Dissecting the Mechanims of Platelet-Fibrin interaction

剖析血小板-纤维蛋白相互作用的机制

• 批准号: 10722537
• 负责人: Claudia Lorena Buitrago
• 金额: $ 13.91万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10722537
• 关键词: Actins; Adhesions; Adult; Affinity; African American population; Agonist; Amino Acid Sequence; Applications Grants; Avidity; Award; Binding; Binding Sites; Biochemical; Biological Assay; Blood Platelets; Blood coagulation; COVID-19 pandemic; Cardiovascular Diseases; Cause of Death; Characteristics; Charge; Chemicals; Clot retraction; Coagulation Process; Collaborations; Collagen; Communities; Complex; Coronary artery; Cryoelectron Microscopy; Cytoskeleton; Data Reporting; Death Rate; Development; Diabetes Mellitus; Elements; Environment; Equipment; Equity; FDA approved; Fab Immunoglobulins; Fibrin; Fibrin fragment D; Fibrinogen; Generations; Goals; Health; Hemorrhage; Hemostatic function; Hispanic Populations; Hospitals; Human; Human Biology; Immunization; Integrins; Knowledge; Laboratories; Link; Maps; Mediating; Mentors; Methods; Minority Groups; Molecular; Molecular Target; Monitor; Monoclonal Antibodies; Myocardial Infarction; Obesity; Parents; Peptides; Persons; Pharmaceutical Preparations; Platelet aggregation; Play; Polymers; Process; Proteomics; Research; Research Proposals; Resources; Risk; Role; Scientist; Senior Scientist; Series; Signal Pathway; Signal Transduction; Stroke; Structure-Activity Relationship; Techniques; Testing; Thrombin Receptor; Thrombosis; Thrombus; Training; Training and Education; Translational Research; Underrepresented Minority; United States; Universities; Woman; abciximab; antagonist; clinically relevant; crosslink; high throughput screening; imaging approach; improved; indexing; inhibiting antibody; inhibitor; insight; lectures; microscopic imaging; miniaturize; mortality; murine monoclonal antibody; novel; novel therapeutics; phase 3 study; physical inactivity; platelet function; polymerization; prevent; protein complex; receptor; response; role model; symposium; tool

Project Summary/Abstract Platelets play a pivotal role in hemostasis and thrombosis as they are required for platelet aggregation which contributes to both the arrest of bleeding and the development of arterial thrombi. The platelet receptor integrin αIIbβ3 plays a non-redundant role in supporting platelet-platelet interactions via binding of fibrinogen (aggregation) while its interaction with polymerized fibrin stabilizes the clot by the process of clot retraction. A fundamental challenge in thrombosis research is to understand precisely how platelets interact with fibrinogen versus polymerized fibrin and, how the latter dynamically retracts the clot. The aim of this proposal is to apply state-of-the-art and novel experimental approaches to obtain new insights and understanding of fibrin-αIIbβ3 binding and signaling mechanisms leading to clot retraction and thrombus stabilization. To that end, I developed a novel assay for assessing the interaction of platelets with fibrin, independent of platelet-fibrinogen interactions, and I recently developed a high-throughput screening assay to identify inhibitors of clot retraction. I screened 408,724 compounds, from which I identified 580 confirmed inhibitory compounds. With colleagues, I have also screened 301 murine monoclonal antibodies (mAb’s) made in response to immunization with human platelets or αIIbβ3 and have identified 4 antibodies that inhibit clot retraction but not platelet adhesion to fibrinogen. Using structural, biochemical, and functional approaches, I now propose to: i) determine the mechanisms of action of the different inhibitors of clot retraction, with the goal of identifying novel fibrin-specific targets for antiplatelet therapy. ii) employ cryo-electron microscopy to obtain structural information on the binding sites on αIIbβ3 of the fibrin-specific mAb’s and thus their mechanisms of interfering with clot-retraction. iii) study the unique αIIbβ3- fibrin cellular interactions and signaling pathways with methods to capture and analyze the protein complexes that form in response to fibrin-αIIbβ3 interaction. My mentor, Dr. Barry Coller is an expert in platelet and αIIbβ3 translational research, having developed the first FDA approved αIIbβ3 antagonist and with a second in a Phase 3 study. My co-mentor, Dr. Alisa Wolberg is a leader in platelet-fibrin interactions and clot retraction. During this award I will continue my technical and scientific education by training in several outstanding collaborating laboratories in techniques that will serve as building blocks for an RO1 research proposal I plan to submit in year 3 of this award, leading to scientific independence at the end of this award. Rockefeller University provides an outstanding research environment, with a wide range of lectures, seminars, and symposia, and access to state- of-the-art equipment and resource centers, led by senior scientists who are charged with training junior scientists. Dr. Coller and Rockefeller University are committed to a more diverse, equitable, and inclusive scientific community and so are delighted to support Dr. Buitrago’s application, because of her demonstrated commitment to mentor and serve as a role model for women scientists and scientists from underrepresented minority groups.

项目摘要/摘要 血小板在止血和血栓形成中起关键作用，因为它们是血小板聚集所必需的 有助于逮捕出血和动脉血栓的发展。血小板受体整合素 αIIBβ3通过结合纤维蛋白原而在支持血小板 - 血小板相互作用中起着不冗余的作用 （聚集）与聚合纤维蛋白相互作用时，通过凝块回缩的过程稳定了凝块。一个 血栓形成研究的基本挑战是确切地了解血小板如何与纤维蛋白原相互作用 与聚合纤维蛋白相比，后者如何动态缩回凝块。该提议的目的是申请 最新的和新颖的实验方法，以获得新的见解和对纤维蛋白-αIIBβ3的理解 结合和信号传导机制，导致克隆回缩和血栓稳定。为此，我发展了 一种评估血小板与纤维蛋白相互作用的新颖测定法，与血小板 - 纤维蛋白原相互作用无关， 我最近开发了一种高通量筛选测定法，以鉴定克隆回缩的抑制剂。我筛选了 408,724种化合物，我从中确定了580种确认的抑制性化合物。与同事，我也有 筛选了301个鼠单克隆抗体（mAb），以响应人类血小板的免疫或 αIIBβ3并鉴定了4种抑制凝块回缩但不抑制纤维蛋白原的粘合剂的抗体。使用 结构，生化和功能方法，我现在建议：i）确定作用机理 凝块回缩的不同抑制剂，目的是识别新型纤维蛋白特异性靶标的抗血小板 治疗。 ii）员工冷冻电子显微镜以获取有关在结合位点的结构信息 纤维蛋白特异性mAb的MAB，因此它们干扰了凝块撤离的机制。 iii）研究独特的αIIBβ3- 纤维蛋白细胞相互作用和信号传导途径使用捕获和分析蛋白质复合物的方法 响应于纤维蛋白-αIIBβ3相互作用的形式。我的心理，巴里·科勒（Barry Coller）博士是血小板和αIIBβ3的专家 转化研究，已经开发了第一个FDA批准的αIIBβ3拮抗剂，并在一个阶段第二次 3研究。我的同事Alisa Wolberg博士是血小板 - 纤维蛋白相互作用和凝块回收的领导者。在此期间 奖项我将通过几个出色的合作培训来继续我的技术和科学教育 我计划在一年中提交的RO1研究建议的技术实验室 该奖项的3个，导致该奖项结束时科学独立。洛克菲勒大学提供 杰出的研究环境，具有广泛的讲座，半手和专题讨论会，并获得州 由负责培训初级科学家的高级科学家领导的艺术设备和资源中心。 科勒博士和洛克菲勒大学致力于更加多样化，公平和包容的科学 社区，因此很高兴支持Buitrago博士的申请，因为她表现出的承诺 为女性科学家和代表性不足的少数群体的科学家和科学家的榜样而成为榜样。