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J. Biol. Chem., Vol. 282, Issue 35, 25367-25375, August 31, 2007

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Proteolysis at Arg⁷⁴⁰ Facilitates Subsequent Bond Cleavages during Thrombin-catalyzed Activation of Factor VIII^*

From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642

Thrombin activates factor VIII by proteolysis at three P1 residues: Arg³⁷², Arg⁷⁴⁰, and Arg¹⁶⁸⁹. Cleavage at Arg³⁷² and Arg¹⁶⁸⁹ are essential for procofactor activation; however cleavage at Arg⁷⁴⁰ has not been rigorously studied. To evaluate the role for cleavage at Arg⁷⁴⁰, we prepared and stably expressed two recombinant B-domainless factor VIII mutants, R740H and R740Q to slow and eliminate, respectively, cleavage at this site. Specific activity values for the variants were 50 and 20%, respectively, that of wild-type factor VIII. Activation of factor VIII R740H by thrombin showed an 40-fold reduction in the rate of A2 subunit generation, which reflected an 20-fold reduction in cleavage rate at Arg³⁷². Similarly, a 40-fold rate reduction in cleavage at Arg¹⁶⁸⁹ and consequent generation of the A3-C1-C2 subunit were observed. Rate values for A2 and A3-C1-C2 subunit generation were reduced by >700-fold and 140-fold, respectively, in the R740Q variant. These results suggest that initial cleavage at Arg⁷⁴⁰ affects cleavage at both Arg³⁷² and Arg¹⁶⁸⁹ sites. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed more modest rate reductions (<10-fold) in generating A2 and A3-C1-C2 subunits from either variant, suggesting that factor Xa-catalyzed activation of factor VIII was significantly less dependent upon prior cleavage at residue 740 than thrombin. Overall, these results support a model whereby cleavage of factor VIII by thrombin is an ordered pathway with cleavage at Arg⁷⁴⁰ facilitating cleavages at Arg³⁷² and Arg¹⁶⁸⁹, which result in procofactor activation.

Received for publication, April 24, 2007 , and in revised form, June 19, 2007.

^* This work was supported by National Institutes of Health Grants HL38199 and HL76213. An account of this work was presented at the 48th meeting of the American Society of Hematology, Orlando, FL on December 9, 2006. 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 Biochemistry and Biophysics, P.O. Box 712, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-6576; Fax: 585-275-6007; E-mail: philip_fay{at}urmc.rochester.edu.

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				J. L. Newell and P. J. Fay Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII J. Biol. Chem., April 24, 2009; 284(17): 11080 - 11089. [Abstract] [Full Text] [PDF]