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J. Biol. Chem., Vol. 279, Issue 4, 3084-3095, January 23, 2004

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The Contribution of Amino Acid Region Asp⁶⁹⁵-Tyr⁶⁹⁸ of Factor V to Procofactor Activation and Factor Va Function^*

Daniel O. Beck, Michael A. Bukys, Lisam S. Singh, Katalin A. Szabo, and Michael Kalafatis¶

From the Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115 and the Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690-699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC₅₀ values of 12 and 10 µM, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with K_i values of 6.3 and 5.3 µM, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. The shared amino acid motif between HC3 and HC4 is composed of Asp⁶⁹⁵-Tyr-Asp-Tyr-Gln⁶⁹⁹ (DYDYQ). A pentapeptide with this sequence inhibited both prothrombinase function with an IC₅₀ of 1.6 µM (with a K_D for prothrombin of 850 nM), and activation of factor V by thrombin. Peptides HC3, HC4, and DYDYQ were also found to interact with immobilized thrombin. A recombinant factor V molecule with the mutations Asp⁶⁹⁵ Lys, Tyr⁶⁹⁶ Phe, Asp⁶⁹⁷ Lys, and Tyr⁶⁹⁸ Phe (factor V^2K2F) was partially resistant to activation by thrombin but could be readily activated by RVV-V activator (factor Va_RVV^2K2F) and factor Xa (factor Va_Xa^2K2F). Factor Va_RVV^2K2F and factor Va_Xa^2K2F had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695-698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase.

Received for publication, June 26, 2003 , and in revised form, September 11, 2003.

^* This work was supported by an undergraduate student summer research fellowship from the American Heart Association, Ohio Valley Affiliate (to K. A. S.), by funds from the Department of Chemistry at Cleveland State University (to M. K.), by Established Investigator Award 0040100N from the American Heart Association (to M. K.), and by Grant HL34575 from the National Institutes of Health. 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.

^¶ To whom all correspondence should be addressed: Dept. of Chemistry, Cleveland State University, 2121 Euclid Ave., Campus Location Science Bldg. SR370, Cleveland, OH 44115. Tel.: 216-687-2460; Fax: 216-687-9298; E-mail: m.kalafatis{at}csuohio.edu.

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