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Blood, 1 October 2008, Vol. 112, No. 7, pp. 2761-2769. Prepublished online as a Blood First Edition Paper on July 23, 2008; DOI 10.1182/blood-2008-02-142158. This Article Full Text Full Text (PDF) All Versions of this Article: blood-2008-02-142158v1 112/7/2761    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 CrossRef Citing Articles via Google Scholar Google Scholar Articles by Wakabayashi, H. Articles by Fay, P. J. Search for Related Content PubMed PubMed Citation Articles by Wakabayashi, H. Articles by Fay, P. J. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface Hironao Wakabayashi1, Fatbardha Varfaj2, Jennifer DeAngelis1, and Philip J. Fay1 1 Department of Biochemistry and Biophysics, University of Rochester School of Medicine, NY; and 2 Department of Pharmaceutical Chemistry, University of California, San Francisco Factor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. The charged residues Glu272, Asp519, Glu665, and Glu1984 appear buried at the interface of the A2 domain with either the A1 or A3 domain, and thus may impact protein stability. To determine the effects of these residues on procofactor/cofactor stability, these residues were individually replaced with either Ala or Val, and stable BHK cell lines expressing the B-domainless proteins were prepared. Specific activity and thrombin generation parameters for 7 of the 8 variants were more than 80% the wild-type value. Factor VIII activity at 52°C to 60°C and the decay of factor VIIIa activity after thrombin activation were monitored. Six of the 7 variants showing wild-type-like activity demonstrated enhanced stability, with the Glu1984Val variant showing a 2-fold increase in thermostability and an approximately 4- to 8-fold increase in stability of factor VIIIa. These results indicate that replacement of buried charged residues is an effective alternative to covalent modification in increasing factor VIII (VIIIa) stability. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

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