Thrombosuppressive mechanisms of novel mutants discovered through an ENU mutagenesis screen

通过 ENU 诱变筛选发现的新型突变体的血栓抑制机制

• 批准号: 9383662
• 负责人: Randal J Westrick
• 金额: $ 37.5万
• 依托单位: OAKLAND UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9383662
• 关键词: ANGPTL2 gene; Affect; Agonist; Amino Acids; Arginine; Blood Coagulation Disorders; Blood Platelets; Blood coagulation; CRISPR/Cas technology; Cardiovascular Diseases; Cause of Death; Defect; Disease; Ethylnitrosourea; Event; Exons; Gene Expression; Generations; Genes; Genetic; Genetic Crosses; Genotype; Glycine; Heterozygote; Human; Knock-in Mouse; Lead; Light; Liver; Mediating; Messenger RNA; Microfilaments; Missense Mutation; Mus; Mutagenesis; Mutation; Names; Perinatal; Pharmacotherapy; Phenotype; Phosphatidylserines; Platelet Activation; Platelet aggregation; Positioning Attribute; Proteins; Regulation; Series; Societies; Stroke; Suppressor Genes; Suppressor Mutations; Susceptibility Gene; System; TFPI; Testing; Thrombin; Thromboembolism; Thrombosis; Thrombus; Venous; Venous Thrombosis; Work; coisogenic; comparative; design; disability; exome sequencing; experimental study; genome editing; in vivo; insight; mRNA Expression; mouse model; mutant; new therapeutic target; novel; novel therapeutics; nuclease; offspring; repaired; transcriptome sequencing

Abstract Venous thromboembolism (VTE) susceptibility genes are largely unknown. We used a sensitized ENU mutagenesis screen of 6,739 mice to identify novel dominant thrombosis suppressor genes of the perinatal lethal F5 Leiden homozygous (F5L/L), tissue factor pathway inhibitor (Tfpi+/-) heterozygous phenotype. These highly penetrant, dominant mutations restored viability to the F5L/L Tfpi+/- mice and were able to be successfully passed on to offspring. We named these suppressor genes MF5L for Modifier of Factor 5 Leiden. In order to identify the MF5L13 suppressor, we used whole exome sequencing to directly identify a single candidate suppressor mutation in exon 7 of the Actr2 gene. This arginine (R) to glycine (G) mutation is in the highly conserved amino acid position 258 of the ARP2 protein (Actr2+/G) and suppresses thrombosis by an as yet unknown mechanism. The studies proposed here seek to functionally characterize the MF5L13 suppressor mutation. Preliminary experimental results on platelet aggregation and platelet dependent thrombus formation of Actr2+/G compared to wildtype Actr2+/+ mice have suggested a platelet functional defect in the Actr2+/G mutants. Comparative analysis of liver Serpine2 mRNA levels via qPCR also revealed a dramatic increase in Serpine2 levels in Actr2+/G compared to Actr2+/+ mice. In the proposed studies, we will first use CRISPR/Cas9 genome editing to re- create the Actr2+/G in a clean (non-mutagenized) mouse genetic background. Using this coisogenic Actr2+/G mouse, we will first test its ability to thrombosuppress F5L/L Tfpi+/-. This mouse model will then be crossed to the Tfpi deficient background (Tfpi-/-) to genetically dissect the thrombosuppressive mechanisms of Actr2+/G relative to F5L/L and Tfpi. To determine whether there are more global expression changes associated with Actr2+/G or if Serpine2 alone is affected, we will perform platelet and liver RNAseq. We will then conduct a series of experiments designed to determine the thrombosuppressive mechanism(s) of the Actr2+/G. We will determine the effects of Actr2+/G on platelet actin filament assembly rates, the effects on agonist induced platelet aggregation and platelet activation events, phosphatidylserine exposure, and thrombin generation, F5 activity and in vivo arterial and venous thrombosis induction. This work will lead to new insights into the mechanisms of action of a unique thrombosuppressor gene and may lead to new therapeutic targets for human thrombotic disease.

抽象的 静脉血栓栓塞（VTE）易感基因很大程度上未知。我们使用了敏化的 ENU 对 6,739 只小鼠进行诱变筛选，以确定新的显性血栓形成抑制基因 围产期致死性 F5 Leiden 纯合子 (F5L/L)、组织因子途径抑制剂 (Tfpi+/-) 杂合子 表型。这些高度渗透的显性突变恢复了 F5L/L Tfpi+/- 小鼠的活力，并且 才能够顺利地遗传给后代。我们将这些抑制基因命名为 MF5L 因子 5 Leiden 的修饰符。为了识别 MF5L13 抑制子，我们使用了整个外显子组 测序直接鉴定 Actr2 基因外显子 7 中的单个候选抑制突变。这 精氨酸 (R) 到甘氨酸 (G) 的突变位于 ARP2 高度保守的氨基酸位置 258 蛋白 (Actr2+/G) 并通过尚不清楚的机制抑制血栓形成。提出的研究 在此寻求对 MF5L13 抑制突变进行功能表征。初步实验结果 与野生型相比，Actr2+/G 对血小板聚集和血小板依赖性血栓形成的影响 Actr2+/+ 小鼠表明 Actr2+/G 突变体存在血小板功能缺陷。对比分析 通过 qPCR 检测肝脏 Serpine2 mRNA 水平也显示 Actr2+/G 中 Serpine2 水平显着增加 与 Actr2+/+ 小鼠相比。在拟议的研究中，我们将首先使用 CRISPR/Cas9 基因组编辑来重新 在干净（未诱变）的小鼠遗传背景中创建 Actr2+/G。使用这种同源 Actr2+/G小鼠，我们首先测试其对F5L/L Tfpi+/-的血栓抑制能力。该鼠标模型将是 交叉到 Tfpi 缺陷背景 (Tfpi-/-) 以基因剖析血栓抑制 Actr2+/G 相对于 F5L/L 和 Tfpi 的机制。判断是否有更多的全局表达式 与 Actr2+/G 相关的变化或如果 Serpine2 单独受到影响，我们将进行血小板和肝脏检查 RNA测序。然后我们将进行一系列旨在确定血栓抑制作用的实验 Actr2+/G 的机制。我们将确定 Actr2+/G 对血小板肌动蛋白丝组装的影响 速率、对激动剂诱导的血小板聚集和血小板激活事件的影响， 磷脂酰丝氨酸暴露、凝血酶生成、F5 活性以及体内动脉和静脉 血栓形成诱导。这项工作将对独特的作用机制产生新的见解 血栓抑制基因并可能为人类血栓性疾病提供新的治疗靶点。