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J. Biol. Chem., Vol. 282, Issue 28, 20264-20272, July 13, 2007

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Residues Surrounding Arg³³⁶ and Arg⁵⁶² Contribute to the Disparate Rates of Proteolysis of Factor VIIIa Catalyzed by Activated Protein C^*

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

Activated Protein C (APC) inactivates factor VIIIa by cleavage at Arg³³⁶ and Arg⁵⁶² within the A1 and A2 subunits, respectively, with reaction at the former site occurring at a rate 25-fold faster than the latter. Recombinant factor VIII variants possessing mutations within the P4-P3' sequences were used to determine the contributions of these residues to the disparate cleavage rates at the two P1 sites. Specific activity values for 336(P4-P3')562, 336(P4-P2)562, and 336(P1'-P3')562 mutants, where indicated residues surrounding the Arg³³⁶ site were replaced with those surrounding Arg⁵⁶², were similar to wild type (WT) factor VIII; whereas 562(P4-P3')336 and 562(P4-P2)336 mutants showed specific activity values <1% the WT value. Inactivation rates for the 336 site mutants were reduced 6–11-fold compared with WT factor VIIIa, and approached values attributed to cleavage at Arg⁵⁶². Cleavage rates at Arg³³⁶ were reduced 100-fold for 336(P4-P3')562, and 9–16-fold for 336(P4-P2)562 and 336(P1'-P3')562 mutants. Inhibition kinetics revealed similar affinities of APC for WT factor VIIIa and 336(P4-P3')562 variant. Alternatively, the 562(P4-P3')336 variant showed a modest increase in cleavage rate (4-fold) at Arg⁵⁶² compared with WT, whereas these rates were increased by 27- and 6-fold for 562(P4-P3')336 and 562(P4-P2)336, respectively, using the factor VIII procofactor form as substrate. Thus the P4-P3' residues surrounding Arg³³⁶ and Arg⁵⁶² make significant contributions to proteolysis rates at each site, apparently independent of binding affinity. Efficient cleavage at Arg³³⁶ by APC is attributed to favorable P4-P3' residues at this site, whereas cleavage at Arg⁵⁶² can be accelerated following replacement with more optimal P4-P3' residues.

Received for publication, February 15, 2007 , and in revised form, April 17, 2007.

^* This work was supported in part by National Institutes of Health Grants HL76213 and HL38199. An account of this work was presented at the 48th annual meeting of the American Society of Hematology, Orlando, FL, 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 Supported by an American Heart Association predoctoral fellowship.

² To whom correspondence should be addressed: P. O. Box 712, University of Rochester School of Medicine, 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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