Dissecting the Mechanims of Platelet-Fibrin interaction - 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.
