Contribution of factor VIIIa A2 and A3-C1-C2 subunits to the affinity for factor IXa in factor Xase.

Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, New York 14642, USA.

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.

PMID: 15109268 [PubMed - indexed for MEDLINE]