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J. Biol. Chem., Vol. 281, Issue 27, 18569-18580, July 7, 2006

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The Structural Integrity of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII^*

Michael A. Bukys, Tivadar Orban, Paul Y. Kim, Daniel O. Beck, Michael E. Nesheim, and Michael Kalafatis^¶1

From the Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, the Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada, and the ^¶Department of Molecular Cardiology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

-Thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. -Thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by -thrombin. We have used plasma-derived -thrombin, -thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg³²⁰ (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with -thrombin was increased by 8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than -thrombin under similar experimental conditions. -Thrombin readily activated factor V following cleavages at Arg⁷⁰⁹, Arg¹⁰¹⁸, and Arg¹⁵⁴⁵ and factor VIII following proteolysis at Arg³⁷², Arg⁷⁴⁰, and Arg¹⁶⁸⁹. Cleavage of both procofactors by-thrombin was significantly inhibited by D5Q1,2. In contrast, -thrombin was unable to cleave factor V at Arg¹⁵⁴⁵ and factor VIII at both Arg³⁷² and Arg¹⁶⁸⁹. The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. -Thrombin was found to cleave factor V at Arg⁷⁰⁹ and factor VIII at Arg⁷⁴⁰, albeit less efficiently than -thrombin. The sulfated pentapeptide inhibited moderately both cleavages by -thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of -thrombin can account for the interaction of both procofactors with -thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of -thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during the early stages of coagulation.

Received for publication, January 25, 2006 , and in revised form, April 17, 2006.

^* This work was supported by Grant HL-73343 from the NHLBI, National Institutes of Health (NIH) (to M. K.), by a predoctoral fellowship from the Cellular and Molecular Medicine Specialization at Cleveland State University (to M. A. B.), and by United States Public Health Services Grant HL-46703-6 from NIH (to M. E. 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.

¹ To whom correspondence should be addressed: Cleveland State University, Dept. of Chemistry, 2351 Euclid Ave., Science Center SR370, Cleveland OH 44115. Tel.: 216-687-2460; Fax: 216-687-9298; E-mail: m.kalafatis{at}csuohio.edu.

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