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, KM33 scFv , and factor VIII mutants with an altered KM33 epitope. We prepared a dual mutant Lys2092/Phe2093 3 Ala/ Ala (fVIII YFP 2092/93) and 2 single mutants Lys2092 3 Ala and Phe2093 3 Ala. KM33 scFv inhibited binding of fluorescein-labeled factor VIII to synthetic membranes and inhibited at least 95% of factor Xase activity. fVIII YFP 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, KM33 scFv 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. KM33 scFv inhibited binding to platelets and fVIII YFP 2092/93 had reduced binding to A23187-stimulated platelets. fVIII YFP 2092 exhibited normal activity at various Ptd-L-Ser concentrations, whereas fVIII YFP 2093 showed a reduction of activity with Ptd-L-Ser less than 12%. fVIII YFP 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. (Blood. 2009;114:3938-3946) Introduction Factor VIII functions as a cofactor in the membrane-bound intrinsic factor Xase complex. Together with the enzyme factor IXa, activated factor VIII binds to phosphatidyl-L-serine (Ptd-L-Ser)-containing membranes 1,2 to form an enzyme complex that cleaves the zymogen factor X to factor Xa. 3,4 Factor Xa is thereafter responsible for catalyzing prothrombin cleavage to thrombin. 5 The importance of the factor Xase complex is illustrated by the disease hemophilia, in which a deficiency of factor VIII (hemophilia A) or factor IX (hemophilia B) leads to life-threatening bleeding. Despite the central importance of membrane binding, this aspect of factor VIII function remains poorly understood. Factor VIII is synthesized as a single polypeptide chain containing 2351 amino acids (molecular weight, 280 kDa) and shows a domain structure of A1-a1-A2-a2-B-a3-A3-C1-C2, where a1, a2, and a3 are spacer regions that separate the domains from each other. 6 Factor VIII is homologous to factor V in amino acid sequence and domain structure. 7 The A domains are homologous with ceruloplasmin, the C domains with discoidin I, and with lactadherin, 8,9 and the B domain is unique to each protein. 10 The A domains mediate the dominant interactions with factor IXa and factor X in the factor Xase complex, whereas binding to Ptd-L-Ser-containing membranes is mediated predominantly by the C2 domain. 11-15 The structure-function relationships of factor V resemble those of factor VIII in that the A domains mediate the dominant interactions with the enzyme and substrate and the C2 domain mediates the dominant membrane-binding inter...
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