Transgenic Analysis of Platelet Receptor Expression

描述（由申请人提供）：本提案的目的是研究控制巨核细胞成熟，血小板生物发生和正常血小板功能的分子事件。实验方案利用转基因动物和变异膜受体的表达。我们的目标是描述巨核细胞生成过程中发生的独特生物事件，以及这些事件对正常血小板功能的影响。通过靶向删除血小板受体GP Ib- ix的亚基GP Ib （GP Ibalpha）的A亚基，建立了人类Bernard-Soulier综合征（BSS）的小鼠模型。小鼠模型显示出严重的出血表型，伴有大量血小板减少症，反映了人类伯纳德-苏利尔综合征。小鼠表型是由人类GP α亚基的表达拯救的。本文提出了新的数据，证实了巨核细胞的胞质尾部控制血小板形态和血小板释放。此外，通过GP Ibalpha尾部的信号转导途径被描述为血小板释放和血小板功能的功能后果。我们提出了实验来验证以下假设：i) GP Ib-IX受体通过GP α亚基内的结构元件控制巨核细胞成熟和血小板释放的各个方面；ii) GP Ib-IX复合物通过与正常血小板功能相关的受控信号通路促进巨核细胞发育。目的1确定了促进正常血小板释放所必需的GP α α结构要求，并定义了BSS中导致巨血小板减少症的分子缺陷。目的2描述GP α α缺失小鼠巨核细胞生成缺陷。目的3描述GP α的信号传导及其在血栓形成中的作用。目的4确定基因工程血小板中GP β细胞质相互作用的止血相关性。尽管GP Ib-IX复合物在血小板形成中的作用非常重要，但人们对其了解甚少，这反映出缺乏适当的体外模型。然而，小鼠BSS等模型为研究这些独特事件提供了机会，并将提供有关巨核细胞生成控制机制、血小板释放和血小板在血栓形成中的作用的新信息。

DESCRIPTION (provided by applicant): The objectives of this proposal are to examine the molecular events controlling maturation of the megakaryocyte, platelet biogenesis and normal platelet function. The experimental plan utilizes transgenic animals and the expression of variant membrane receptor. Our goals are to characterize the unique biological events occurring during megakaryocytopoiesis and the consequences of these events on normal platelet function. A murine model of the human Bernard-Soulier syndrome (BSS) has been established via a targeted deletion of the a-subunit of GP Ib (GP Ibalpha), a subunit of the platelet receptor, GP Ib-IX. The mouse model displays a severe bleeding phenotype with macrothrombocytopenia mirroring the human Bernard-Soulier syndrome. The murine phenotype is rescued by expression of a human GP Ibalpha subunit. New data is presented establishing the cytoplasmic tail of GP Ibalpha controls platelet morphology and platelet release from the megakaryocyte. Moreover, a signal transduction pathway via the tail of GP Ibalpha is described with functional consequences for platelet release and platelet function. Experiments are proposed to test the hypotheses: i) The GP Ib-IX receptor controls aspects of megakaryocyte maturation and platelet release via structural elements within the GP Ibalpha subunit and ii) the GP Ib-IX complex contributes to megakaryocyte development via controlled signaling pathways that also become relevant for normal platelet function. Aim 1 determines the GP Ibalpha structural requirements necessary for facilitating normal platelet release and defines the molecular defect responsible for the macrothrombocytopenia in BSS. Aim 2 characterizes the megakaryocytopoiesis defect in the GP Ibalpha null mouse. Aim 3 characterizes signaling through GP Ibalpha and its role in thrombopoiesis. Aim 4 determines the hemostastic relevance of GP Ibalpha cytoplasmic interactions in genetically engineered platelets. Although clearly important, the role of the GP Ib-IX complex in thrombopoiesis is poorly understood reflecting the lack of appropriate in vitro models. However, models, such as the murine BSS, present an opportunity to study these unique events and will provide new information on the mechanisms controlling megakaryocytopoiesis, the release of blood platelets and the role of platelets in thrombus formation.