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J. Biol. Chem., Vol. 283, Issue 21, 14417-14429, May 23, 2008

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Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin^*

Benjamin Richard, Richard Swanson, Sophia Schedin-Weiss, Ben Ramirez^¶, Gonzalo Izaguirre, Peter G. W. Gettins^¶||, and Steven T. Olson^¶1

From the Center for Molecular Biology of Oral Diseases, the ^¶Center for Structural Biology, and the ^||Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60612 and the Department of Medical Biochemistry and Microbiology, Uppsala University, SE-751 23 Uppsala, Sweden

A conformationally altered prelatent form of antithrombin that possesses both anticoagulant and antiangiogenic activities is produced during the conversion of native to latent antithrombin (Larsson, H., Akerud, P., Nordling, K., Raub-Segall, E., Claesson-Welsh, L., and Björk, I. (2001) J. Biol. Chem. 276, 11996–12002). Here, we show that the previously characterized prelatent antithrombin is a mixture of native antithrombin and a modified, true prelatent antithrombin that are resolvable by heparin-agarose chromatography. Kinetic analyses revealed that prelatent antithrombin is an intermediate in the conversion of native to latent antithrombin whose formation is favored by stabilizing anions of the Hofmeister series. Purified prelatent antithrombin had reduced anticoagulant function compared with native antithrombin, due to a reduced heparin affinity and consequent impaired ability of heparin to either bridge prelatent antithrombin and coagulation proteases in a ternary complex or to induce full conformational activation of the serpin. Significantly, prelatent antithrombin possessed an antiangiogenic activity more potent than that of latent antithrombin, based on the relative abilities of the two forms to inhibit endothelial cell growth. The prelatent form was conformationally altered from native antithrombin as judged from an attenuation of tryptophan fluorescence changes following heparin activation and a reduced thermal stability. The alterations are consistent with the limited structural changes involving strand 1C observed in a prelatent form of plasminogen activator inhibitor-1 (Dupont, D. M., Blouse, G. E., Hansen, M., Mathiasen, L., Kjelgaard, S., Jensen, J. K., Christensen, A., Gils, A., Declerck, P. J., Andreasen, P. A., and Wind, T. (2006) J. Biol. Chem. 281, 36071–36081), since the ¹H NMR spectrum, electrophoretic mobility, and proteolytic susceptibility of prelatent antithrombin most resemble those of native rather than those of latent antithrombin. Together, these results demonstrate that limited conformational alterations of antithrombin that modestly reduce anticoagulant activity are sufficient to generate antiangiogenic activity.

Received for publication, December 19, 2007 , and in revised form, March 20, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants HL-39888 (to S. T. O.), HL-49234 (to P. G. W. G.), and P41 GM68944 (for the 900-MHz NMR spectrometer). 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: 801 S. Paulina St., Rm. 530C, Chicago, IL 60612. Fax: 312-413-1604; E-mail: stolson{at}uic.edu.

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