Identification of the genetic regulators of Plasminogen Activator Inhibitor-1

摘要 血栓形成(血管内血栓)是大多数地区发病率和死亡率的主要原因。 世界。血栓的形成发生在动脉和静脉中，是一个动态的过程。这两个 血栓的形成(凝血)和溶解(纤溶)必须严格控制，以 防止严重出血或血栓形成。一种重要的纤溶抑制物水平升高， 血浆中纤溶酶原激活物抑制物-1(PAI-1，基因名称：SERPINE1)与 静脉血栓形成和心肌梗死。血小板PAI-1水平的个体内差异可能 对血栓性疾病有重大影响。我们之前发现一条闭塞的动脉 在PAI-1完全缺乏的小鼠模型中不能形成血栓。超过90%的流通股 PAI-1位于血小板α颗粒中。我们检测了12名患者的血小板PAI-1抗原水平 近交系小鼠品系。C57BL/6J小鼠品系基本上没有血小板PAI-1，而 Lewis/EIJ株的血小板PAI-1水平是C57BL/6J株的100倍左右。我们使用了十字架 刘易斯/EIJ和C57BL/6J品系产生24个遗传信息丰富的F2代并通过QTL 分析确定了一个调节血小板PAI-1水平的主要位点。这个14.4兆基的区域 5号染色体含有PAI-1基因。我们假设顺式调控表达变体是 负责控制血小板PAI-1的表达(PPAI1High)。在下面的研究中，我们建议 通过对额外的F2小鼠的分析和基因组的产生来鉴定这个表达变体 编辑小鼠以验证已确定的候选变异体(S)。我们还将确定这是否 表达变异体通过分析血小板影响PAI-1以外的血小板特异性基因 Lewis/EIJ和C57BL/6J小鼠的转录本。我们还确定，在 Lewis/EIJ小鼠肝脏、心脏和脂肪组织中PAI-1表达和抗原水平的比较 C57BL/6J。这些差异可能是由于相同或不同的表达变体造成的 控制血小板纤溶酶原激活物-1。我们的假设是内皮细胞PAI-1的表达是PAI-1的主要来源。 1从这些组织中提取。细胞类型和内皮细胞相对比例的异质性 组织可能会对这些组织中的PAI-1水平产生重大影响，从而损害我们的绘画能力 关于PAI-1的细胞来源的结论。为了剖析导致这些问题的细胞类型 表达差异，我们将使用Ribottag小鼠模型来特异性地拔出内皮细胞 来自Lewis/EIJ和C57BL/6J的消息，并进行比较转录分析。这个 拟议的PAI-1调节研究将提供对血小板和内皮特异性的洞察 基因表达，并提供新的治疗靶点来调节血栓形成和 纤溶作用。

Abstract Thrombosis (intravascular blood clots) is a major cause of morbidity and mortality in most areas of the world. Thrombus formation occurs in both arteries and veins and is a dynamic process. Both the development (blood coagulation) and dissolution (fibrinolysis) of thrombi must be exquisitely controlled to prevent either severe bleeding or thrombosis. Elevated levels of an important fibrinolysis inhibitor, plasminogen activator inhibitor-1 (PAI-1, gene name: Serpine1) in plasma have been associated with both venous thrombosis and myocardial infarction. Intra-individual variations in platelet PAI-1 levels could have a major impact on thrombotic disease. We have previously found that an occlusive arterial thrombus could not be formed in a mouse model with complete PAI-1 deficiency. Over 90% of circulating PAI-1 is located in the platelet alpha granules. We measured platelet PAI-1 antigen levels among 12 inbred mouse strains. The C57BL/6J mouse strain has essentially no platelet PAI-1, while the LEWES/EiJ strain has ~100 fold higher platelet PAI-1 than the C57BL/6J strain. We used crosses of the LEWES/EiJ and C57BL/6J strains to generate 24 genetically informative F2 progeny and through QTL analysis identified a major locus for the regulation of platelet PAI-1 levels. This 14.4 megabase region on chromosome 5 contains the PAI-1 gene. We hypothesize that a cis-regulatory expression variant is responsible for controlling platelet PAI-1 expression (pPAI1High). In the studies below, we propose to identify this expression variant through the analysis of additional F2 mice and the production of genome- edited mice for validation of the identified candidate variant(s). We will also determine whether this expression variant affects platelet specific genes other than PAI-1 by analyzing the platelet transcriptomes of LEWES/EiJ and C57BL/6J mice. We have also determined that there are differences in PAI-1 expression and antigen levels in liver, heart and adipose tissue in LEWES/EiJ compared to C57BL/6J. These differences could be due to the same or different expression variants as those controlling platelet PAI-1. Our hypothesis is that endothelial PAI-1 expression is the major source of PAI- 1 from these tissues. The heterogeneity of cell types and relative fraction of endothelial cells in each tissue could have a major impact on PAI-1 levels in these tissues, compromising our ability to draw conclusions regarding the cellular origin of PAI-1. In order to dissect the cell types responsible for these expression differences, we will use a Ribotagged mouse model to specifically pull out endothelial messages from LEWES/EiJ and C57BL/6J and perform a comparative transcriptomic analyses. The proposed investigations of PAI-1 regulation will provide insights into platelet and endothelial specific gene expression as well as providing novel therapeutic targets for modulating thrombosis and fibrinolysis.