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Blood, 29 October 2009, Vol. 114, No. 18, pp. 3938-3946. Prepublished online as a Blood First Edition Paper on August 17, 2009; DOI 10.1182/blood-2009-01-197707. This Article Full Text Full Text (PDF) All Versions of this Article: blood-2009-01-197707v1 114/18/3938    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 Google Scholar Articles by Meems, H. Articles by Gilbert, G. E. PubMed PubMed Citation Articles by Meems, H. Articles by Gilbert, G. E. Related Collections Thrombosis and Hemostasis Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  THROMBOSIS AND HEMOSTASIS Factor VIII C1 domain residues Lys 2092 and Phe 2093 contribute to membrane binding and cofactor activity Henriët Meems1,2, Alexander B. Meijer1,3, David B. Cullinan4, Koen Mertens13, and Gary E. Gilbert4 1 Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands; 2 Van Creveld Laboratory of UMC-Utrecht and Sanquin, Amsterdam, The Netherlands; 3 Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands; and 4 Departments of Medicine and Research, Veterans Administration Boston Healthcare System, Brigham and Women's Hospital and Harvard Medical School, Boston, MA Binding of factor VIII to membranes containing phosphatidyl-L-serine (Ptd-L-Ser) is mediated, in part, by a motif localized to the C2 domain. We evaluated a putative membrane-binding role of the C1 domain using an anti-C1 antibody fragment, KM33scFv, and factor VIII mutants with an altered KM33 epitope. We prepared a dual mutant Lys2092/Phe2093 Ala/Ala (fVIIIYFP 2092/93) and 2 single mutants Lys2092 Ala and Phe2093 Ala. KM33scFv inhibited binding of fluorescein-labeled factor VIII to synthetic membranes and inhibited at least 95% of factor Xase activity. fVIIIYFP 2092/93 had 3-fold lower affinity for membranes containing 15% Ptd-L-Ser but more than 10-fold reduction in affinity for membranes with 4% Ptd-L-Ser. In a microtiter plate, KM33scFv was additive with an anti-C2 antibody for blocking binding to vesicles of 15% Ptd-L-Ser, whereas either antibody blocked binding to vesicles of 4% Ptd-L-Ser. KM33scFv inhibited binding to platelets and fVIIIYFP 2092/93 had reduced binding to A23187 [GenBank] -stimulated platelets. fVIIIYFP 2092 exhibited normal activity at various Ptd-L-Ser concentrations, whereas fVIIIYFP 2093 showed a reduction of activity with Ptd-L-Ser less than 12%. fVIIIYFP 2092/93 had a greater reduction of activity than either single mutant. These results indicate that Lys 2092 and Phe 2093 are elements of a membrane-binding motif on the factor VIII C1 domain. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

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