title: Contribution of factor VIIIa A2 and A3-C1-C2 subunits to the affinity for factor IXa in factor Xase.
abstract: Contributions of factor (F) VIIIa subunits to cofactor association with FIXa were evaluated. Steady-state fluorescence resonance energy transfer using an acrylodan-labeled A3-C1-C2 subunit and fluorescein-Phe-Phe-Arg-FIXa yielded K(d) values of 52 +/- 10 and 197 +/- 55 nM in the presence and absence of phospholipid vesicles, respectively. A3-C1-C2 was an effective competitor of FVIIIa binding to FIXa as judged by inhibition of FXa generation performed in the absence of vesicles (K(i) approximately 1.6K(d) for FVIIIa-FIXa). However, the capacity for A3-C1-C2 to inhibit FVIIIa-dependent FXa generation in the presence of phospholipid was poor with a K(i) values (approximately 400 nM) that were approximately 100-fold greater than the K(d) for FVIIIa-FIXa interaction (4.2 +/- 0.6 nM). These results indicated that a significant component of the interprotein affinity is contributed by FVIIIa subunits other than A3-C1-C2 in the membrane-dependent complex. The isolated A2 subunit of FVIIIa interacts weakly with FIXa, and recent modeling studies have implicated a number of residues that potentially contact the FIXa protease domain (Bajaj et al. (2001) J. Biol. Chem. 276, 16302-16309). Site-directed mutagenesis of candidate residues in the A2 domain was performed, and recombinant proteins were stably expressed and purified. Functional affinity determinations demonstrated that one mutant, FVIII/Asp712Ala exhibited an 8-fold increased K(d) (35 +/- 1.5 nM) relative to wild-type suggesting a contribution by this residue of approximately 10% of the FVIIIa-FIXa binding energy. Thus both A2 and A3-C1-C2 subunits contribute to the affinity of FVIIIa for FIXa in the membrane-dependent FXase.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, New York 14642, USA.
authors.forename: P Vincent
authors.initials: PV
authors.lastname: Jenkins
authors.forename: Julie L
authors.initials: JL
authors.lastname: Dill
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhou
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D001667:Binding, Competitive; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015949:Factor IXa / Q000378:metabolism*; D015944:Factor VIIIa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008567:Membranes, Artificial; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D008567:Membranes, Artificial; D009363:Neoplasm Proteins; D010743:Phospholipids; D021122:Protein Subunits; D011994:Recombinant Proteins; D015944:Factor VIIIa; D015949:Factor IXa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant