title: Cofactor activities of factor VIIIa and A2 subunit following cleavage of A1 subunit at Arg336.
abstract: Factor VIIIa consists of three subunits designated A1, A2, and A3-C1-C2. The isolated A2 subunit possesses limited cofactor activity in stimulating factor IXa-catalyzed activation of factor X. This activity is markedly enhanced by the A1 subunit (inter-subunit K(d) = 1.8 microm). The C-terminal region of A1 subunit (residues 337-372) is thought to represent an A2-interactive site. This region appears critical to factor VIIIa, because proteolysis at Arg(336) by activated protein C or factor IXa is inactivating. A truncated A1 (A1(336)) showed similar affinity for A2 subunit (K(d) = 0.9 microm) and stimulated its cofactor activity to approximately 50% that observed for native A1. However, A1(336) was unable to reconstitute factor VIIIa activity in the presence of A2 and A3-C1-C2 subunits. Fluorescence anisotropy of fluorescein (Fl)-FFR-factor IXa was differentially altered by factor VIIIa trimers containing either A1 or A1(336). Fluorescence energy transfer demonstrated that, although Fl-A1(336)/A3-C1-C2 bound acrylodan-A2 with similar affinity as the native dimer, an increased inter-fluorophore separation was observed. These results indicate that the C-terminal region of A1 appears necessary to properly orient A2 subunit relative to factor IXa in the cofactor rather than directly stimulate A2 and elucidate the mechanism for cofactor inactivation following cleavage at this site.
authors.affiliation: Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Koszelak Rosenblum
authors.forename: Kyla
authors.initials: K
authors.lastname: Schmidt
authors.forename: Jan
authors.initials: J
authors.lastname: Freas
authors.forename: Maria
authors.initials: M
authors.lastname: Mastri
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D016880:Anisotropy; D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D002417:Cattle; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015944:Factor VIIIa / Q000737:chemistry*; D005170:Factor X / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D015944:Factor VIIIa; C038518:acrylodan; D005170:Factor X; D001120:Arginine; D015081:2-Naphthylamine; D015951:Factor Xa