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J. Biol. Chem., Vol. 281, Issue 32, 23066-23074, August 11, 2006

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Heparin Modulates the 99-Loop of Factor IXa

EFFECTS ON REACTIVITY WITH ISOLATED KUNITZ-TYPE INHIBITOR DOMAINS^*

Pierre F. Neuenschwander¹, Stephen R. Williamson, Armen Nalian, and Kimberly J. Baker-Deadmond

From the Department of Biochemistry, Biomedical Research Program, The University of Texas Health Science Center, Tyler, Texas 75708 and the Department of Biotechnology, Stephen F. Austin State University, Nacogdoches, Texas 75962

Reactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (>10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor domain of protease Nexin-2 (1.4-fold). Modeling studies of factor IXa with basic pancreatic trypsin inhibitor suggest that binding of this inhibitor is sterically hindered by the 99-loop of factor IXa, specifically residue Lys⁹⁸. Slow-binding kinetic studies support the formation of a weak initial enzyme-inhibitor complex between factor IXa and basic pancreatic trypsin inhibitor that is facilitated by enoxaparin binding. Mutation of Lys⁹⁸ to Ala in factor IXa results in enhanced reactivity with all inhibitors examined, whereas almost completely abrogating the enhancing effects of enoxaparin. The results implicate Lys⁹⁸ and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite.

Received for publication, April 19, 2006 , and in revised form, June 2, 2006.

^* This work was supported by National Institutes of Health Grant HL075696 (to P. F. N.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1-S3.

¹ To whom correspondence should be addressed: Biomedical Research Lab C8, The University of Texas Health Science Center at Tyler, 11937 US Hwy. 271, Tyler, TX 75708. Tel.: 903-877-7678; Fax: 903-877-7679; E-mail: Pierre.Neuenschwander{at}uthct.edu.

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