Regulators of von Willebrand Factor Levels

摘要 血管性血友病因子 (VWF) 是一种多聚体糖蛋白，在 内皮下层附着在损伤部位的血小板上并稳定血浆中的因子 VIII。缺乏 VWF 会导致血管性血友病 (VWD)，这是最常见的遗传性出血性疾病 在人类中。许多导致 VWD 的 VWF 基因突变已得到表征。尽管如此 尽管 VWD 的遗传学研究取得了进展，但关于导致 VWD 的修饰基因的信息有限。 I 型 VWD，患者的 VWF 水平相当低。同样，具有高 VWF 水平存在血栓形成的风险。因此，对相关基因的了解存在差距。 调节 VWF 水平。识别解释分子基础的修饰剂/调节剂 了解 1 型 VWD 的机制需要 1 型 VWD。同样，调控基因 高 VWF 水平可能有助于缓解血栓形成。我们最近创建了 斑马鱼 VWD 模型通过使用杂合子对各种基因进行功能性敲低来识别基因 VWD 鱼。斑马鱼对于该提案最重要的优势是它能够快速 使用我们实验室开发的背负式敲低方法筛选基因，以用于监管 修饰控制 VWF 水平的基因。这次筛选应该为我们提供以下名单： 已在斑马鱼中进行功能验证的调节基因。这些结果应该是 可用于对 I 型 VWD 患者的相应基因进行测序。他们还应该 补充 I 型 VWD 患者的全基因组测序工作，初步了解 与上述斑马鱼筛选中鉴定的基因相对应的序列。同样，这些结果 将有助于咨询血栓形成的风险。我们在本提案中的目标是确定新颖的 控制 VWF 水平的因素。 为了识别参与改变 Vwf 水平的新基因，我们提出了一个具体目标。在这个 目标，我们将建立一个筛选方案，通过检测来识别斑马鱼中的 Vwf 修饰基因 通过 ELISA 检测 Vwf 水平。随后，我们将敲除一个已知基因，该基因已被发现为 人类 VWF 修饰基因，并确认该测定在筛选控制基因中的效用 人类 VWF 水平。然后我们将对基因进行全面的全基因组敲除， 可能调节斑马鱼的 Vwf 水平。这一目标的结果将确定迄今为止未知的 调节 Vwf 水平的因素。我们还将针对选定的基因生成突变斑马鱼 进行结构功能研究。上述斑马鱼修饰基因的鉴定将是有用的 作为导致 I 型 VWD 和高 VWF 水平的候选基因。此外，全基因组 这里开发的敲低策略将为研究其他生理途径提供一种方法。

ABSTRACT Von Willebrand Factor (VWF) is a multimeric glycoprotein that acts as an adhesive in the subendothelium to the platelets at the injury site and stabilizes factor VIII in the plasma. Deficiency of VWF results in von Willebrand disease (VWD), which is the most prevalent inherited bleeding disorder in humans. Many mutations in the VWF gene causing VWD have been characterized. Despite this progress in genetic studies on VWD, only limited information is available on modifier genes that cause type I VWD, where the patients have considerably lower VWF levels. Similarly, individuals with high VWF levels are at risk for thrombosis. Thus, there is a gap in the knowledge of the genes involved in regulating the levels of VWF. Identification of modifiers/regulators that explain the molecular basis for type 1 VWD is needed for understanding the mechanisms of type 1 VWD. Likewise, regulatory genes for high VWF levels might help alleviate the thrombotic episodes. We have recently created the zebrafish VWD model to identify genes by functional knockdowns of various genes using heterozygote VWD fish. The most important advantage of the zebrafish for this proposal is its amenability to rapidly screen for genes using the piggyback knockdown method developed in our laboratory for the regulatory genes that modifier control the VWF levels. This screening should provide us with the list of the regulatory genes that would have been functionally validated in zebrafish. These results should be useful in sequencing the corresponding genes from patients with type I VWD. They should also complement the whole genome sequencing efforts of type I VWD patients in taking a first look at the sequences corresponding to genes identified in the above zebrafish screening. Similarly, these results will be useful in counseling for the risk of thrombosis. Our goal in this proposal will be to identify novel factors that control the levels of the VWF. To identify novel genes involved in modifying the levels of Vwf, we propose one specific aim. In this aim, we will establish a screening protocol for identifying Vwf modifier genes in zebrafish by assaying for the Vwf levels by ELISA. Subsequently, we will knockdown a known gene that is already found as a modifier gene for VWF in humans and confirm the assay's utility in screening for the genes controlling human VWF levels. Then we will comprehensively perform genome-wide knockdowns of genes that may regulate the Vwf levels in zebrafish. The results from this aim will identify the hitherto unknown factor(s) that regulate the Vwf levels. We will also generate mutant zebrafish for selected genes to pursue structure-function studies. The above identification of modifier genes in zebrafish will be useful as candidate genes causing the type I VWD and high VWF levels. Furthermore, the genome-wide knockdown strategies developed here will provide an approach to study other physiological pathways.