Small GTPases in the biology of platelets and megakaryocytes

摘要 哺乳动物血小板是一种小的无核血细胞，专门用于持续监测和保存 心血管系统的完整性(止血)。它们是由骨骼中的巨核细胞(MK)产生的 并释放到血液中，在人类体内循环10天，在老鼠体内循环5天，直到它们 被吞噬细胞清除。血小板动态平衡，即建立定义的外周血小板计数， 需要严格控制血小板的生成和清除。为了完成止血功能，血小板 依靠一种非常敏感的信号机制，在剪切力下促进血小板粘连。这么高 然而，敏感性带来了不必要的血小板激活的风险，这可能导致血小板清除和/或 血栓形成。我们工作的首要目标是更好地理解分子 调节MK发育和血小板反应性的机制，特别关注Small的作用 GTP酶在这些过程中的作用。此R35 OIA应用程序是三个资助的NHLBI R01赠款的扩展： 巨核细胞生物学中的小GTP酶；血小板稳态和血管止血中的RAP1信号； Podoplan in-Clec2信号对血小板激活的空间调节我们的MK研究使用了独特的 生物传感器建立小分子GTP酶活性(Rho和Rap GTP酶)的分子特征 发育的最后阶段，包括从增殖到原血小板形成的过渡。一次 ，我们将建立原则证明，通过以下方式精确定位GTP酶活性的扰动 光遗传工具是优化体外血小板产生的一种可行策略，这是输血领域的一个热门话题 医学。我们的血小板工作更具体地关注Rap GTP酶作为主要调节因子的作用 细胞活化和止血栓的形成。我们利用独特的小鼠模型建立了 RAP1信号在血小板活化过程中的重要性和关键调节因子。此外，我们已经展示了 RAP1的活性必须在静止的、循环的和止血活性的血小板中保持紧密平衡， 而这一途径的紊乱会导致出血或血小板减少/血栓形成。在持续和未来 工作中，我们将对我们的细胞生物学和生化/物理研究提供全面的 了解Rap信号如何控制血小板功能，它是如何调节的，以及它是否/如何参与 其他病理生理过程，如发育/炎症过程中的血管完整性和静脉 血栓形成。我们将使用我们独特的生化分析来筛选Rap信号的抑制物。我们的临床 研究将调查Rap1信号是否在不同的病理中改变，以及是否存在个体间的 健康受试者和患病受试者这一途径的可变性？总而言之，这些研究有望导致 一些遗传性和获得性血小板减少症的诊断和治疗的新策略 出血障碍，以及更个性化的抗血小板治疗方法。

ABSTRACT Mammalian platelets are small anucleate blood cells specialized to continuously monitor and preserve the integrity of the cardiovascular system (hemostasis). They are produced by megakaryocytes (MKs) in the bone marrow and released into blood, where they circulate for ten days in humans and five days in mice until they get cleared by phagocytes. Platelet homeostasis, i.e. the establishment of a defined peripheral platelet count, requires tight regulation of both platelet production and clearance. To fulfill their hemostatic function, platelets depend on a very sensitive signaling machinery that facilitates platelet adhesion under shear stress. This high sensitivity, however, poses a risk for unwanted platelet activation that can lead to platelet clearance and/or thrombosis. The overarching goal of our work is to achieve a better understanding of the molecular mechanisms regulating MK development and platelet reactivity, with a specific focus on the role of small GTPases in these processes. This R35 OIA application is an extension to three funded NHLBI R01 grants: Small GTPases in Megakaryocyte Biology; Rap1 Signaling in Platelet Homeostasis and Vascular Hemostasis; Spatial Regulation of Platelet Activation by Podoplanin-Clec2 Signaling. Our MK studies utilize unique biosensors to establish a molecular signature of small GTPase activity (both Rho and Rap GTPases) during the final stages of development, including the transition from proliferation to proplatelet formation. Once established, we will establish proof-of-principle that precisely targeted perturbation of GTPase activity by optogenetic tools is a viable strategy to optimize in vitro platelet production, a hot topic in Transfusion Medicine. Our platelet work focuses more specifically on the role of Rap GTPases as master regulators of cellular activation and hemostatic plug formation. We have utilized unique mouse models to establish the importance and the key regulators of Rap1 signaling during platelet activation. Furthermore, we have shown that Rap1 activity has to be tightly balanced both in quiescent, circulating and in hemostatically active platelets, and that disturbance of this pathway leads to bleeding or thrombocytopenia/ thrombosis. In ongoing and future work, we will expand on our cell biological and biochemical/-physical studies to provide a comprehensive understanding of how Rap signaling controls platelet function, how it is regulated, and if/ how it contributes to other patho-physiological processes such as vascular integrity in development/ inflammation and venous thrombosis. We will use our unique biochemical assays to screen for inhibitors of Rap signaling. Our clinical studies will investigate if Rap1 signaling is altered in various pathologies, and whether there is interindividual variability in this pathway in healthy and diseased subjects? Together, these studies are expected to lead to novel strategies for the diagnosis and management of some inherited and acquired thrombocytopenias and bleeding disorders, and to a more personalized approach to anti-platelet therapy.