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J. Biol. Chem., Vol. 282, Issue 44, 31891-31899, November 2, 2007

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A Novel Allosteric Pathway of Thrombin Inhibition

EXOSITE II MEDIATED POTENT INHIBITION OF THROMBIN BY CHEMO-ENZYMATIC, SULFATED DEHYDROPOLYMERS OF 4-HYDROXYCINNAMIC ACIDS^*

Brian L. Henry, Bernhard H. Monien, Paul E. Bock, and Umesh R. Desai¹

From the Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia 23298 and the Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Thrombin and factor Xa, two important pro-coagulant proteinases, can be regulated through direct and indirect inhibition mechanisms. Recently, we designed sulfated dehydropolymers (DHPs) of 4-hydroxycinnamic acids that displayed interesting anticoagulant properties (Monien, B. H., Henry, B. L., Raghuraman, A., Hindle, M., and Desai, U. R. (2006) Bioorg. Med. Chem. 14, 7988–7998). To better understand their mechanism of action, we studied the direct inhibition of thrombin, factor Xa, factor IXa, and factor VIIa by CDSO3, FDSO3, and SDSO3, three analogs of sulfated DHPs. All three sulfated DHPs displayed a 2–3-fold preference for direct inhibition of thrombin over factor Xa, whereas this preference for inhibiting thrombin over factor IXa and factor VIIa increased to 17–300-fold, suggesting a high level of selectivity. Competitive binding studies with a thrombin-specific chromogenic substrate, a fluorescein-labeled hirudin peptide, bovine heparin, enoxaparin, and a heparin octasaccharide suggest that CDSO3 preferentially binds in or near anion-binding exosite II of thrombin. Studies of the hydrolysis of H-D-hexahydrotyrosol-Ala-Arg-p-nitroanilide indicate that CDSO3 inhibits thrombin through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. Overall, designed sulfated DHPs appear to be the first molecules that bind primarily in the region defined by exosite II and allosterically induce thrombin inhibition. The molecules are radically different in structure from all the current clinically used anticoagulants and thus represent a novel class of potent dual thrombin and factor Xa inhibitors.

Received for publication, May 23, 2007 , and in revised form, September 4, 2007.

^* This work was supported by the Grants RO1 HL069975 and R41 HL081972 from the National Institutes of Health, Grant EIA 0640053N from the American Heart Association National Center (to U. R. D.), and Grant HL038779 from the National Institutes of Health (to P. E. B.). 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: Dept. of Medicinal Chemistry, Virginia Commonwealth University, 410 N. 12th St., P. O. Box 980540, Richmond, VA 23298-0540. Tel.: 804-828-7328; Fax: 804-827-3664; E-mail: urdesai{at}vcu.edu.

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