Functional genetics of tissue factor in bleeding and thrombotic risk

出血和血栓风险中组织因子的功能遗传学

• 批准号: 10249178
• 负责人: Sol Schulman
• 金额: $ 43.75万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-16 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10249178
• 关键词: Address; Advisory Committees; Anticoagulant therapy; Anticoagulants; Area; Award; Biochemistry; Biological Assay; Blood Coagulation Disorders; Blood Coagulation Factor; Blood Vessels; Blood coagulation; Budgets; Cell surface; Cells; Cellular biology; Choristoma; Clinic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Coagulation Process; Collaborations; Conflict (Psychology); Core Facility; Custom; Data; Diagnostic; Disease; Disease susceptibility; Disseminated Intravascular Coagulation; Electron Transport; Endothelium; Ensure; Equipment; Etiology; Explosion; Faculty; Fellowship; Foundations; Future; Genes; Genetic; Genetic Models; Genetic Variation; Genomic approach; Goals; Grant; Hematology; Hemorrhage; Hemostatic Disorders; Hemostatic function; Hereditary Disease; Heritability; Human; Human Genetics; Human Resources; Individual; Inherited; Injury; Institutional Review Boards; International; Investigation; Israel; Laboratories; Lead; Leadership; Medical center; Medicine; Mentors; Mentorship; Metabolism; Morbidity - disease rate; Mus; Myocardial Infarction; Ownership; Oxidation-Reduction; Pathogenesis; Pathologic; Pathway interactions; Patient Care; Physicians; Population; Positioning Attribute; Principal Investigator; Prize; Protein Biochemistry; Proteins; Protocols documentation; Pulmonary Embolism; Quantitative Trait Loci; Readiness; Reporting; Research; Research Activity; Risk; Rodent Model; Scientist; Secure; Seminal; Sickle Cell Anemia; Smooth Muscle Myocytes; Stroke; Structure; Students; Testing; Therapeutic; Thromboplastin; Thrombosis; Training; Variant; Vascular Smooth Muscle; Vision; Vitamin K; Vocational Guidance; Wages; Warfarin; career development; cell type; design; disorder risk; functional genomics; genetic approach; genetic association; genome-wide; human population study; improved; in vivo; induced pluripotent stem cell; innovation; insight; intravital microscopy; kindred; loss of function; medical schools; mortality; personalized medicine; professor; recruit; senior faculty; square foot; stem cell differentiation; thrombotic; ubiquitin-protein ligase; vascular injury; venous thromboembolism

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.

项目概要/摘要 组织因子（TF）是体内凝血的主要引发剂，是确保止血所必需的 然而，损伤后，不适当的 TF 促凝血活性会造成严重的人类痛苦， 包括由于心肌梗死 (MI)、静脉血栓栓塞 (VTE)、中风和镰状细胞病 因为任何常规临床血液检测均未捕获 TF 的细胞贡献。 凝血过程中，影响 TF 表达和促凝血活性的机制仍然很大程度上未知。 中心假设是细胞转录因子及其修饰因子导致了无法解释的大量遗传性 止血和血栓性疾病，包括静脉血栓栓塞（VTE）、心肌梗死（MI）和中风，我们将共同努力实现这一目标。 功能遗传学和人类遗传学方法来识别有助于 TF 依赖性的修饰因子 出血和凝血风险的遗传性 TF（“因子 III”）是唯一具有遗传性的凝血因子。 缺陷尚未被描述，但在目标 1 中，我们将研究具有相关出血素质的亲属 使用蛋白质生物化学、CRISPR 编辑的诱导多能干细胞，发现杂合 TF 缺陷 （iPSC）分化为人类无法获得的内皮细胞和血管平滑肌细胞，以及 重要的是，这些努力提供了在血管损伤后通过活体显微镜对小鼠进行基因改造； 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。 我们将使用基因组规模的阵列功能丧失筛选来识别 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位点相交叉 现有的人口数据捕获常见和罕见的人类遗传变异，以识别交叉区域， 提供一种通用策略，使用功能筛选超越遗传关联 可能导致出血和凝血的 TF 依赖性遗传性的修饰因素。 将使用定制的 CRISPR 阵列对风险进行大规模机械评估，然后在 iPSC 中进行评估， 在目标 3 中，我们将研究一个这样的发现作为代表性例子， 一种 E3 泛素连接酶，强烈负调节 TF 蛋白稳定性和促凝血活性， 利用细胞生物学、生物化学、啮齿动物模型和人类遗传学来机械地展示如何 人类遗传变异可能会导致 VTE 风险，我们的研究结果将强调这一关键但临床意义。 细胞 TF 对广义出血性疾病的发病机制和遗传的贡献尚未测量 这些发现将具有直接转化的诊断潜力。 捕获这种风险以进行个性化指导治疗，并建议新的抗凝疗法的前景 选择性地针对导致不适当的 TF 促凝血活性的途径，降低出血风险。