Platelet ?IIb?3 plays a central role in hemostasis, but also contributes to thrombosis. ?V?3 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 ?IIb?3 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 ?V?3. We also provided the highest resolution 3D reconstruction of intact ?IIb?3 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 ?IIb?3 and fibrinogen beyond the interaction between the C-terminal ?-chain peptide ?404-411 with the RGD-binding site on ?IIb?3, we demonstrated that activated, but not unactivated, ?IIb?3 could mediate adhesion to fibrinogen fragment D98, which lacks ?404-411. 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 ?IIb?3 ligand binding and initiate translational studies with our new ?V?3 antagonists.
Specific aim 1. To provide further insight into ?IIb?3 receptor activation and ligand binding: a. Identify ancillary interactions between the fibrinogen ? module and ?IIb?3 beyond the interaction of ?- 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 ?IIb?3 in a nanodisc lipid bilayer, 2) Obtain additional 3D models of ?IIb?3 activated by the talin head domain (THD) and THD-activated, fibrinogen-liganded ?IIb?3, 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 ?V?3 antagonists: a. Further characterize the pure ?V?3 antagonists we have synthesized for specificity for different ?V- 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 ?V?3 antagonists, and then compare their impact to those of current RGD-based partial agonists on the pathophysiology of animal models of disease.

Public Health Relevance

This project is designed to learn more about two related cell surface receptors (?V?3 and ?IIb?3) and to develop novel compounds to inhibit the function of these receptors. Past studies from this grant produced the drug abciximab, which is used to treat patients with heart attacks, and tests to monitor the effects of antiplatelet drugs like clopidogrel (Plavix). Our new studies are designed to develop new therapies for heart attacks, sickle cell disease, osteoporosis, herpes virus infection, and disease associated with fibrosis (scarring) of the liver and other organs.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL019278-43
Application #
9427998
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Kindzelski, Andrei L
Project Start
1976-09-01
Project End
2021-05-31
Budget Start
2018-06-01
Budget End
2019-05-31
Support Year
43
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Biology
Type
Graduate Schools
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Zafar, Hina; Shang, Yi; Li, Jihong et al. (2017) ?IIb?3 binding to a fibrinogen fragment lacking the ?-chain dodecapeptide is activation dependent and EDTA inducible. Blood Adv 1:417-428
Wang, Wei; Burg, Nathalie; Vootukuri, Spandana et al. (2016) Increased Smad2/3 phosphorylation in circulating leukocytes and platelet-leukocyte aggregates in a mouse model of aortic valve stenosis: Evidence of systemic activation of platelet-derived TGF-?1 and correlation with cardiac dysfunction. Blood Cells Mol Dis 58:1-5
Buitrago, Lorena; Rendon, Augusto; Liang, Yupu et al. (2015) ?IIb?3 variants defined by next-generation sequencing: predicting variants likely to cause Glanzmann thrombasthenia. Proc Natl Acad Sci U S A 112:E1898-907
Coller, Barry S (2015) Blood at 70: its roots in the history of hematology and its birth. Blood 126:2548-60
Coller, B S (2015) ?IIb?3: structure and function. J Thromb Haemost 13 Suppl 1:S17-25
Jiang, Jian-kang; McCoy, Joshua G; Shen, Min et al. (2014) A novel class of ion displacement ligands as antagonists of the ?IIb?3 receptor that limit conformational reorganization of the receptor. Bioorg Med Chem Lett 24:1148-53
Li, Jihong; Vootukuri, Spandana; Shang, Yi et al. (2014) RUC-4: a novel ?IIb?3 antagonist for prehospital therapy of myocardial infarction. Arterioscler Thromb Vasc Biol 34:2321-9
Provasi, Davide; Negri, Ana; Coller, Barry S et al. (2014) Talin-driven inside-out activation mechanism of platelet ?IIb?3 integrin probed by multimicrosecond, all-atom molecular dynamics simulations. Proteins 82:3231-3240
Wang, Wei; Vootukuri, Spandana; Meyer, Alexander et al. (2014) Association between shear stress and platelet-derived transforming growth factor-?1 release and activation in animal models of aortic valve stenosis. Arterioscler Thromb Vasc Biol 34:1924-32
Coller, Barry S (2014) The platelet: life on the razor's edge between hemorrhage and thrombosis. Transfusion 54:2137-46

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