title: Covalent inactivation of factor VIII antibodies from hemophilia A patients by an electrophilic FVIII Analog.
abstract: 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.
authors.affiliation: Chemical Immunology Research Center, Department of Pathology, University of Texas-Houston Medical School, Houston, Texas 77030, USA.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Planque
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Escobar
authors.forename: Keri C
authors.initials: KC
authors.lastname: Smith
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Taguchi
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Nishiyama
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Donnachie
authors.forename: Kathleen P
authors.initials: KP
authors.lastname: Pratt
authors.forename: Sudhir
authors.initials: S
authors.lastname: Paul
mesh_terms: D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000276:immunology; D002648:Child; D002675:Child, Preschool; D005169:Factor VIII / Q000737:chemistry / Q000276:immunology*; D006467:Hemophilia A / Q000276:immunology*; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D009942:Organometallic Compounds / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011489:Protein Denaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000276:immunology; D012997:Solvents; D017186:Titrimetry; D014230:Triazoles / Q000494:pharmacology
chemical_list: C517041:(4-amino-1,4-dihydro-3-(2-pyridyl)-5-thioxo-1,2,4-triazole)copper(II); D000906:Antibodies; D000936:Antigen-Antibody Complex; D009942:Organometallic Compounds; D010455:Peptides; D011994:Recombinant Proteins; D012997:Solvents; D014230:Triazoles; D005169:Factor VIII