Cardiovascular disease is the major cause of mortality in the United States, primarily due to myocardial infarction resulting from rupture of an atherosclerotic plaque and subsequent thrombosis within a coronary artery. A key event that stimulates thrombus formation is platelet aggregation, which is mediated by the prototypical integrin aIIb?3. Integrins are a family of heterodimeric transmembrane receptors which modulate cell adhesion, such as platelet aggregation, as well as other important biological processes such as cell migration, differentiation, proliferation and programmed cell death. Integrins accomplish these diverse functions by mediating dynamic linkages between extracellular adhesion molecules and the intracellular environment. Integrin functions are regulated by transmembrane signaling, which can occur as a consequence of binding extracellular ligands (""""""""outside-in"""""""" signaling), as well as the binding of molecules to the cytoplasmic domains (""""""""inside-out"""""""" signaling). We propose to use electron cryo-microscopy, image reconstruction and molecular modeling to examine the structure of the human platelet integrin aIIb?3. Electron cryo-microscopy (cryo-EM) is a powerful technique by which macromolecular complexes such as membrane proteins can be examined in a native, physiological state. We have completed a 3D structure of the full-length, human platelet integrin aIIb?3 in the low-affinity, inactive conformation. The X-ray structure of the aV?3 ectodomain was then docked into the low resolution cryo-EM map. This combined approach allowed us to propose a model for the structural rearrangements associated with integrin activation. The overall goal of this project is to test this model for integrin activation, and our experiments are organized according to the following specific aims:
Aim 1 : Image analysis will be continued in order to improve the resolution of the 3D reconstruction of aIIb?3 in the low-affinity state;
Aim 2 : A 3D map of the high-affinity state will be derived by analysis of aIIb?3 with bound ligands;
Aim 3 : Antibody labeling will be used to localize specific sites within the high- and low-affinity states;
Aim 4 : Molecular models of the high- and low- affinity states will be derived by combining the cryo-EM maps, site-specific antibody labeling data, and homology models for individual integrin domains. The structural details revealed by these studies will provide insight into the molecular basis of integrin activation, relevant for the design of new therapeutic agents.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-ICI-G (01))
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Flicker, Paula F
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University of Virginia
Schools of Medicine
United States
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