Identifying Thrombosis Modifier Genes and Novel Anticoagulants in Zebrafish

斑马鱼血栓形成修饰基因的鉴定及新型抗凝剂的研究 该项目将利用斑马鱼强大的遗传工具进行大规模的 基因组筛选止血调节基因，可以改变人类出血性和 血栓性疾病该系统还将促进用于高通量筛选的新方法， 化学化合物库，以确定新的候选抗凝剂和止血剂 治疗学在初步研究中，我们已经定位克隆了一个新的ENU诱导的突变， 斑马鱼pak2a基因导致脑内出血，可能是由于内皮细胞缺陷 功能和血管完整性。我们还设计了一种与以下疾病相关的特定突变（M385L）： 人类蛋白C缺乏症的基因转移到斑马鱼蛋白C基因中。其他初步研究包括高 通过化学筛选，成功鉴定了调节链激酶基因的化合物 在致病性A组链球菌中表达。在具体目标I中，我们将描述M385L和其他 工程改造的斑马鱼蛋白C突变体开发体内血栓形成模型，可用作基础 用于阳性和阴性遗传和药理学筛查。《Specific Aim II》将大规模上映 化学文库筛选鉴定部分蛋白C诱导血栓形成的“增强剂”化合物 缺陷鱼和“抑制”化合物，“拯救”致命的血栓形成模型。Aim III将使用相同的 斑马鱼模型进行全基因组END诱变筛选，以鉴定血栓前和血栓形成前的基因。 抗凝修饰基因总之，这些研究应该确定多个候选基因， 人出血性和血栓性疾病的修饰剂，提供了新的见解， 止血，并提出了新的药物制剂，用于治疗出血和血栓形成， 人类

Identifying Thrombosis Modifier Genes and Novel Anticoagulants in Zebrafish This project will take advantage of the powerful genetic tools available in zebrafish to conduct a large scale genomic screen for hemostasis regulatory genes that could modify the severity of human hemorrhagic and thrombotic disorders. This system will also facilitate a novel approach for high-throughput screening of chemical compound libraries in an effort to identify new candidate anticoagulant and hemostatic therapeutics. In preliminary studies, we have positionally cloned a novel ENU-induced mutation in the zebrafish pak2a gene that results in intracerebral hemorrhage, likely due to a defect in endothelial cell function and vascular integrity. We have also engineered a specific mutation (M385L) associated with human protein C deficiency into the zebrafish protein C gene. Additional preliminary studies include a high throughput chemical screen that successfully identified compounds that modulate streptokinase gene expression in pathogenic group A streptococci. In Specific Aim I we will characterize M385L and additional engineered zebrafish protein C mutants to develop in vivo thrombosis models that can be used as the basis for both positive and negative genetic and pharmacologic screens. Specific Aim II will perform large scale chemical library screens to identify "enhancer" compounds that induce thrombosis in partially protein C deficient fish and "suppressor" compounds that "rescue" a lethal thrombosis model. Aim III will use the same zebrafish models to perform whole genome END mutagenesis screens to identify both prothrombotic and anticoagulant modifier genes. Taken together, these studies should identify multiple candidate genes for modifiers of human hemorrhagic and thrombotic disorders, provide new insight into the regulation of hemostasis in vivo, and suggest novel pharmaceutical agents for the treatment of bleeding and thrombosis in humans.