title: Residues surrounding Arg336 and Arg562 contribute to the disparate rates of proteolysis of factor VIIIa catalyzed by activated protein C.
abstract: Activated Protein C (APC) inactivates factor VIIIa by cleavage at Arg(336) and Arg(562) within the A1 and A2 subunits, respectively, with reaction at the former site occurring at a rate approximately 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 Arg(336) site were replaced with those surrounding Arg(562), 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 approximately 6-11-fold compared with WT factor VIIIa, and approached values attributed to cleavage at Arg(562). Cleavage rates at Arg(336) were reduced approximately 100-fold for 336(P4-P3')562, and approximately 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 ( approximately 4-fold) at Arg(562) compared with WT, whereas these rates were increased by approximately 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 Arg(336) and Arg(562) make significant contributions to proteolysis rates at each site, apparently independent of binding affinity. Efficient cleavage at Arg(336) by APC is attributed to favorable P4-P3' residues at this site, whereas cleavage at Arg(562) can be accelerated following replacement with more optimal P4-P3' residues.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, NY 14642, USA.
authors.forename: Fatbardha
authors.initials: F
authors.lastname: Varfaj
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D015944:Factor VIIIa / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D020125:Mutation, Missense; D011486:Protein C / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011486:Protein C; D021122:Protein Subunits; D011994:Recombinant Proteins; D015944:Factor VIIIa; D001120:Arginine