Mouse Genes that Regulate Hemostasis and/or Thrombosis

描述（由申请人提供）：血小板GPVI是一种关键受体，可在小鼠和男性体内启动与胶原蛋白的粘附并促进血栓形成。GPVI的表达和/或活性在两个物种中都有显著的差异。小鼠基因Gp6的消除在体外导致血小板对胶原反应的统一和预期缺陷，但在混合背景下，体内表型是二分的，要么是正常的血栓形成，要么是严重的止血受损。使用这些“致敏”小鼠，我已经确定了一个单一的显性位点，即止血修饰因子（MH），它与出血表型相关，并映射到4号染色体的8 Mb区域。我提出一个或多个修饰基因在MH调节体内表型。为了鉴定这个基因座上的修饰基因，我在Specific Aim 1.1中提出，通过重组克隆的方法，定位克隆对体内表型有贡献的修饰基因。在获得候选基因之一可能是kruppel样因子4基因Klf4的初步数据后，我设计了一种策略来评估体内Klf4和任何其他已确定的候选基因内皮表达的相关性。因此，Specific Aim 1.2旨在开发能够在体内评估候选修饰基因相关性的转基因小鼠。这些体内模型将使诱导（tetON）、条件（内皮细胞）敲低或过表达Klf4作为原型靶点。该模型适用于其他相关的血管细胞类型，如平滑肌细胞或成纤维细胞。在Specific Aim 2中，我建议使用这些转基因小鼠来评估修饰基因产物在体内血栓形成模型中的相关性。我将重点介绍颈动脉损伤模型的结果，这是我最有经验的，但也可以采用其他模型。MH和负责出血/血栓表型的复合基因的特征将对我们理解体内止血调节做出重大贡献。GPVI和其他血小板受体与许多人类血栓形成病例有关。然而，疾病表型的严重程度是可变的，这表明存在修饰基因性状。鉴定可以改变血栓性疾病发病率和严重程度的基因对一般血液学和心血管疾病具有重要价值。公共卫生相关性：血小板是血液中最小的细胞成分，在人类和大多数动物（包括小鼠）中负责血栓的形成。当以研究为目的在小鼠身上进行实验时，我们有能力添加或删除基因，从而发现这些基因如何影响血小板形成凝块的能力。这些信息对我们了解人类冠状动脉疾病和中风的风险和预防具有潜在的价值。我将利用这只老鼠去除我们认为控制血小板黏性的关键基因之一，然后我将把相同的人类基因或修改过的老鼠基因加回到同一只老鼠身上。然后，当适当的系统到位时，我可以在整个动物的特定细胞中，用化学方法诱导老鼠或人类基因的增加或减少，并测量这种基因操作对活老鼠体内血栓形成的影响。人类冠状动脉疾病和中风的严重程度是可变的，这表明这些修饰基因在个体之间存在差异。我的目的是发现老鼠与人类共有的基因差异，这将有助于我们预测甚至预防人类冠状动脉疾病或中风的风险。

DESCRIPTION (provided by applicant): Platelet GPVI is a pivotal receptor that initiates adhesion to collagen and propagates thrombus formation in vivo in both mice and men. A significant variability in GPVI expression and/or activity has been noted in both species. Elimination of the mouse gene Gp6 causes a uniform and expected defect in platelet responses to collagen in vitro, but on a mixed background, the in vivo phenotype is dichotomous, with either normal thrombus formation or severely impaired hemostasis. Using these "sensitized" mice, I have identified a single dominant locus, Modifier of hemostasis (MH), which is in linkage with the bleeding phenotype and maps to an 8 Mb region of chromosome 4. I propose that one or more modifier genes in MH regulate the in vivo phenotypes. To identify the modifier genes in this locus, I propose in Specific Aim 1.1, to positionally clone modifier gene(s) contributing to the in vivo phenotypes, through recombination cloning. Having obtained preliminary data that one of the candidate genes may be Klf4, the gene for Kruppel-like Factor 4, I have designed a strategy to evaluate in vivo the relevance of endothelial expression of Klf4 and any additional candidate genes that are identified. For this reason, Specific Aim 1.2 is to develop transgenic mice in which the relevance of the candidate modifier gene(s) can be assessed in vivo. These in vivo models will enable inducible (tetON), conditional (endothelial cell) knockdown or over expression of Klf4, as the prototype target. The models are adaptable to other relevant vascular cell types, such as smooth muscle cells or fibroblasts. In Specific Aim 2, I propose to use these transgenic mice to assess the relevance of the modifier gene products using in vivo thrombosis models. Emphasis will be placed on the results of the carotid artery injury model, with which I have the most experience, but alternative models can also be employed. The characterization of MH and the composite genes responsible for the bleeding/thrombotic phenotype will make a significant contribution to our understanding of the in vivo regulation of hemostasis. GPVI and other platelet receptors are implicated in many cases of human thrombosis. However, the severity of the disease phenotype is variable, suggesting the existence of modifying gene traits. Identification of genes that can modify the incidence and severity of thrombotic disease is of significant value to general hematology and cardiovascular disease. PUBLIC HEALTH RELEVANCE: Blood platelets are the smallest cellular component of the blood and are responsible for the formation of clots in humans and most animals, including mice. When working on mice for research purposes, we have the ability to add or remove genes and to thus find out how these genes influence the ability of platelets to form clots. This information is potentially valuable to our understanding of risk for and prevention of coronary artery disease and stroke in humans. I will take advantage of the mouse to remove one of the key genes that we believe controls platelet stickiness, and then I will add back into the same mice either the same human gene or a modified mouse gene. Then, when the appropriate system is in place, I can chemically induce an increase or a decrease in either the mouse or the human gene in a specific cell in the whole animal and measure the effect that this gene manipulation has on the formation of clots in that live mouse. The severity of coronary artery disease and stroke in man is variable, suggesting that there are differences between individuals in these modifying genes. It is my intention to discover gene differences in the mouse that are shared by humans and will help us predict the risk for or even prevent coronary artery disease or stroke in man.