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、血小板生物生成和正常血小板功能的分子事件。该实验计划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。通过靶向缺失GP Ib(GP Ibalpha)的a亚单位，建立了人类Bernard-Soulier综合征(BSS)的小鼠模型。GP Ibalpha是血小板受体GP Ib-IX的一个亚单位。小鼠模型表现为严重出血表型，并伴有人类Bernard-Soulier综合征的大血小板减少症。人类GP Ibalpha亚单位的表达挽救了小鼠的表型。提供了新的数据，建立了GP Ibalpha的细胞质尾巴，控制着血小板的形态和从巨核细胞释放的血小板。此外，还描述了通过GP Ibalpha尾部的信号转导途径对血小板释放和血小板功能的功能影响。实验被用来检验假设：i)GP Ib-IX受体通过GP Ibalpha亚单位内的结构元件控制巨核细胞成熟和血小板释放；ii)GP Ib-IX复合体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的发育。目的1确定促进正常血小板释放所必需的GP Ibalpha结构要求，并确定导致血瘀证患者大血小板减少的分子缺陷。目的2研究GP Ibalpha缺失小鼠的巨核细胞生成缺陷。目的3研究GP Ibalpha信号转导途径及其在血小板生成中的作用。目的4确定基因工程血小板中GP Ibalpha胞浆相互作用的止血相关性。尽管GP Ib-IX复合体显然很重要，但由于缺乏合适的体外模型，人们对其在血栓形成中的作用知之甚少。然而，模型，如小鼠血瘀证，提供了一个机会来研究这些独特的事件，并将提供关于控制巨核细胞生成、血小板释放和血小板在血栓形成中的作用的机制的新信息。

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.