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J. Biol. Chem., Vol. 283, Issue 18, 11876-11886, May 2, 2008

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

Stephanie Planque, Miguel A. Escobar, Keri C. Smith, Hiroaki Taguchi, Yasuhiro Nishiyama, Elizabeth Donnachie, Kathleen P. Pratt, and Sudhir Paul¹

From the Chemical Immunology Research Center, Departments of Pathology and Medicine and Gulf States Hemophilia and Thrombophilia Center, University of Texas-Houston Medical School, Houston, Texas 77030 and Puget Sound Blood Center and Division of Hematology, University of Washington, Seattle, Washington 98104-1256

The antigen-binding sites of antibodies (Abs) can express enzyme-like nucleophiles that react covalently with electrophilic compounds. We examined the irreversible and specific inactivation of antibodies (Abs) to Factor VIII (FVIII) responsible for failure of FVIII replacement therapy in hemophilia A (HA) patients. Electrophilic analogs of FVIII (E-FVIII) and its C2 domain (E-C2) were prepared by placing the strongly electrophilic phosphonate groups at surface-exposed Lys side chains of diverse antigenic epitopes. IgG Abs to FVIII from HA patients formed stable immune complexes with E-FVIII and E-C2 that were refractory to 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.

Received for publication, January 23, 2008 , and in revised form, March 5, 2008.

^* This work was supported by grants from the Hemophilia Associations of New York and Georgia and National Institutes of Health Grant AI31268. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Pathology, University of Texas, Houston Medical School, 6431 Fannin, Houston, TX 77030. E-mail: Sudhir.Paul{at}uth.tmc.edu.

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