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) d omain of factor V and interfere with phospholipid binding. To define specif ic residues recognized by inhibitors from four patients (one bovine thrombi n-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 CZ domain (FV des B C2 mutant s). FV des B C2 mutants located in the region between Lys(2060) and Glu(206 9) were resistant to inhibition by three IgG preparations including the bov ine thrombin-induced antibody in both prothrombinase and phospholipid-bindi ng assays. In contrast, mutations at Lys(2087) and Lys(2092)/ Glu(2096) wer e significantly resistant to inhibition by the fourth Ige preparation in bo th prothrombinase and phospholipid-binding assays. These results confirm in terference of phospholipid binding by hemorrhagic factor V inhibitors and s upport the role(s) of these residues in phospholipid binding.