Circulating factor VIII immune complexes in patients with type 2 acquired hemophilia A and protection from activated protein C-mediated proteolysis

Factor VIII (FVIII) inhibitor antibodies are classified into 2 groups accor ding to the kinetic pattern of FVIII inactivation, Type 2 antibodies are mo re commonly observed in patients with acquired hemophilia A and do not comp letely inhibit FVIII activity; in most cases, substantial levels of circula ting FVIII are detected, Three type 2 autoantibodies from patients who had normal levels of FVIII antigen despite having low levels of FVIII activity were studied. The antibodies reacted exclusively with the light chain of FV III but not with the C2 domain, and their epitopes were therefore ascribed to the regions in the A3-C1 domains, Heavy and light chains of FVIII were d etected in plasma-derived immune complexes extracted by using protein G Sep harose, Direct binding assays using anhydro-activated protein C (anhydro-AP C), a catalytically inactive derivative of activated protein C (APC) in whi ch the active-site serine is converted to dehydroalanine, were used to exam ine the relation between immune complexes and APC. The intact FVIII, 80-kd light chain, and 72-kd light chain bound in a dose-dependent manner to anhy dro-APC, with K-d values of 580, 540, and 310 nM, respectively, whereas no appreciable binding was detected for the heavy chain, The 3 autoantibodies blocked FVIII binding to anhydro-APC by approximately 80% and consequently inhibited APC-induced FVIII proteolytic inactivation. These antibodies also bound to a synthetic peptide, His2009-Val2018, which contains the APC bind ing site, The findings suggest that binding of type 2 autoantibodies, recog nizing residues His2009 to Val2018, protects FVIII from APC-mediated proteo lysis and might contribute to the presence of FVIII immune complexes in the circulation. (C) 2001 by The American Society of Hematology.