title: The role of P4-P3' residues flanking Arg336 in facilitating activated protein C-catalyzed cleavage and inactivation of factor VIIIa.
abstract: BACKGROUND
  Activated protein C (APC) inactivates factor VIIIa (FVIIIa) through cleavages at Arg336 in the A1 subunit and Arg562 in the A2 subunit. Proteolysis at Arg336 occurs 25-fold faster than at Arg562. Replacing residues flanking Arg336 en bloc with the corresponding residues surrounding Arg562 markedly reduced the rate of cleavage at Arg336, indicating a role for these residues in the catalysis mechanism.
  
  
  METHODS
  To assess the contributions of individual P4-P3' residues flanking the Arg336 site to cleavage efficiency, point mutations were made based upon those flanking Arg562 of FVIIIa (Pro333Val, Gln334Asp, Leu335Gln, Met337Gly, Lys338Asn, Asn339Gln) and selected residues flanking Arg506 of FVa (Leu335Arg, and Lys338Ile). APC-catalyzed inactivation of the FVIII variants and cleavage of FVIIIa subunits were monitored by FXa generation assays and Western blotting.
  
  
  RESULTS
  Specific activity values of the variants were 60-135% of the wild type (WT) value. APC-catalyzed rates of cleavage at Arg336 remained similar to WT for the Pro333Val and Lys338Ile variants and was modestly increased for the Asn339Gln variant; while rates were reduced ~2-3-fold for the Gln334Asp, Leu335Gln, Leu335Arg, and Lys338Asn variants, and 5-fold for the Met337Gly variant. Rates for cofactor inactivation paralleled cleavage at the A1 site. APC slowly cleaves Arg372 in FVIII, a site responsible for procofactor activation. Using FVIII as substrate for APC, the Met337Gly variant yielded significantly greater activation compared with WT FVIII.
  
  
  CONCLUSIONS
  These results show that individual P4-P3' residues surrounding Arg336 are in general more favorable to cleavage than those surrounding the Arg562 site.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY 14642, USA.
authors.forename: Jennifer P
authors.initials: JP
authors.lastname: DeAngelis
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: D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D055162:Biocatalysis; D015944:Factor VIIIa / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000032:analysis; D017354:Point Mutation; D011486:Protein C / Q000378:metabolism*; D059748:Proteolysis
chemical_list: D010446:Peptide Fragments; D011486:Protein C; D015944:Factor VIIIa; D001120:Arginine