Fine mapping of inhibitory anti-factor V antibodies using factor V C2 domain mutants: Identification of two antigenic epitopes involved in phospholipid binding

Hemorrhagic factor V inhibitors frequently bind to the second C-type (C2) domain of factor V and interfere with phospholipid binding. To define specific residues recognized by inhibitors from four patients (one bovine thrombin-induced and three spontaneous antibodies), epitope mapping was performed using recombinant human factor V lacking most of the B-type domain (FV des B) and alanine-substituted mutants within the C2 domain (FV des B C2 mutants). FV des B C2 mutants located in the region between Lys²⁰⁶⁰ and Glu²⁰⁶⁹ were resistant to inhibition by three IgG preparations including the bovine thrombin-induced antibody in both prothrombinase and phospholipid-binding assays. In contrast, mutations at Lys²⁰⁸⁷ and Lys²⁰⁹²/Glu²⁰⁹⁶ were significantly resistant to inhibition by the fourth IgG preparation in both prothrombinase and phospholipid-binding assays. These results confirm interference of phospholipid binding by hemorrhagic factor V inhibitors and support the role(s) of these residues in phospholipid binding.