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，在血管内皮细胞中起粘附性作用。 损伤部位的血小板的内皮下层，稳定血浆中的第VIII因子。不足之处 VWF导致von Willebrand病(VWD)，这是最常见的遗传性出血疾病 在人类身上。引起VWD的VWF基因的许多突变已经被鉴定。尽管如此 VWD的遗传学研究进展，目前关于引起VWD的修饰基因的信息有限 I型VWD，患者的VWF水平明显较低。同样，拥有高收入的个人 VWF水平存在血栓形成的风险。因此，对与此相关的基因的了解存在差距。 调节VWF水平。确定解释分子基础的修饰剂/调节剂 1型VWD是了解1型VWD发病机制所必需的。同样，调控基因 因为高VWF水平可能有助于减轻血栓发作。我们最近创建了 斑马鱼杂合子功能敲除多种基因识别基因的VWD模型 VWD鱼。对于这一提议，斑马鱼最重要的优势是它的顺应性 使用本实验室开发的用于调节基因的背负基因敲除方法进行基因筛查 修饰基因控制着VWF的水平。这次放映应该会给我们提供一份 本可以在斑马鱼身上得到功能验证的调控基因。这些结果应该是 对I型VWD患者的相应基因进行测序很有用。他们还应该 补充I型VWD患者的全基因组测序工作，首先了解 与上述斑马鱼筛选中确定的基因相对应的序列。同样，这些结果 对血栓形成风险的咨询将是有用的。我们在这项提案中的目标将是确定小说 控制VWF水平的因素。 为了识别与调节VWF水平有关的新基因，我们提出了一个特定的目标。在这 目的建立斑马鱼vwf修饰基因的筛选方法。 用ELISA法测定VWF水平。随后，我们将敲除一个已知的基因，该基因已被发现为 并证实该方法在筛选VWF控制基因中的作用 人体VWF水平。然后我们将全面进行全基因组的基因敲除， 可能调节斑马鱼体内的VWF水平。这一目标的结果将确定迄今未知的 调节VWF水平的因素(S)。我们还将为选定的基因产生突变斑马鱼 继续进行结构功能研究。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 这里开发的基因敲除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.