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着关键作用，因为它们是血小板聚集所必需的，而 有助于止血和形成动脉血栓。血小板受体整合素 αIIbβ3在通过结合纤维蛋白原支持血小板-血小板相互作用中的非冗余作用 (聚集)，而它与聚合纤维蛋白的相互作用通过凝块回缩过程稳定凝块。一个 血栓形成研究的根本挑战是准确了解血小板与纤维蛋白原的相互作用。 对比聚合纤维蛋白，以及后者如何动态收缩凝块。这项提案的目的是适用于 最新和新颖的实验方法以获得对纤维蛋白-αIIbβ3的新见解和理解 导致血栓回缩和血栓稳定的结合和信号机制。为此，我开发了 一种评估血小板与纤维蛋白相互作用的新方法，不依赖于血小板-纤维蛋白原相互作用， 我最近开发了一种高通量筛选试验，以确定凝块回缩的抑制物。我放映了 408,724个化合物，从中鉴定出580个已确认的抑制活性化合物。与同事们一起，我还 筛选出301株抗人血小板免疫的小鼠单抗 αIIbβ3，并已确定4种抗体可抑制凝块回缩，但不能抑制血小板与纤维蛋白原的粘连。vbl.使用 结构、生化和功能方法，我现在建议：i)确定 不同的凝块回缩抑制剂，目的是确定抗血小板的新的纤维蛋白特异性靶点 心理治疗。Ii)使用冷冻电子显微镜获得关于αIIbβ3上结合位点的结构信息 纤维蛋白特异性单抗及其干扰凝块回缩的机制。Iii)研究独特的αIIbβ3- 纤维蛋白细胞相互作用和信号通路及其捕获和分析蛋白质复合体的方法 这种形式是对纤维蛋白-αIIbβ3相互作用的反应。我的导师巴里·科勒博士是血小板和αIIbβ3方面的专家 翻译研究，已经开发出美国食品和药物管理局批准的第一个αIIbβ3拮抗剂，第二个正在开发阶段 3研究。我的合作导师，Alisa Wolberg博士是血小板-纤维蛋白相互作用和凝块回缩方面的领导者。在此期间 我将继续我的技术和科学教育，在几个优秀的合作伙伴中接受培训 技术实验室将作为我计划在年内提交的RO1研究提案的构建块 3这一奖项，导致在这一奖项结束时的科学独立性。洛克菲勒大学提供 卓越的研究环境，有广泛的讲座、研讨会和专题讨论会，并有机会获得国家- 最先进的设备和资源中心，由负责培训初级科学家的资深科学家领导。 科勒博士和洛克菲勒大学致力于更多样化、公平和包容性的科学 因此很高兴支持Buitrago博士的申请，因为她表现出了承诺 指导女科学家和来自代表性不足的少数群体的科学家，并为他们树立榜样。