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J. Biol. Chem., Vol. 280, Issue 31, 28711-28720, August 5, 2005

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A Mechanism for Assembly of Complexes of Vitronectin and Plasminogen Activator Inhibitor-1 from Sedimentation Velocity Analysis^*

Kenneth H. Minor, Christine R. Schar, Grant E. Blouse¶, Joseph D. Shore¶, Daniel A. Lawrence||, Peter Schuck**, and Cynthia B. Peterson

From the Department of Biochemistry, Cellular, and Molecular Biology and the Center of Excellence in Structural Biology, University of Tennessee, Knoxville, Tennessee 37996, the Department of Pathology, Division of Biochemical Research, Henry Ford Health System, Detroit, Michigan 48202, the ^¶Department of Pharmacology and Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, the ^||Departments of Surgery and Physiology, University of Maryland School of Medicine, Rockville, Maryland 20855, the ^**Protein Biophysics Resource, Division of Bioengineering and Physical Science, Office of Research Services, Office of the Director, National Institutes of Health, Bethesda, Maryland 20892

Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and pericellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complexes form in a stepwise and concentration-dependent fashion via 1:1 and 2:1 intermediates, with the 2:1 complex serving a key role in assembly of higher order complexes. To test this hypothesis, sedimentation velocity experiments in the analytical ultracentrifuge were performed to identify different PAI-1-vitronectin complexes. Analysis of sedimentation data invoked a novel multisignal method to discern the stoichiometry of the two proteins in the higher-order complexes formed (Balbo, A., Minor, K. H., Velikovsky, C. A., Mariuzza, R. A., Peterson, C. B., and Schuck, P. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 81-86). Our results demonstrate that PAI-1 and vitronectin assemble into higher order forms via a pathway that is triggered upon saturation of the two PAI-1-binding sites of vitronectin to form the 2:1 complex. This 2:1 PAI-1-vitronectin complex, with a sedimentation coefficient of 6.5 S, is the key intermediate for the assembly of higher order complexes.

Received for publication, January 14, 2005 , and in revised form, April 20, 2005.

^* This work was supported by NHLBI, National Institutes of Health Grant HL50676 (to C. B. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Cellular and Molecular Biology, M407 Walters Life Sciences Bldg., University of Tennessee, Knoxville, TN 37996. Tel.: 865-974-4083; Fax: 865-974-6306; E-mail: cynthia_peterson{at}utk.edu.

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