Rap1 signaling in platelet homeostasis and vascular hemostasis

项目总结 哺乳动物的血小板是一种小的无核血细胞，专门用于持续监测和维持 心血管系统(止血)。它们由骨髓中的巨核细胞(MK)产生， 释放到血液中，在人类体内循环10天，在老鼠体内循环5天。血小板动态平衡，即 建立明确的外周血小板计数(PPC)，需要两个过程-血小板产生和 通关--受到严格监管。同时，血小板依赖于一种非常敏感的信号机制 在剪切力作用下，促进血小板黏附和止血栓的形成。这种高度的敏感性， 然而，这会带来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。 我们和其他人确定了小GTP酶Rap1在血小板激活中的关键作用。我们进一步 证明了血小板中的Rap1活性受鸟嘌呤核苷酸交换因子的调节， CalDAG-GEFI(CD-GEFI，RasGRP2)和GTP酶激活蛋白Rasa3(GAP1IP4BP)。CD-GEFI 感知细胞内钙的微小变化，并对RAP1在细胞内的快速激活至关重要 刺激。在止血过程中，Rasa3对抑制循环中的血小板CD-GEFI/Rap1信号至关重要 然而，塞子的形成在与血小板ADP受体结合后其活性被下调， P2Y12是抗血小板治疗的靶点。缺乏功能性Rasa3的小鼠表现出严重的血小板减少症，引起 由于生产受损和过早清除血小板所致。基于这些和其他研究，我们得出结论 血小板动态平衡和血管止血都依赖于对Rap信号的严格调控，而且这是一种更好的 对这些基本过程的了解可能对临床诊断和治疗具有重要意义。 影响血小板数量和功能的疾病。利用独特的小鼠模型，原始的和永生的MK， 和临床相关的人类血小板样本，我们将研究与Rap1信号转导有关的关键问题 巨核细胞和血小板：CD-GEFI和Rasa3之间拮抗平衡的改变如何影响 血小板存活？RAP1信号在巨核细胞发育和血小板生成中的作用是什么？是他们的 调节巨噬细胞和血小板中特定细胞反应的不同的Rap1蛋白池？鼠标有多相似？ 和人类血小板有关的Rap1信号吗？控制Rasa3活性的分子机制是什么？ 在2012年的下游？为了测试我们的发现的临床相关性，我们将调查RAP1信号在 由钙稳态受损引起的血小板和巨噬细胞集落刺激因子是显著的 在Stormorken综合征中观察到了血小板减少，我们将确定在 RAP1信号通路参与了健康个体和2型患者对P2Y12抑制剂的抵抗 糖尿病。如果成功，这些研究可能为诊断和管理一些新的战略铺平道路 遗传性和获得性血小板减少症，以及更个性化的抗血小板治疗方法。

PROJECT SUMMARY 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. Platelet homeostasis, i.e. the establishment of a defined peripheral platelet count (PPC), requires that both processes - platelet production and clearance - are tightly regulated. At the same time, platelets depend on a very sensitive signaling machinery that facilitates platelet adhesion and hemostatic plug formation under shear stress. This high sensitivity, however, poses a risk for unwanted platelet activation that can lead to platelet clearance and/or thrombosis. We and others identified a critical role for the small GTPase Rap1 in platelet activation. We further demonstrated that Rap1 activity in platelets is regulated by the guanine nucleotide exchange factor, CalDAG-GEFI (CD-GEFI, RasGRP2), and the GTPase-activating protein, Rasa3 (GAP1IP4BP). CD-GEFI senses small changes in intracellular calcium and is crucial for the rapid activation of Rap1 upon cellular stimulation. Rasa3 is critical to restrain CD-GEFI/Rap1 signaling in circulating platelets; during hemostatic plug formation, however, its activity is downregulated after engagement of the platelet ADP receptor, P2Y12, the target of antiplatelet therapy. Mice lacking functional Rasa3 exhibit severe thrombocytopenia, caused by impaired production and premature clearance of platelets. Based on these and other studies we concluded that both platelet homeostasis and vascular hemostasis depend on a tight regulation of Rap signaling, and that a better understanding of these fundamental processes may have important implications in the diagnosis and treatment of disorders that affect platelet number and function. Utilizing unique mouse models, primary and immortilized MKs, and clinically relevant human platelet samples, we will study key questions concerning Rap1 signaling in megakaryocytes and platelets: how does a shift in the antagonistic balance between CD-GEFI and Rasa3 affect platelet survival? What is the role of Rap1 signaling in megakaryocyte development and platelet production? Are their different pools of Rap1 protein that regulate specific cellular responses in MKs and platelets? How similar are mouse and human platelets with regard to Rap1 signaling? What are the molecular mechanisms controlling Rasa3 activity downstream of P2Y12? To test the clinical relevance of our findings, we will investigate if increased Rap1 signaling in platelets and MKs, induced by impaired calcium homeostasis, is the underlying cause of the marked thrombocytopenia observed in Stormorken syndrome, and we will determine whether interindividual variability in the Rap1 signaling pathway contributes to P2Y12 inhibitor resistance in healthy individuals and patients with type 2 diabetes. If successful, these studies could pave the way to novel strategies for diagnosing and managing some of the inherited and acquired thrombocytopenias, and to a more personalized approach to anti-platelet therapy.