Covalent Inactivation of Factor VIII Antibodies from Hemophilia A Patients by an Electrophilic FVIII Analog

Abstract

dissociation by SDS treatment and boiling, procedures that dissociate noncovalent Ab-antigen complexes. The rate-limiting step in the reaction was formation of the initial noncovalent complexes. Conversion of the initial complexes to the irreversible state occurred rapidly. The antigenic epitopes of E-FVIII were largely intact, and most of the Abs were consumed covalently. E-FVIII expressed poor FVIII cofactor activity in clotting factor assays. Nonspecific interference by E-FVIII in clotting factor function was not evident. Treatment with E-FVIII, and to a lesser extent E-C2, irreversibly relieved the FVIII inhibitory effect of HA IgG in clotting factor assays. Small FVIII peptides did not display useful reactivity, highlighting the diverse epitope specificities of the Abs and the conformational character of FVIII epitopes. E-FVIII is a prototype reagent able to attain irreversible and specific inactivation of pathogenic Abs. Specific antibodies (Abs)2 to individual antigens are thought to cause harmful effects in autoimmune diseases, transfusion of incompatible blood products, and organ transplantation. Inhibitory Abs to Factor VIII (FVIII) in hemophilia A (HA) are a well characterized example. HA is a chromosome X-linked genetic disorder characterized by the synthesis of functionally inactive FVIII. This impairs the intrinsic pathway of blood coagulation. The primary therapy for control of bleeding in HA patients is infusion of recombinant or plasma-derived FVIII (1). About 20 -30% of patients receiving FVIII replacement therapy produce antibodies (Abs) to FVIII that inhibit FVIII cofactor activity. These are referred to clinically as "inhibitors." The inhibitory effect is thought to derive from reversible steric hindrance of FVIII interactions with phospholipids and other coagulation factors, including thrombin, Factor IXa (FIXa), and von Willebrand factor (2). In addition, some Abs inactivate FVIII permanently by catalyzing its proteolytic breakdown (3). Epitope mapping studies using FVIII fragments (heavy chain, light chains, and A2, A3, C1, and C2 domains) and FVIII hybrid molecules have suggested that many Abs are directed to conformational epitopes (4, 5). Most inhibitor positive patients mount a highly diverse immune response consisting of Abs to multiple FVIII epitopes located in the A2, C1, C2, and A3 domains (2, 6, 7).The Abs pose major problems in managing acute bleeding episodes and surgical procedures in the patients. Short term bleeding in inhibitor-positive patients can be controlled by infusing activated prothrombin complex concentrates or recombinant factor VIIa, agents that bypass the requirement for FVIII in the coagulation pathway (8, 9). Refractory bleeds occur in about 20% of inhibitor-positive HA patients receiving bypass therapy, and an overdose carries the risk of inducing thrombotic events (9). In principle, FVIII itself could be infused to saturate the Abs and restore the coagulation pathway. However, massive quantities of FVIII are required to overcome the inhibitory effect of the ...

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