title: Exosite-dependent regulation of factor VIIIa by activated protein C.
abstract: Activated protein C (APC) is a natural anticoagulant serine protease in plasma that down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. Recent results have indicated that basic residues of 2 surface loops known as the 39-loop (Lys37-Lys39) and the Ca2+-binding 70-80-loop (Arg74 and Arg75) are critical for the anticoagulant function of APC. Kinetics of factor Va degradation by APC mutants in purified systems have demonstrated that basic residues of these loops are involved in determination of the cleavage specificity of the Arg506 scissile bond on the A2 domain of factor Va. In this study, we characterized the properties of the same exosite mutants of APC with respect to their ability to interact with factor VIIIa. Time course of the factor VIIIa degradation by APC mutants suggested that the same basic residues of APC are also critical for recognition and degradation of factor VIIIa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the factor VIIIa cleavage reactions revealed that these residues are involved in determination of the specificity of both A1 and A2 subunits in factor VIIIa, thus facilitating the cleavages of both Arg336 and Arg562 scissile bonds in the cofactor.
authors.affiliation: Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, MO 63104, USA.
authors.forename: Chandrashekhara
authors.initials: C
authors.lastname: Manithody
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
authors.forename: Alireza R
authors.initials: AR
authors.lastname: Rezaie
mesh_terms: D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D015944:Factor VIIIa / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D011486:Protein C / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D011486:Protein C; D011994:Recombinant Proteins; D015944:Factor VIIIa; D001120:Arginine; D002118:Calcium