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J. Biol. Chem., Vol. 282, Issue 12, 9288-9296, March 23, 2007

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Mechanism of Inactivation of Plasminogen Activator Inhibitor-1 by a Small Molecule Inhibitor^*

Natalia V. Gorlatova¹, Jacqueline M. Cale¹, Hassan Elokdah^¶, Donghua Li^||, Kristi Fan^¶, Mark Warnock, David L. Crandall^**, and Daniel A. Lawrence²

From the Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0644, ^**Cardiovascular and Metabolic Disease Research and ^¶Chemical and Screening Sciences, Wyeth Research, Collegeville, Pennsylvania 19426, the Center for Advanced Research in Biotechnology, University of Maryland, Rockville, Maryland 20850, and the ^||Department of Vascular Biology, the Holland Laboratory, American Red Cross, Rockville, Maryland 20855

The inactivation of plasminogen activator inhibitor-1 (PAI-1) by the small molecule PAI-1 inhibitor PAI-039 (tiplaxtinin) has been investigated using enzymatic analysis, direct binding studies, site-directed mutagenesis, and molecular modeling studies. Previously PAI-039 has been shown to exhibit in vivo activity in various animal models, but the mechanism of inhibition is unknown. PAI-039 bound specifically to the active conformation of PAI-1 and exhibited reversible inactivation of PAI-1 in vitro. SDS-PAGE indicated that PAI-039 inactivated PAI-1 predominantly through induction of PAI-1 substrate behavior. Preincubation of PAI-1 with vitronectin, but not bovine serum albumin, blocked PAI-039 activity while analysis of the reciprocal experiment demonstrated that preincubation of PAI-1 with PAI-039 blocked the binding of PAI-1 to vitronectin. Together, these data suggest that the site of interaction of the drug on PAI-1 is inaccessible when PAI-1 is bound to vitronectin and may overlap with the PAI-1 vitronectin binding domain. This was confirmed by site-directed mutagenesis and molecular modeling studies, which suggest that the binding epitope for PAI-039 is localized adjacent to the previously identified interaction site for vitronectin. Thus, these studies provide a detailed characterization of the mechanism of inhibition of PAI-1 by PAI-039 against free, but not vitronectin-bound PAI-1, suggesting for the first time a novel pool of PAI-1 exists that is vulnerable to inhibition by inactivators that bind at the vitronectin binding site.

Received for publication, December 19, 2006 , and in revised form, February 2, 2007.

^* This work was supported by National Institutes of Health Grants HL55374, HL55747, and P01HL54710 and by Wyeth Research. 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.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Internal Medicine, University of Michigan Medical School, 7301 MSRB III, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0644. Tel.: 734-763-7838; Fax: 734-936-2641; E-mail dlawrenc{at}umich.edu.

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