Osteopontin, an acidic phosphorylated glycoprotein, is a component of arteriosclerotic plaques where it serves as an adhesive substrate for smooth muscle and endothelial cells by an interacting with alphav-containing integrins. We have shown that activated, but not unactivated, platelets interact with immobilized osteopontin and that this interaction is mediated by alphavbeta3, not by alphaIIbbeta3. These are the first data that demonstrate a specific function for platelet alphavbeta3 and indicate that the adhesive properties of alphavbeta3 can be modulated in a manner analogous to alphaIIbbeta3. Further, they suggest that the osteopontin surrounding foci of calcification in atherosclerotic plaques could provide a substrate for the occlusive thrombi that form after plaque disruption. The goal of this project is to understand the structural basis for the interaction of osteopontin with alphav-containing integrins. The ability of osteopontin to interact with alphavbeta1, alphavbeta3, alphavbeta5, but not alphaIIbbeta3, suggests that sequences in the alphav subunit are responsible for osteopontin recognition.
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 osteopontin. Studies will focus on the role of cytoplasmic domain sequences, RGD-peptide crosslinking sites, and calcium-binding domain sequences in this process.
In Specific Aim 2, we will determine the conformational and structural features of osteopontin required for its recognition by alphav-containing integrins; the binding affinities of various osteopontin fragments for alphavbeta3 will be measured using the BIAcore technique to determine the minimal fragment showing the full binding affinity of the native protein. The structure of this fragment with then be determined by NMR methods. No information is available regarding the ultrastructure of osteopontin.
In Specific Aim 3, the ultrastructure of osteopontin, osteopontin fragments, and osteopontin: integrin complexes will be examined by rotary- shadowed electron microscopy. In addition, optical tweezers will be used to investigate the strength of the interaction of osteopontin with various integrins. Our results suggest that it may be possible to inhibit the formation of platelet thrombi in arteries involved by atherosclerosis by preventing the interaction of platelet alphavbeta3 with osteopontin.
| Litvinov, Rustem I; Bennett, Joel S; Weisel, John W et al. (2005) Multi-step fibrinogen binding to the integrin (alpha)IIb(beta)3 detected using force spectroscopy. Biophys J 89:2824-34 |
| Litvinov, Rustem I; Nagaswami, Chandrasekaran; Vilaire, Gaston et al. (2004) Functional and structural correlations of individual alphaIIbbeta3 molecules. Blood 104:3979-85 |
| Weisel, J W; Litvinov, R I; Shuman, H et al. (2004) Measuring the binding strength of single ligand-receptor pairs on cells: rebuttal. J Thromb Haemost 2:372-4; author reply 374-7 |
| Litvinov, Rustem I; Vilaire, Gaston; Shuman, Henry et al. (2003) Quantitative analysis of platelet alpha v beta 3 binding to osteopontin using laser tweezers. J Biol Chem 278:51285-90 |
| Litvinov, Rustem I; Shuman, Henry; Bennett, Joel S et al. (2002) Binding strength and activation state of single fibrinogen-integrin pairs on living cells. Proc Natl Acad Sci U S A 99:7426-31 |
