Functional genetics of tissue factor in bleeding and thrombotic risk

项目摘要/摘要 组织因子是体内凝血的主要起始剂，是确保止血所必需的。 在受伤之后。然而，不适当的转铁蛋白促凝剂活性是造成大量人类痛苦的基础， 包括心肌梗死(MI)、静脉血栓栓塞症(VTE)、中风和镰状细胞 疾病。因为在任何常规的血液临床化验中都不能捕捉到转铁蛋白的细胞贡献 凝血，影响TF表达和促凝血剂活性的机制在很大程度上仍不清楚。我们的 中心假说是细胞转铁蛋白及其修饰物导致了大量无法解释的遗传性。 止血和血栓形成障碍，包括VTE、MI和中风。为了实现这一目标，我们将相交 功能遗传学和人类遗传学方法，以确定有助于Tf依赖的修饰因子 出血和凝血风险的遗传性。Tf(“因子III”)是唯一一个遗传性凝血因子 缺陷没有被描述，但在目标1中，我们将研究一个与出血素质相关的家系。 用蛋白质生物化学方法检测杂合性TF缺乏症，CRISPR编辑的诱导多能干细胞 (IPSCs)分化为人类无法获得的内皮细胞和血管平滑肌细胞，以及 血管损伤后通过活体显微镜进行转基因小鼠；重要的是，这些努力提供了 概念证明，将TF表达降低50%的修饰剂可以影响体内出血风险。在目标2中， 我们将使用基因组水平的阵列功能丧失筛查来鉴定TF细胞表面的修饰物 表达和促凝血活性，并将这些发现与表达数量性状基因座和 现有的种群数据捕捉常见和罕见的人类遗传变异，以识别相交的区域， 提供一种可推广的策略，使用功能筛查超越基因关联，迈向 因果关系和机制。调节剂可能导致出血和凝血的TF依赖遗传性 风险将使用定制CRISPR阵列在规模上进行机械评估，然后在IPSC中进行评估， 老鼠，如果可能的话，还有人类。在目标3中，我们将作为一个代表性的例子来研究这样的发现， 一种E3泛素连接酶，它强烈负向调节TF蛋白的稳定性和促凝血活性， 使用细胞生物学、生物化学、啮齿动物模型和人类遗传学来机械演示 人类基因变异可能是VTE风险的原因之一。我们的发现将突出关键但临床上的 细胞因子在广义出血性发病机制和遗传中的不可测量作用 和血栓性疾病。这些发现将立即对诊断具有潜在的翻译能力 捕捉这一风险以指导个性化治疗，并提出新的抗凝治疗的承诺 降低选择性靶向导致不适当的TF促凝活性的通路的出血风险。

Project Summary/Abstract Tissue factor (TF) is the primary initiator of blood coagulation in vivo and is required to ensure hemostasis following injury. However, inappropriate TF procoagulant activity underlies substantial human suffering, including that due to myocardial infarction (MI), venous thromboembolism (VTE), stroke, and sickle cell disease. Because the cellular contribution of TF is not captured in any routine clinical assay of blood coagulation, the machinery influencing TF expression and procoagulant activity remains largely unknown. Our central hypothesis is that cellular TF and its modifiers contribute to the substantial unexplained heritability of hemostatic and thrombotic disorders, including VTE, MI, and stroke. To achieve this goal, we will intersect functional genetic and human genetic approaches to identify modifiers that contribute to TF-dependent heritability of bleeding and clotting risk. TF (“factor III”) is the only coagulation factor for which a hereditary deficiency has not been described, but in Aim 1 we will study a kindred with a bleeding diathesis in association with heterozygous TF deficiency using protein biochemistry, CRISPR-edited induced pluripotent stem cells (iPSCs) differentiated into endothelial and vascular smooth muscle cells not accessible in humans, and genetically modified mice via intravital microscopy following vascular injury; importantly, these efforts provide proof of concept that modifiers reducing TF expression by 50% can influence bleeding risk in vivo. In Aim 2, we will use an arrayed loss-of-function screen at genome-scale to identify modifiers of TF cell surface expression and procoagulant activity and intersect these findings with expression quantitative trait loci and existing population data capturing common and rare human genetic variation to identify areas of intersection, offering a generalizable strategy to use a functional screen to move beyond genetic association towards causation and mechanism. Modifiers likely contributing to TF-dependent heritability of bleeding and clotting risk will be mechanistically evaluated at scale using a custom CRISPR array and then evaluated in iPSCs, mice, and, where possible, humans. In Aim 3, we will study as a representative example one such discovery, an E3 ubiquitin ligase that strongly negatively regulates TF protein stability and procoagulant activity, employing cell biology, biochemistry, rodent models, and human genetics to mechanistically demonstrate how human genetic variation may contribute to VTE risk. Our findings will highlight the critical but clinically unmeasured contribution of cellular TF to the pathogenesis and inheritance of broadly defined hemorrhagic and thrombotic diseases. The findings will have immediate translational potential for diagnostics able to capture this risk to guide personalized treatment and suggest promise for new anticoagulant therapies with reduced bleeding risk that selectively target pathways leading to inappropriate TF procoagulant activity.