Transgenic analysis of platelet receptor expression

描述（由申请人提供）：血小板如何支持血管系统的稳态是止血和血栓形成的核心问题。血小板糖蛋白（GP）Ib-IX/von Willebrand因子轴在此过程中的重要性是公认的，代表了血小板粘附到受损血管表面的起始事件。我们的长期目标是建立血小板受体功能的分子机制，特别是GP Ib-IX（粘附）和胶原蛋白受体GP VI（活化），维持正常生理学中的血管稳态并促进疾病状态。已经产生了具有靶向基因缺失和表达的变体表面受体的小鼠，以检查体内巨核细胞和血小板的独特生物学特性。这些模型实用性的一个例子在于，几十年来，人们对血小板GP Ib-IX和凝血酶之间的相互作用进行了描述，但对这种结合的生理相关性（如果有的话）却知之甚少。我们提出了新的数据表征的小鼠模型与Tyr276 Phe276取代内的GP Ib/凝血酶接触位点的关键残基。初步研究确定了该残留物在形成富血小板血栓中的体内重要性。我们建议使用血栓形成模型来表征凝血酶与血小板GP Ib-IX结合的体内相关性，以测试GP Ib-IX/凝血酶相互作用稳定生长血栓的假设（目的1）。我们提出了一种血小板型血管性血友病（Pt-vWD）的动物模型，以检验Pt-vWD模拟GP Ib-IX阻断并呈现新表位的假设，该新表位类似于血小板整合素受体的配体诱导的结合位点（目的2）。血小板GP Ib-IX的先天性缺失导致Bernard-Soulier综合征（BSS），并呈现出血表型和循环巨血小板。我们提出了一种蛋白质组学的方法来定义独特的巨核细胞和血小板表型产生的BSS（目的3）。这些研究测试的假设，BSS巨核细胞和血小板含有改变的蛋白质谱，导致继发多效性，这是BSS，并可能有助于阐明未知的分子基础，为其他巨大的血小板表型。最后，我们建议使用LDL受体缺陷模型确定GP Ib-IX（血小板粘附）和GP VI（血小板活化）在动脉粥样硬化病变和血栓形成发展中的相关性（目的4）。我们建议测试的假设，血小板粘附和激活介导的GP Ib-IX和/或GP VI是炎症的重要贡献者。每个目标的完成进一步定义了GP Ib-IX和GP VI在血栓形成和巨核细胞生成中的生物学相关性，但也考虑了血小板在止血和血栓形成中的相关性。血小板循环的作用是防止失血。然而，在疾病中，血小板可引起血液的病理性破坏，导致器官损伤和衰竭。最常见的事件是心脏心肌梗死，这是美国死亡的主要原因。我们的研究使用小鼠模型来模拟导致致命血栓形成的事件，并表征与心脏病发作和中风相关的分子事件。了解血小板在这一过程中的作用将有助于制定更好的治疗策略，改善美国的健康状况。

DESCRIPTION (provided by applicant): How platelets support homeostasis in the vascular system is a paradigm-central question in hemostasis and thrombosis. The importance of the platelet glycoprotein (GP) Ib-IX/von Willebrand factor axis in this process is well-established and represents an initiating event for platelet adhesion to a damaged vascular surface. Our long-term goals are to establish the molecular mechanism(s) by which platelet receptor function, specifically GP Ib-IX (adhesion) and the collagen receptor, GP VI (activation), maintain vascular homeostasis in normal physiology and contribute to disease states. Mice with targeted gene deletions and expressed variant surface receptors have been generated to examine the unique biological properties of the megakaryocyte and platelet in vivo. An example of the utility of these models resides in the fact that for several decades an interaction between platelet GP Ib-IX and thrombin has been described with little insight as to the physiologic relevance, if any, of the binding. We present new data characterizing a mouse model with a Tyr276 to Phe276 substitution within a key residue of the GP Ib/thrombin contact site. Preliminary studies establish the in vivo importance of this residue in forming a platelet-rich thrombus. We propose to characterize the in vivo relevance of thrombin binding to platelet GP Ib-IX using models of thrombus formation testing the hypothesis that GP Ib-IX/thrombin interactions stabilize a growing thrombus (Aim 1). We present an animal model of platelet-type von Willebrand disease (Pt-vWD) to test the hypothesis that Pt-vWD mimics GP Ib-IX blockade and presents neoepitopes, analogous to the ligand-induced binding sites characterized for platelet integrin receptors (Aim 2). The congenital absence of platelet GP Ib-IX results in the Bernard-Soulier syndrome (BSS) and presents with a bleeding phenotype and circulating giant platelets. We propose a proteomics approach to define the unique megakaryocyte and platelet phenotypes generated by a BSS (Aim 3). These studies test the hypothesis that the BSS megakaryocyte and platelet contain altered protein profiles leading to a secondary pleiotropy that is the BSS and may help elucidate the unknown molecular basis for other giant platelet phenotypes. Finally we propose to determine the relevance of GP Ib-IX (platelet adhesion) and GP VI (platelet activation) in development of atherosclerotic lesions and thrombosis using a model of LDL-receptor deficiency (Aim 4). We propose to test the hypothesis that platelet adhesion and activation mediated by GP Ib-IX and/or GP VI are important contributors to inflammation. Completion of each aim further defines the biological relevance of GP Ib-IX and GP VI in thrombosis and megakaryocytopoiesis but also considers platelet relevance beyond the platelet paradigm in hemostasis and thrombosis. PROJECT NARRATIVE The role of circulating blood platelets is to prevent blood loss. However, in disease platelets can cause the pathologic disruption of blood that leads to organ damage and failure. The most common event is a myocardial infarction in the heart and this is the leading cause of death in the U.S. Our studies use mouse models to mimic those events leading to fatal thrombosis and characterize the molecular events associated with heart attack and stroke. An understanding of how platelets function in the process will lead to better therapeutic strategies and improved health in the U.S.