Dissecting the Mechanims of Platelet-Fibrin interaction

项目总结/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， 有助于止血和动脉血栓的形成。血小板受体整合素 αIIbβ3通过与纤维蛋白原结合，在支持血小板-血小板相互作用中发挥非冗余作用 （聚集），而其与聚合的纤维蛋白的相互作用通过凝块收缩过程使凝块稳定。一 血栓形成研究的基本挑战是精确地了解血小板与纤维蛋白原的相互作用 与聚合的纤维蛋白，以及后者如何动态地收缩凝块。这项建议的目的是适用于 最先进和新颖的实验方法，以获得对纤维蛋白-αIIbβ3的新见解和理解 结合和信号传导机制导致凝块收缩和血栓稳定。为此，我开发了 一种用于评估血小板与纤维蛋白的相互作用的新测定法，其独立于血小板-纤维蛋白原相互作用， 我最近开发了一种高通量筛选试验来识别血栓收缩的抑制剂。我筛选 408，724种化合物，从中我鉴定了580种确认的抑制化合物。与同事们，我也 筛选了301种鼠单克隆抗体（mAb），这些抗体是响应于用人血小板或 αIIbβ3，并已鉴定出4种抑制凝块收缩但不抑制血小板与纤维蛋白原粘附的抗体。使用 结构，生物化学和功能的方法，我现在建议：i）确定的作用机制， 不同的血块收缩抑制剂，目的是确定新的纤维蛋白特异性抗血小板靶点 疗法ii）使用冷冻电子显微镜获得αIIbβ3上结合位点的结构信息， 纤维蛋白特异性单克隆抗体，因此它们干扰凝块收缩的机制。iii）研究独特的αIIbβ3- 纤维蛋白细胞相互作用和信号传导途径以及捕获和分析蛋白复合物的方法 在纤维蛋白-αIIbβ3相互作用下形成。我的导师巴里科勒博士是血小板和αIIbβ3的专家 转化研究，已开发出第一个FDA批准的αIIbβ3拮抗剂，第二个在一个阶段 3研究。我的共同导师Alisa Wolberg博士是血小板-纤维蛋白相互作用和凝块收缩的领导者。在此 我将继续我的技术和科学教育的培训，在几个优秀的合作 这些实验室的技术将作为我计划在年内提交的RO 1研究提案的基石 3这个奖项，导致科学独立在这个奖项结束。洛克菲勒大学提供了一个 杰出的研究环境，广泛的讲座，研讨会和座谈会，并获得国家- 最先进的设备和资源中心，由负责培训初级科学家的高级科学家领导。 博士科勒和洛克菲勒大学致力于建立一个更加多样化，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 指导女科学家和来自代表性不足的少数群体的科学家并作为她们的榜样。

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.