Rap1 signaling in platelet homeostasis and vascular hemostasis

Rap1 信号在血小板稳态和血管止血中的作用

• 批准号: 9330204
• 负责人: Wolfgang Bergmeier
• 金额: $ 41.39万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9330204
• 关键词: 1-Phosphatidylinositol 3-Kinase; ADP Receptors; Address; Adhesions; Adhesives; Affect; Agonist; Alpha Granule; Blood; Blood Cells; Blood Circulation; Blood Platelets; Blood Vessels; Bone Marrow; Calcium; Cardiovascular system; Cell Adhesion; Cell membrane; Cell physiology; Cells; Clinical; Collagen; Complement Factor B; Coupled; Cytoplasmic Granules; Development; Diagnosis; Diglycerides; Disease; Equilibrium; Event; Exhibits; Fibrinogen; G-substrate; GTP-Binding Proteins; GTPase-Activating Proteins; Generations; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Half-Life; Hemostatic Agents; Hemostatic function; Heterogeneity; Homeostasis; Human; Human Pathology; ITAM; Impairment; In Vitro; Individual; Inherited; Injury; Inositol; Integrins; Lead; Link; Mediating; Megakaryocytes; Modeling; Molecular; Monitor; Monomeric GTP-Binding Proteins; Mus; Non-Insulin-Dependent Diabetes Mellitus; Nucleotides; Pathology; Pathway interactions; Peripheral; Phospholipase C; Platelet Activation; Platelet Count measurement; Process; Production; Protein Isoforms; Protein Kinase C; Proteins; Regulation; Research; Resistance; Risk; Role; Sampling; Signal Pathway; Signal Transduction; Site; Syndrome; Testing; Thrombin; Thrombocytopenia; Thrombosis; Thromboxane A2; Thrombus; Time; Translating; Work; base; chemical genetics; clinically relevant; clopidogrel; genetic approach; in vivo; individual patient; inhibitor/antagonist; mouse model; novel strategies; personalized approach; podoplanin; premature; prevent; receptor; response; shear stress

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.

项目摘要 哺乳动物血小板是小的无核血细胞，专门用于持续监测和保持血小板的完整性。 心血管系统（止血）。它们由骨髓中的巨核细胞（MK）产生， 它们被释放到血液中，在人体内循环10天，在小鼠体内循环5天。血小板稳态，即 建立一个明确的外周血小板计数（PPC），需要两个过程-血小板的生产和 间隙-受到严格管制。同时，血小板依赖于一种非常敏感的信号机制， 在剪切应力下促进血小板粘附和止血栓形成。这种高灵敏度， 然而，这造成了不希望的血小板活化的风险，其可导致血小板清除和/或血栓形成。 我们和其他人确定了小GTAP 1在血小板活化中的关键作用。我们进一步 证明血小板中Rap 1活性受鸟嘌呤核苷酸交换因子调节， CalDAG-GEFI（CD-GEFI，RasGRP 2）和GTP酶激活蛋白Rasa 3（GAP 1 IP 4 BP）。CD-GEFI 感觉细胞内钙离子的微小变化，并对细胞内Rap 1的快速激活至关重要。 刺激. Rasa 3对抑制循环血小板中的CD-GEFI/Rap 1信号传导至关重要;在止血期间 然而，栓形成，其活性在血小板ADP受体接合后下调， P2 Y12是抗血小板治疗的靶点。缺乏功能性Rasa 3的小鼠表现出严重的血小板减少症， 血小板生成受损和过早清除。根据这些和其他研究，我们得出结论， 血小板稳态和血管止血都依赖于Rap信号的严格调节， 了解这些基本过程可能对诊断和治疗 影响血小板数量和功能的疾病。利用独特的小鼠模型，原代和永生化的MK， 和临床相关的人类血小板样本，我们将研究Rap 1信号转导的关键问题， 巨核细胞和血小板：CD-GEFI和Rasa 3之间拮抗平衡的变化如何影响 血小板存活率？Rap 1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 不同的Rap 1蛋白池调节MK和血小板中的特异性细胞反应？老鼠有多相似 和人类血小板之间的关系控制Rasa 3活性的分子机制是什么 P2 Y12的下游为了检验我们发现的临床相关性，我们将研究是否增加了Rap 1信号， 由钙稳态受损诱导的血小板和MK是显著性血小板减少的根本原因。 血小板减少症中观察到的Stormorken综合征，我们将确定是否在个体间的变异性， Rap 1信号通路导致健康个体和2型糖尿病患者对P2 Y12抑制剂耐药 糖尿病如果成功的话，这些研究可以为诊断和管理一些疾病的新策略铺平道路。 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。