Identifying Thrombosis Modifier Genes and Novel Anticoagulants in Zebrafish

斑马鱼血栓调节因子基因和新型抗凝剂的鉴定 该项目将利用斑马鱼可用的强大遗传工具进行大规模 从基因组中筛选可改变人类出血严重程度的止血调节基因 血栓性疾病。该系统还将促进一种新的方法来高通量筛选 化合物文库努力寻找新的候选抗凝剂和止血剂 治疗学。在初步研究中，我们已经定位地克隆了一种新的ENU诱导的突变 斑马鱼pak2a基因导致脑出血，可能是由于内皮细胞缺陷所致 功能和血管完整性。我们还设计了一种特定的突变(M385L)，这种突变与 将人类蛋白C缺乏症转化为斑马鱼蛋白C基因。其他初步研究包括高 通过化学筛选成功鉴定调控链激酶基因的化合物 A组致病性链球菌的表达。在具体目标I中，我们将对M385L和其他 转基因斑马鱼蛋白C突变体体内血栓形成模型的建立 对于阳性和阴性的遗传和药理学筛查。特定目标II将进行大规模演出 化学文库筛选鉴定在部分蛋白C中诱导血栓形成的“增强剂”化合物 缺乏鱼类和“抑制物”的化合物可以“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末端突变筛选以识别血栓前和 抗凝剂修饰基因。综上所述，这些研究应该确定多个候选基因 人类出血性和血栓性疾病的修饰物，为调节 体内止血，并提出了治疗出血和血栓形成的新药物。 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.