的研究将 证实了小鼠的发现。目的2描绘介导MK之间新连接的机制 机械传感器、整合素受体活化、细胞骨架变化和MK机械敏感性转录 因素这一建议是重要的，因为需要确定新的和替代的血小板生成途径 以及调节血小板计数的药剂。除了概念创新，在技术层面， 分析我们开发的删除MK中特定机械传感器的新小鼠模型，并将应用于 在流动下进行最先进的成像和测量，以跟踪细胞过程。拟议的研究包括 有望对MK在控制MK细胞骨架中的选择性ECM传感作用产生新的见解， 粘附和血小板生成，具有显著的影响我们调节血小板水平的能力的潜力。

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.