Document Detail

Contribution of factor VIIIa A2 and A3-C1-C2 subunits to the affinity for factor IXa in factor Xase.
MedLine Citation:
PMID:  15109268     Owner:  NLM     Status:  MEDLINE    
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.
P Vincent Jenkins; Julie L Dill; Qian Zhou; Philip J Fay
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  43     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2004 May 
Date Detail:
Created Date:  2004-04-27     Completed Date:  2004-08-12     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  5094-101     Citation Subset:  IM    
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, New York 14642, USA.
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MeSH Terms
Binding, Competitive
Cysteine Endopeptidases / metabolism*
Factor IXa / metabolism*
Factor VIIIa / chemistry,  genetics,  metabolism*
Membranes, Artificial
Mutagenesis, Site-Directed
Neoplasm Proteins / metabolism*
Phospholipids / metabolism
Point Mutation
Protein Structure, Tertiary
Protein Subunits
Recombinant Proteins / chemistry,  isolation & purification,  metabolism
Spectrometry, Fluorescence
Substrate Specificity
Grant Support
Reg. No./Substance:
0/Membranes, Artificial; 0/Neoplasm Proteins; 0/Phospholipids; 0/Protein Subunits; 0/Recombinant Proteins; 72175-66-7/Factor VIIIa; EC IXa; EC 3.4.22.-/Cysteine Endopeptidases; EC procoagulant

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