Platelet αIIbβ3 plays a central role in hemostasis, but also contributes to thrombosis. αVβ3 has wider tissue distribution and is implicated in contributing to the pathogenesis of osteoporosis, sickle cell disease, tumor angiogenesis, viral invasion, fibrosis, tumor angiogenesis, and supravalvular aortic stenosis. We have developed novel αIIbβ3 pure antagonists that lock the receptor in the inactive conformation in contrast to existing RGD- based antagonists that induce the receptor to undergo a major conformational change that places it in a high affinity ligand binding state and thus are partial agonists. We have brought one of our pure antagonists (RUC-4) forward for clinical development. We then built on this experience to synthesize non-peptide, small molecule, orally available, potent pure antagonists of αVβ3. We also provided the highest resolution 3D reconstruction of intact αIIbβ3 in a nanodisc that unexpectedly demonstrated an upward tilt of the headpiece and separation of the leg regions. In a search for ancillary binding site(s) on αIIbβ3 and fibrinogen beyond the interaction between the C-terminal γ-chain peptide γ404-411 with the RGD-binding site on αIIbβ3, we demonstrated that activated, but not unactivated, αIIbβ3 could mediate adhesion to fibrinogen fragment D98, which lacks γ404-411. In the upcoming grant period we will bring advanced computational and cryo-electron microscopy (cryo-EM) analysis in collaboration with Drs. Marta Filizola and Thomas Walz to extend these studies to address important unresolved aspects of αIIbβ3 ligand binding and initiate translational studies with our new αVβ3 antagonists. Specific aim 1. To provide further insight into αIIbβ3 receptor activation and ligand binding: a. Identify ancillary interactions between the fibrinogen γ module and αIIbβ3 beyond the interaction of γ- 404-411 with the RGD binding pocket by: 1) Functional ligand binding assays, 2) Molecular modeling, 3) Cryo- EM, 4) High throughput screening to identify small molecule and monoclonal antibody (mAb) inhibitors. b. Use cryo-EM and computational analyses to: 1) Improve the resolution of our 3D reconstruction of unliganded, full length purified αIIbβ3 in a nanodisc lipid bilayer, 2) Obtain additional 3D models of αIIbβ3 activated by the talin head domain (THD) and THD-activated, fibrinogen-liganded αIIbβ3, 3) Define the activation pathway and transformation among the inactive receptor; the inactive, unliganded receptor; and the active, liganded receptor. Specific aim 2. To develop and test the biologic effects and potential clinical use of novel pure αVβ3 antagonists: a. Further characterize the pure αVβ3 antagonists we have synthesized for specificity for different αV- containing receptors, binding characteristics, and cellular effects, and make them available as tool compounds. b. As a prelude to potential clinical testing, assess the species specificity of the pure αVβ3 antagonists, and then compare their impact to those of cu...