Thrombosuppressive mechanisms of novel mutants discovered through an ENU mutagenesis screen

摘要 静脉血栓栓塞症(VTE)的易感基因在很大程度上是未知的。我们使用了致敏的ENU 对6,739只小鼠进行突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)、组织因子途径抑制物(TFP I+/-)杂合子 表型。这些高渗透性、显性突变恢复了F5L/L TFP I+/-小鼠的生存能力 能够成功地传给后代。我们将这些抑制基因命名为MF5L 因子5的修饰符莱顿。为了鉴定MF5L13抑制子，我们使用了整个外显子 直接测序以确定Actr2基因外显子7的单个候选抑制子突变。这 精氨酸(R)到甘氨酸(G)突变位于Arp2的高度保守的氨基酸位置258 蛋白(Actr2+/G)，并通过一种未知的机制抑制血栓形成。这项研究建议 这里试图从功能上表征MF5L13抑制子突变。初步实验结果 Actr2+/G与野生型比较对血小板聚集和血小板依赖性血栓形成的影响 Actr2+/+小鼠提示Actr2+/G突变中存在血小板功能缺陷。比较分析与对比分析 定量聚合酶链式反应显示Actr2+/G细胞中Serpine2基因表达水平显著升高 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(非诱变)的小鼠遗传背景中创建Actr2+/G。利用这种同源基因 Actr2+/G小鼠，我们将首先检测其对F5L/L TFPI+/-的抑制血栓能力。然后，这个鼠标模型将被 与TFPI缺陷性背景(TFPI-/-)杂交，对血栓抑制进行遗传学分析 Actr2+/G与F5L/L和TFPI的作用机制以确定是否存在更多全局表达式 与Actr2+/G相关的变化，或者如果Serpine2单独受到影响，我们将进行血小板和肝脏手术 RNAseq.然后，我们将进行一系列实验，以确定血栓抑制 Actr2+/G的作用机制(S)我们将确定Actr2+/G对血小板肌动蛋白细丝组装的影响 对激动剂诱导的血小板聚集和血小板激活事件的影响， 磷脂酰丝氨酸暴露与凝血酶生成、F5活性以及活体动静脉的关系 血栓形成诱导。这项工作将导致对一种独特的 血栓抑制基因，并可能为人类血栓性疾病带来新的治疗靶点。

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.