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J. Biol. Chem., Vol. 283, Issue 26, 17789-17796, June 27, 2008

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Thrombin Hydrolysis of Human Osteopontin Is Dependent on Thrombin Anion-binding Exosites^*

Timothy Myles¹ and Lawrence L. K. Leung

From the Division of Hematology, Stanford University School of Medicine, Stanford, California 94305 and the Palo Alto Veterans Affairs Health Care System, Palo Alto, California 94304

The cytokine osteopontin (OPN) can be hydrolyzed by thrombin exposing a cryptic ₄β₁/₉β₁ integrin-binding motif (SVVYGLR), thereby acting as a potent cytokine for cells bearing these activated integrins. We show that purified milk OPN is a substrate for thrombin with a k_cat/K_m value of 1.14 x 10⁵ M^–1 s^–1. Thrombin cleavage of OPN was inhibited by unsulfated hirugen (IC₅₀ = 1.2 ± 0.2 µM), unfractionated heparin (IC₅₀ = 56.6 ± 8.4 µg/ml) and low molecular weight (5 kDa) heparin (IC₅₀ = 31.0 ± 7.9 µg/ml), indicating the involvement of both anion-binding exosite I (ABE-I) and anion-binding exosite II (ABE-II). Using a thrombin mutant library, we mapped residues important for recognition and cleavage of OPN within ABE-I and ABE-II. A peptide (OPN-(162–197)) was designed spanning the OPN thrombin cleavage site and a hirudin-like C-terminal tail domain. Thrombin cleaved OPN-(162–197) with a specificity constant of k_cat/K_m = 1.64 x 10⁴ M^–1 s^–1. Representative ABE-I mutants (K65A, H66A, R68A, Y71A, and R73A) showed greatly impaired cleavage, whereas the ABE-II mutants were unaffected, suggesting that ABE-I interacts principally with the hirudin-like OPN domain C-terminal and contiguous to the thrombin cleavage site. Debye-Hückel slopes for milk OPN (–4.1 ± 1.0) and OPN-(162–197) (–2.4 ± 0.2) suggest that electrostatic interactions play an important role in thrombin recognition and cleavage of OPN. Thus, OPN is a bona fide substrate for thrombin, and generation of thrombin-cleaved OPN with enhanced pro-inflammatory properties provides another molecular link between coagulation and inflammation.

Received for publication, October 17, 2007 , and in revised form, April 2, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant R01 HL057530. 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 To whom correspondence should be addressed: Maxygen Inc., 515 Galveston Dr., Redwood City, CA 94063. E-mail: timothy.myles{at}maxygen.com.

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