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J. Biol. Chem., Vol. 279, Issue 17, 17914-17920, April 23, 2004

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Conservation of Critical Functional Domains in Murine Plasminogen Activator Inhibitor-1^*

Zhi Xu, Rashna D. Balsara, Natalia V. Gorlatova¶, Daniel A. Lawrence¶, Francis J. Castellino, and Victoria A. Ploplis||

From the W. M. Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 and ^¶Department of Vascular Biology, The Holland Laboratory, American Red Cross, Rockville, Maryland, 20855

Plasminogen activator inhibitor-1 is the main physiological regulator of tissue-type plasminogen activator in normal plasma. In addition to its critical function in fibrinolysis, plasminogen activator inhibitor-1 has been implicated in roles in other physiological and pathophysiological processes. To investigate structure-function aspects of mouse plasminogen activator inhibitor-1, the recombinant protein was expressed in Escherichia coli and purified. Five variant recombinant murine proteins (R76E, Q123K, R346A, R101A, and Q123K/R101A) were also generated using site-directed mutagenesis. The variant (R346A) was found to be defective in its inhibitory activity against tissue plasminogen activator relative to its wild-type counterpart. Enzyme-linked immunosorbent assay and surface plasmon resonance experiments demonstrated reduced vitronectin-binding affinity of the (Q123K) variant (K_D = 1800 nM) relative to the wild-type protein (K_D = 5.4 nM). Kinetic analyses indicated that the (Q123K) variant had a slower association (k_on = 2.92 x 10⁴ M^-1 s^-1) to, and a faster dissociation from, vitronectin (k_off = 5.3 x 10^-2 s^-1), (wild-type k_on = 1.03 x 10⁶ M^-1 s^-1 and k_off = 5.27 x 10^-3 s^-1). The Q123K/R101A variant demonstrated an even lower vitronectin-binding ability. Low density lipoprotein receptor-related protein binding was decreased for the (R76E) variant. It was also demonstrated that the plasminogen activator inhibitor-1/vitronectin complex decreased the interaction of plasminogen activator inhibitor-1 with low density lipoprotein receptor-related protein. These results indicate that the complex interactions traditionally associated with different plasminogen activator inhibitor-1 functions apply to the murine system, thus showing a commonality of subtle functions among different species and evolutionary conservation of this protein. Further, this study provides additional evidence that the human hemostasis system can be studied effectively in the mouse, which is a great asset for investigations with gene-altered mice.

Received for publication, December 26, 2003 , and in revised form, February 11, 2004.

^* This work was supported by National Institutes of Health Grants HL-63682 (to V. A. P.) and HL-13423 (to F. J. C.), the Kleiderer-Pezold Family Endowed Professorship (to F. J. C.), and the Krizmanich Family Fellowship (to Z. X.). 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: W. M. Keck Center for Transgene Research and the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556. Tel.: 574-631-4017; Fax: 574-631-8017; E-mail: vploplis{at}nd.edu.

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