Platelet Integrin AlphaIIbBeta3 Structure, Activation, and Ligand Binding: Fibrinogen, Fibrin, D-dimer, and von Willebrand Factor

点击翻译按钮获取中文摘要

Project Summary/Abstract Platelets play a key role in both hemostasis and thrombosis and contribute to COVID-19 pathology. Platelet αIIbβ3 is the paradigmatic integrin receptor and a validated drug target, with abciximab, developed under this grant, the first FDA-approved αIIbβ3 antagonist. In the current grant period, we rationally designed and synthesized novel αIIbβ3 and αVβ3 antagonists that lock the receptor in the inactive conformation, which may confer therapeutic benefits. The αIIbβ3 antagonist (RUC-4; zalunfiban) is now in Phase 2 human studies for pre- hospital therapy of heart attacks and the αVβ3 antagonists are being developed for pre-clinical testing. We also began studying key gaps in our understanding of how αIIbβ3: 1. Binds its medically important ligands, including fibrinogen, polymerizing fibrin, von Willebrand factor (vWf), and cross-linked fibrin. 2. Initiates clot retraction to confer resistance to thrombolysis. 3. Transitions from its inactive to its active state. Thus, we studied the interaction of αIIbβ3 with fragment D-dimer and used a novel functional assay to dissect the interaction of αIIbβ3 with polymerizing fibrin. In addition, we obtained high-resolution cryo-electron microscopy (EM) structures of αIIbβ3 in complex with abciximab and with the activating monoclonal antibody (mAb) PT25-2, providing data on each antibody’s mechanism of activation. We also initiated hydrogen-deuterium exchange-mass spectroscopy (HDX) studies of αIIbβ3 to provide peptide-level dynamic structural information on ligand binding and receptor activation to complement cryo-EM data. The new Specific Aims build on the PI’s long-standing collaborations with Dr. Marta Filizola, an expert in computational methods, and Dr. Thomas Walz, an expert in cryo-EM. Specific Aim 1. A. To determine high-resolution cryo-EM structures of D-dimer and of αIIbβ3 in complex with: 1) fibrinogen fragment D100 and fibrinogen γ-module; 2) vWf C4 domain; 3) D-dimer. B. To obtain peptide- level solvent-exposed area data by HDX on αIIbβ3 alone and in complex with the ligands. C. To obtain complementary structural and dynamics information from computer simulations to guide mutational analysis and mAb production to validate the proposed mechanisms of binding, as well as to develop novel ligand-specific small-molecule antagonists. Specific Aim 2. To utilize currently available and future cryo-EM data sets in concert with computational and HDX data to define intermediate structures of the integrin leg domains along the αIIbβ3 activation pathway. Specific Aim 3. To utilize our novel functional polymerizing fibrin assays using platelets and HEK293 cells expressing native and mutant forms of αIIbβ3, in concert with mAbs and small-molecule αIIbβ3 antagonists, to define the unique αIIbβ3 ligand-specific binding mechanisms and develop ligand-specific inhibitors. A. To produce mAbs to D-dimer that inhibit platelet-fibrin, but not platelet-fibrinogen interactions. B. To identify mutations that selectively impair fibrinogen, fibrin, or vWf binding, and examine their impact on clot retraction. C. To test predictions derived from the structural and computational studies with mAb and small- molecule ligand-specific inhibitors as a prelude to animal and ultimately human studies.