Cell adhesion plays a prominent role in the initiation and evolution of atherosclerosis. For example, platelet-matrix and platelet-platelet adhesion are responsible for the ultimate events in the atherosclerotic process, acute coronary occlusion and stroke. We have shown that activated, but not resting, platelets adhere to osteopontin (OPN), a component of the extracellular matrix of calcified atherosclerotic plaques, but not of the normal arterial wall. Thus, platelet adherence to OPN could be part of the process whereby platelets form thrombi on disrupted atherosclerotic plaques. Platelet adherence to OPN is predominantly mediated by the integrin alphavbeta3. Thus, alphavbeta3, like alphaIIbbeta3, is present on the surface of resting platelets in an inactive state and is activated by platelet agonists. A second potential receptor for OPN on platelets is CD44. CD44 is a receptor for hyaluronic acid on many cells and has been reported to interact with OPN in a cation and RGD-independent manner. The goal of this project is to understand the structural basis for the interaction of OPN with platelet various receptors.
In Specific Aim 1, we will use in vitro mutagenesis to exchange selected segments of alphav and alphaIIb and measure constitutive and agonist-stimulated integrin binding to purified OPN. Studies will focus on the role of cytoplasmic domain sequences and RGD-peptide cross-linking sites. In addition, studies of the role of CD44 in platelet adhesion to OPN will be performed.
In Specific Aim 2, we will identify signaling pathways that activate alphavbeta3. Proposed experiments will focus on the role of protein and lipid kinases, the actin cytoskeleton, and members of the Rho family of small GTP-binding proteins in regulating alphavbeta3 ligand binding activity.
In Specific Aim 3, we will study the structural basis for the recognition of matrix proteins by alphavbeta3. The features of OPN required for its recognition by alphav-containing integrins, as well as the binding affinities of various OPN fragments for alphavbeta3 will be determined. Simple and complex flow systems will be used to study the ability of OPN to support the adherence of both activated and unactivated platelets in the presence of shear stress. Lastly, we determine the structural and dynamic properties of various OPN fragment by NMR methods and the information derived for these studies will be used to synthesize constrained alphavbeta3 peptides and peptidomimetics.
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