Platelets play a central role in both hemostasis and thrombosis, and contribute to a wide range of related phenomena, including inflammation and metastasis formation. The ultimate goal of this grant proposal is to understanding the way in which platelets interact with the blood vessel wall and with other platelets via the glycoprotein receptors on their surface, and to use that knowledge to improve human health. Despite advances in analyzing the structure and function of the ?IIb?3 receptor, which is crucial for normal hemostasis and a validated target of antithrombotic therapy, major gaps remain in understanding ligand binding and its impact on platelet physiology. Moreover, there is a need for improved potent antiplatelet therapies that cn be administered in the pre-hospital phase of myocardial infarction. The data obtained from these studies will inform attempts to develop novel inhibitors of ?IIb?3 that have therapeutic advantages over existing agents.
In Specific Aim 1 we propose to improve our understanding of ligand binding by: a) Using functional ligand binding data and new crystal structures of ?IIb?3 as inputs to state-of-the art computational methods to identify interactions between the fibrinogen ?-module and ?IIb?3 in addition to those made by the fibrinogen ?- chain C-terminal dodecapeptide (?C-12), b) Using electron cryomicroscopy (cryo-EM) and negative stain EM in conjunction with random conical tilt reconstructions, molecular dynamics (MD)-based flexible fitting and steered MD to obtain atomic resolution 3-dimensional (3D) representations of intact ?IIb?3 in a nanodisc lipid bilayer in the absence of detergent. The inactive receptor, the receptor activated by talin head-domain (THD) in the absence of ligand, and THD-activated receptor in the presence of fibrinogen will each be studied. c) Using zinc finger nuclease-mediated gene editing to evaluate mutations of ?3 in murine platelets rather than in cell lines that lack the platelet's signaling machinery, focusing initially on a mutation we hypothesize will enhance the binding of filamin to the ?3 cytoplasmic tail and thus diminish platelet sensitivity to activation. d) Using enhanced MD techniques to characterize ?IIb?3 activation pathways and testing the hypothesis that the new ?IIb?3 antagonists identified in the past grant period (RUC-1, RUC-2, MSSM-1, MSSM-2) stabilize a closed, inactive conformation, thus accounting for their reduced ability to activate ?IIb?3 compared to ?IIb?3 antagonists patterned on the Arg-Gly-Asp (RGD) cell recognition sequence.
In Specific Aim 2 we propose to identify new compounds that will provide insights into ?IIb?3 structure-function and may have therapeutic potential by characterizing the 57 compounds (out of 126,000 tested) that most potently inhibit ?IIb?3-mediated platelet adhesion and aggregation. We will also perform a new screen to selectively identify compounds that target ancillary fibrinogen binding sites since these may lead to new therapeutics that are safer and more efficacious.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01HL019278-39
Application #
8722585
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
Project End
Budget Start
Budget End
Support Year
39
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Biology
Type
Graduate Schools
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10065
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-β1 and correlation with cardiac dysfunction. Blood Cells Mol Dis 58:1-5
Buitrago, Lorena; Rendon, Augusto; Liang, Yupu et al. (2015) αIIbβ3 variants defined by next-generation sequencing: predicting variants likely to cause Glanzmann thrombasthenia. Proc Natl Acad Sci U S A 112:E1898-907
Coller, B S (2015) αIIbβ3: structure and function. J Thromb Haemost 13 Suppl 1:S17-25
Coller, Barry S (2015) Blood at 70: its roots in the history of hematology and its birth. Blood 126:2548-60
Li, Jihong; Vootukuri, Spandana; Shang, Yi et al. (2014) RUC-4: a novel αIIbβ3 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 αIIbβ3 integrin probed by multimicrosecond, all-atom molecular dynamics simulations. Proteins 82:3231-40
Jiang, Jian-kang; McCoy, Joshua G; Shen, Min et al. (2014) A novel class of ion displacement ligands as antagonists of the αIIbβ3 receptor that limit conformational reorganization of the receptor. Bioorg Med Chem Lett 24:1148-53
Coller, Barry S (2014) The platelet: life on the razor's edge between hemorrhage and thrombosis. Transfusion 54:2137-46
Wang, Wei; Vootukuri, Spandana; Meyer, Alexander et al. (2014) Association between shear stress and platelet-derived transforming growth factor-β1 release and activation in animal models of aortic valve stenosis. Arterioscler Thromb Vasc Biol 34:1924-32
Cheng, Ming; Li, Jihong; Negri, Ana et al. (2013) Swing-out of the β3 hybrid domain is required for αIIbβ3 priming and normal cytoskeletal reorganization, but not adhesion to immobilized fibrinogen. PLoS One 8:e81609

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