Factor VIII C1 domain residues Lys 2092 and Phe 2093 contribute to membrane binding and cofactor activity

Abstract

AbstractBinding 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-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.