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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 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 Citation Map 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. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 July 2002, Vol. 100, No. 2, pp. 501-508 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY 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 From the Department of Biochemistry and Biophysics and the Department of Medicine, University of Rochester Medical Center, NY. 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 (dissociation constant [Kd] values ~5 nM-20 nM and ~100 nM-250 nM in the presence 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 FVIIIArg562Ala showed catalytic rate constant (kcat) values of approximately 60 minute1 in the presence of phospholipid, whereas the hemophilia A-associated mutants showed kcat values ranging from 3.3 minute1 to 7.5 minute1. In the absence of phospholipid, all kcat values were reduced but FVIIIWT and FVIIIArg562Ala retained higher activities as compared with 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. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: J. L. Newell and P. J. Fay Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII J. Biol. Chem., April 24, 2009; 284(17): 11080 - 11089. [Abstract] [Full Text] [PDF] M. Steen, S. Tran, L. Autin, B. O. Villoutreix, A.-L. Tholander, and B. Dahlback Mapping of the Factor Xa Binding Site on Factor Va by Site-directed Mutagenesis J. Biol. Chem., July 25, 2008; 283(30): 20805 - 20812. [Abstract] [Full Text] [PDF] J. L. Newell and P. J. Fay Proteolysis at Arg740 Facilitates Subsequent Bond Cleavages during Thrombin-catalyzed Activation of Factor VIII J. Biol. Chem., August 31, 2007; 282(35): 25367 - 25375. [Abstract] [Full Text] [PDF] A. J. Gale, S. Yegneswaran, X. Xu, J.-L. Pellequer, and J. H. Griffin Characterization of a Factor Xa Binding Site on Factor Va near the Arg-506 Activated Protein C Cleavage Site J. Biol. Chem., July 27, 2007; 282(30): 21848 - 21855. [Abstract] [Full Text] [PDF] F. Varfaj, H. Wakabayashi, and P. J. Fay Residues Surrounding Arg336 and Arg562 Contribute to the Disparate Rates of Proteolysis of Factor VIIIa Catalyzed by Activated Protein C J. Biol. Chem., July 13, 2007; 282(28): 20264 - 20272. [Abstract] [Full Text] [PDF] J. P. Sheehan and E. N. Walke Depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic tenase complex by a common antithrombin-independent mechanism Blood, May 15, 2006; 107(10): 3876 - 3882. [Abstract] [Full Text] [PDF] K. Nogami, Q. Zhou, H. Wakabayashi, and P. J. Fay Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site Blood, June 1, 2005; 105(11): 4362 - 4368. [Abstract] [Full Text] [PDF] K. Nogami, Q. Zhou, T. Myles, L. L. K. Leung, H. Wakabayashi, and P. J. Fay Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain J. Biol. Chem., May 6, 2005; 280(18): 18476 - 18487. [Abstract] [Full Text] [PDF] R. J. Kerschbaumer, K. Riedrich, M. Kral, K. Varadi, F. Dorner, J. Rosing, and F. Scheiflinger An Antibody Specific for Coagulation Factor IX Enhances the Activity of the Intrinsic Factor X-activating Complex J. Biol. Chem., September 24, 2004; 279(39): 40445 - 40450. [Abstract] [Full Text] [PDF] K. Nogami, K. A. Lapan, Q. Zhou, H. Wakabayashi, and P. J. Fay Identification of a Factor Xa-interactive Site within Residues 337-372 of the Factor VIII Heavy Chain J. Biol. Chem., April 16, 2004; 279(16): 15763 - 15771. [Abstract] [Full Text] [PDF] H. Wakabayashi, J. Freas, Q. Zhou, and P. J. Fay Residues 110-126 in the A1 Domain of Factor VIII Contain a Ca2+ Binding Site Required for Cofactor Activity J. Biol. Chem., March 26, 2004; 279(13): 12677 - 12684. [Abstract] [Full Text] [PDF]

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