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...

Vinyl chloride reacts with cellular DNA producing 3,N4-etheno-2'-deoxycytidine (epsilonC) along with other exocyclic adducts. The solution structure of an oligodeoxynucleotide duplex containing an epsilonC.dG base pair was determined by high-resolution NMR spectroscopy and molecular dynamics simulations. NMR data indicated that the duplex adopts a right-handed helical structure having all residues in anti orientation around the glycosylic torsion angle. The epsilonC adduct has a sugar pucker in the C3'-endo/C4'-exo region while the rest of the residues are in the C2'-endo/C3'-exo range. NOE interactions established Watson-Crick alignments for canonical base pairs of the duplex. The imino proton of the lesion-containing base pair resonated as a sharp signal that was resistant to water exchange, suggesting hydrogen bonding. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. The refined structures are slightly bent at the lesion site without major perturbations of the sugar-phosphate backbone. The adduct is displaced and shifted toward the major groove of the helix while its partner on the complementary strand remains stacked. The epsilonC(anti).dG(anti) base pair alignment is sheared and stabilized by the formation of hydrogen bonds. The biological implications of structures of epsilonC-containing DNA duplexes are discussed.