Small GTPases in the biology of platelets and megakaryocytes

摘要 哺乳动物血小板是专门用于持续监测和保存血小板的小无核血细胞。 心血管系统的完整性（止血）。它们由骨骼中的巨核细胞（MK）产生 骨髓中，并释放到血液中，在那里他们循环十天，在小鼠中五天，直到他们 被吞噬细胞清除血小板稳态，即确定外周血小板计数， 需要严格调节血小板的产生和清除。为了实现其止血功能，血小板 依赖于在剪切应力下促进血小板粘附的非常敏感的信号机制。这种高 然而，敏感性造成了不需要的血小板活化的风险，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了解分子 调节MK发育和血小板反应性的机制，特别关注小分子的作用。 在这些过程中的GTP酶。这个R35 OIA申请是三个资助NHLBI R 01赠款的扩展： 巨核细胞生物学中的小GTP酶; Rap 1信号在血小板稳态和血管止血中的作用; Podoplanin-Clec 2信号通路对血小板活化的空间调控我们的MK研究利用独特的 生物传感器，以建立小GTP酶活性（Rho和Rap GTP酶）的分子特征， 发育的最后阶段，包括从增殖到前血小板形成的过渡。一旦 建立，我们将通过以下方法建立精确靶向干扰GTdR活性的原理验证： 光遗传学工具是优化体外血小板生产的可行策略，这是输血中的热门话题 药我们的血小板工作更具体地关注Rap GTP酶作为血小板活化的主要调节因子的作用。 细胞活化和止血栓形成。我们利用独特的小鼠模型来建立 重要性和血小板活化过程中Rap 1信号传导的关键调节因子。此外，我们还表明， Rap 1活性必须在静止、循环和止血活性血小板中紧密平衡， 并且该途径的紊乱导致出血或血小板减少症/血栓形成。目前和今后 工作，我们将扩大我们的细胞生物学和生物化学/物理研究，提供全面的 了解Rap信号传导如何控制血小板功能，它是如何调节的，以及它是否/如何有助于 其他病理生理过程，如发育/炎症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检测来筛选Rap信号传导的抑制剂。我们的临床 研究将调查Rap 1信号传导是否在各种病理中改变，以及是否存在个体间 在健康和患病受试者中该途径的变异性？总之，这些研究预计将导致 用于诊断和管理某些遗传性和获得性血小板减少症的新策略， 出血性疾病，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.