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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-12-0361. This Article Full Text (PDF) All Versions of this Article: 2001-12-0361v1 100/2/501    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Jenkins, P. V. Articles by Fay, P. J Search for Related Content PubMed PubMed Citation Articles by Jenkins, P. V. Articles by Fay, P. J Social Bookmarking                   What's this? Submitted December 28, 2001 Accepted February 1, 2002 Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex P. Vincent Jenkins, Jan Freas, Kyla M Schmidt, Qian Zhou, and Philip J Fay* Biochemistry and Biophysics, P. O. Box 610, Rochester, NY, USA * Corresponding author; email: philip_fay{at}urmc.rochester.edu. The 558-565 loop region in the A2 subunit of Factor (F) VIIIa forms a direct interface with FIXa. We have expressed and purified B domainless FVIII (FVIIIWT) and B domainless FVIII containing the hemophilia A-associated mutations Ser558Phe, Val559Ala, Asp560Ala, Gln565Arg, and the activated protein C cleavage site mutant Arg562Ala. Titration of FVIIIa in FXa generation assays showed that the mutant and wild type proteins had similar functional affinities for FIXa (Kd values ~5-20 nM and ~100-250 nM in the presence of and absence of phospholipid, respectively). The catalytic activities of the factor Xase complex composed of the hemophilia A-associated FVIII species were markedly reduced both in the presence and absence of phospholipid. FVIIIWT and FVIIIR562A showed kcat values of approximately 60 min-1 in the presence of phospholipid, whereas the hemophilia A-associated mutants showed kcat values ranging from 3.3-7.5 min-1. In the absence of phospholipid all kcat values were reduced but FVIIIWT and FVIIIR562A retained higher activities as compared to the hemophilic mutant FVIII forms. Fluorescence anisotropy experiments using fluorescein-modified FIXa confirmed that all FVIII forms interacted with FIXa. However, the presence of factor X yielded minimal increases in anisotropy observed with the mutant factor VIII forms, consistent with their reduced activity. These results show that residues within the 558-565 loop are critical in modulating FIXa enzymatic activity but do not contribute significantly to the affinity of FVIIIa for FIXa. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: I. Jagannathan, H. T. Ichikawa, T. Kruger, and P. J. 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