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• W2073294017 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. 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. Specific antibodies (Abs) 2The abbreviations used are: Ab, antibody; APTT, activated partial thromboplastin time; E-C2, C2-domain analog containing electrophilic phosphonates; E-hapten, electrophilic hapten; E-FVIII, electrophilic Factor VIII; FVIII, Factor VIII; FIXa, activated Factor IX; FX, Factor X; FXa, activated Factor X; HA, hemophilia A; VIP, vasoactive intestinal peptide; ELISA, enzyme-linked immunosorbent assay; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Z, benzyloxycarbonyl; PBS, phosphate-buffered saline; ESI, electrospray ionization. 2The abbreviations used are: Ab, antibody; APTT, activated partial thromboplastin time; E-C2, C2-domain analog containing electrophilic phosphonates; E-hapten, electrophilic hapten; E-FVIII, electrophilic Factor VIII; FVIII, Factor VIII; FIXa, activated Factor IX; FX, Factor X; FXa, activated Factor X; HA, hemophilia A; VIP, vasoactive intestinal peptide; ELISA, enzyme-linked immunosorbent assay; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Z, benzyloxycarbonyl; PBS, phosphate-buffered saline; ESI, electrospray ionization. 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 (1Ettingshausen C.E. Kreuz W. Haemophilia. 2006; 12: 102-106Crossref PubMed Scopus (54) Google Scholar). 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 (2Scandella D. Vox Sang. 1999; 77: 17-20Crossref PubMed Scopus (28) Google Scholar). In addition, some Abs inactivate FVIII permanently by catalyzing its proteolytic breakdown (3Lacroix-Desmazes S. Moreau A. Sooryanarayana Bonnemain C. Stieltjes N. Pashov A. Sultan Y. Hoebeke J. Kazatchkine M.D. Kaveri S.V. Nat. Med. 1999; 5: 1044-1047Crossref PubMed Scopus (174) Google Scholar). 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 (4Spiegel Jr., P.C. Jacquemin M. Saint-Remy J.M. Stoddard B.L. Pratt K.P. Blood. 2001; 98: 13-19Crossref PubMed Scopus (119) Google Scholar, 5Villard S. Piquer D. Raut S. Leonetti J.P. Saint-Remy J.M. Granier C. J. Biol. Chem. 2002; 277: 27232-27239Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). 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 (2Scandella D. Vox Sang. 1999; 77: 17-20Crossref PubMed Scopus (28) Google Scholar, 6Lavigne-Lissalde G. Schved J.F. Granier C. Villard S. Thromb. Haemostasis. 2005; 94: 760-769PubMed Google Scholar, 7Kopecky E.M. Greinstetter S. Pabinger I. Buchacher A. Romisch J. Jungbauer A. J. Immunol. Methods. 2006; 308: 90-100Crossref PubMed Scopus (26) Google Scholar).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 (8Lloyd Jones M. Wight J. Paisley S. Knight C. Haemophilia. 2003; 9: 464-520Crossref PubMed Scopus (79) Google Scholar, 9Berntorp E. Gringeri A. Leissinger C. Negrier C. Key N. Semin. Thromb. Hemostasis. 2006; 32: 22-27Crossref PubMed Scopus (7) Google Scholar). Refractory bleeds occur in about 20% of inhibitor-positive HA patients receiving bypass therapy, and an overdose carries the risk of inducing thrombotic events (9Berntorp E. Gringeri A. Leissinger C. Negrier C. Key N. Semin. Thromb. Hemostasis. 2006; 32: 22-27Crossref PubMed Scopus (7) Google Scholar). 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 circulating Abs even for a short duration. An important clinical advance has been the development of immune tolerance protocols in which high dose FVIII infusions are administered over prolonged periods to suppress Ab production by memory B lymphocytes (10Brackmann H.H. Prog. Clin. Biol. Res. 1984; 150: 181-195PubMed Google Scholar, 11Brackmann H.H. Gormsen J. Lancet. 1977; 2: 933Abstract PubMed Scopus (218) Google Scholar). Experimental peptides (5Villard S. Piquer D. Raut S. Leonetti J.P. Saint-Remy J.M. Granier C. J. Biol. Chem. 2002; 277: 27232-27239Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 12Villard S. Lacroix-Desmazes S. Kieber-Emmons T. Piquer D. Grailly S. Benhida A. Kaveri S.V. Saint-Remy J.M. Granier C. Blood. 2003; 102: 949-952Crossref PubMed Scopus (48) Google Scholar) and anti-idiotypic Abs (13Gilles J.G. Grailly S.C. De Maeyer M. Jacquemin M.G. VanderElst L.P. Saint-Remy J.M. Blood. 2004; 103: 2617-2623Crossref PubMed Scopus (32) Google Scholar) have been reported to block FVIII inhibitory Abs by mimicking the structure of certain FVIII epitopes. Regrettably, there is no single immunodominant FVIII epitope, and these approaches do not adequately address the problem of diverse epitope reactivities of the Abs.The combining sites of certain Abs contain enzyme-like activated nucleophiles. The Ab nucleophilic reactivities were evident from formation of covalent complexes with electrophilic phosphonate diesters (14Planque S. Taguchi H. Burr G. Bhatia G. Karle S. Zhou Y.X. Nishiyama Y. Paul S. J. Biol. Chem. 2003; 278: 20436-20443Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 15Nishiyama Y. Bhatia G. Bangale Y. Planque S. Mitsuda Y. Taguchi H. Karle S. Paul S. J. Biol. Chem. 2004; 279: 7877-7883Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 16Paul S. Planque S. Zhou Y.X. Taguchi H. Bhatia G. Karle S. Hanson C. Nishiyama Y. J. Biol. Chem. 2003; 278: 20429-20435Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), compounds that were originally developed as class-specific inhibitors of serine proteases (17Oleksyszyn J. Powers J.C. Methods Enzymol. 1994; 244: 423-441Crossref PubMed Scopus (93) Google Scholar). The phosphonates react with activated nucleophiles generated by intramolecular interactions between certain amino acids. For instance, the Ser side chain acquires enhanced nucleophilicity by virtue of the hydrogen-bonded network in the Ser-His-Asp catalytic triads of serine proteases (18Bertrand J.A. Oleksyszyn J. Kam C.M. Boduszek B. Presnell S. Plaskon R.R. Suddath F.L. Powers J.C. Williams L.D. Biochemistry. 1996; 35: 3147-3155Crossref PubMed Scopus (56) Google Scholar). The nucleophilic sites permit certain Abs to catalyze the hydrolysis of their cognate antigens (19Paul S. Volle D.J. Beach C.M. Johnson D.R. Powell M.J. Massey R.J. Science. 1989; 244: 1158-1162Crossref PubMed Scopus (380) Google Scholar). Ser-His-Asp and Ser-Arg-Glu catalytic triads have been identified in proteolytic Abs by site-directed mutagenesis (20Gao Q.S. Sun M. Rees A.R. Paul S. J. Mol. Biol. 1995; 253: 658-664Crossref PubMed Scopus (85) Google Scholar) and crystallography studies (21Ramsland P.A. Terzyan S.S. Cloud G. Bourne C.R. Farrugia W. Tribbick G. Geysen H.M. Moomaw C.R. Slaughter C.A. Edmundson A.B. Biochem. J. 2006; 395: 473-481Crossref PubMed Scopus (40) Google Scholar). Nucleophilic catalytic Abs that hydrolyze FVIII and inhibit FVIII cofactor activity are found in HA patients (3Lacroix-Desmazes S. Moreau A. Sooryanarayana Bonnemain C. Stieltjes N. Pashov A. Sultan Y. Hoebeke J. Kazatchkine M.D. Kaveri S.V. Nat. Med. 1999; 5: 1044-1047Crossref PubMed Scopus (174) Google Scholar). However, only a subset of nucleophilic Abs displays catalytic activity (14Planque S. Taguchi H. Burr G. Bhatia G. Karle S. Zhou Y.X. Nishiyama Y. Paul S. J. Biol. Chem. 2003; 278: 20436-20443Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), indicating that additional events in the catalytic cycle occurring after the initial nucleophilic attack on the peptide bond carbonyl group can be rate-limiting (e.g. water attack and product release).We hypothesize that electrophilic FVIII (E-FVIII) analogs may relieve the anti-coagulant effect of Abs by reacting specifically and covalently with their nucleophilic sites. The covalent reaction is predicted to preclude dissociation of the immune complexes, thereby permitting prolonged Ab inactivation. We describe here E-FVIII analogs that relieve the FVIII inhibitory effect of Abs from patients with HA. E-FVIII is a prototypic therapeutic reagent for control of bleeding in inhibitor-positive HA patients. The observed properties of E-FVIII suggest that electrophilic antagonism can potentially be developed as a general basis for attaining specific inactivation of various antigen-specific pathogenic Abs found in immunological diseases.EXPERIMENTAL PROCEDURESElectrophilic FVIII Analogs—Recombinant FVIII (Helixate, CSL Behring) in 10 mm HEPES, 150 mm NaCl, 0.025% Tween 20 was derivatized at Lys residues with the 3-sulfosuccinimidyl ester of diphenyl N-suberoyl-amino(4-amidinophenyl)methanephosphonate, and unincorporated phosphonate was removed by gel filtration (16Paul S. Planque S. Zhou Y.X. Taguchi H. Bhatia G. Karle S. Hanson C. Nishiyama Y. J. Biol. Chem. 2003; 278: 20429-20435Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The phosphonate content of three E-FVIII preparations employed in this study was 52–76 mol of phosphonate/mol of FVIII determined by fluorescamine labeling of the residual amine groups (16Paul S. Planque S. Zhou Y.X. Taguchi H. Bhatia G. Karle S. Hanson C. Nishiyama Y. J. Biol. Chem. 2003; 278: 20429-20435Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To prepare biotinylated E-proteins, biotin was first introduced into recombinant FVIII and C2 protein (22Pratt K.P. Shen B.W. Takeshima K. Davie E.W. Fujikawa K. Stoddard B.L. Nature. 1999; 402: 439-442Crossref PubMed Scopus (283) Google Scholar) by partial acylation in 10 mm HEPES, 150 mm NaCl, 0.1 mm CHAPS (14Planque S. Taguchi H. Burr G. Bhatia G. Karle S. Zhou Y.X. Nishiyama Y. Paul S. J. Biol. Chem. 2003; 278: 20436-20443Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). This yielded FVIII and C2, respectively, containing 8.8 and 0.6 mol of biotin/mol of protein. Phosphonate labeling of biotinylated FVIII was as above (81 mol of phosphonate/mol of protein). Fast protein liquid chromatography-gel filtration of E-FVIII was on a Superose-6 column at a flow rate of 0.3 ml/min as described (23Planque S. Bangale Y. Song X.T. Karle S. Taguchi H. Poindexter B. Bick R. Edmundson A. Nishiyama Y. Paul S. J. Biol. Chem. 2004; 279: 14024-14032Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Initial attempts to prepare E-C2 as described above for E-FVIII resulted in precipitation of the protein (>90%). Therefore, a phosphonate reagent with a more hydrophilic linker was prepared by treating diphenyl amino(4-amidinophenyl)methanephosphonate with di(N-succinimidyl) ethylene glycol disuccinate (Sigma), followed by reversed phase HPLC purification (observed m/z 722.5 (MH+) and calculated MH+ 722.2). Treatment of biotinylated C2 with this reagent yielded E-C2 preparations containing 6.7–8.2 mol of phosphonate/mol of C2. The E-VIP preparation has been described (15Nishiyama Y. Bhatia G. Bangale Y. Planque S. Mitsuda Y. Taguchi H. Karle S. Paul S. J. Biol. Chem. 2004; 279: 7877-7883Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The following peptides were prepared by Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based solid phase synthesis: FVIII residues 484–508 with a Cys residue at the N terminus (N-acetyl-CRPLYSRRLPKGVKHLKDFPILPGEI); residues 1804–1819 (KNFVKPNETKTYFWKV); FVIII residues 2303–2332 with lysyl-biotin at the C terminus (TRYLRIHPQSWVHQIALKMEVLGCEAQDLYK-biotinamidohexanoyl); and the mimotope of a C2 epitope recognized by monoclonal Ab BO2C11 (SCHAWSNRRTCR) (12Villard S. Lacroix-Desmazes S. Kieber-Emmons T. Piquer D. Grailly S. Benhida A. Kaveri S.V. Saint-Remy J.M. Granier C. Blood. 2003; 102: 949-952Crossref PubMed Scopus (48) Google Scholar). The peptides were purified by HPLC (>95% purity) and characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) or electrospray ionization (ESI) mass spectroscopy (FVIII-(484–508), m/z (MALDI) 3076.0 (MH+; calculated MH+ 3075.7); FVIII-(2303–2332), m/z (ESI) 1356.2 (MH33+, calculated MH33+ 1356.4), 1018.0 (MH44+, 1017.5), and 814.5 (MH55+, 814.2); FVIII-(1804–1819), m/z (ESI) 677.4 (MH33+, calculated MH33+ 677.4), and 508.5 (MH44+; 508.3); BO2C11 epitope: m/z (MALDI) 1474.8 (MH+; calculated MH+ 1474.7)). E-(2303–2332) was prepared by regiospecific acylation (15Nishiyama Y. Bhatia G. Bangale Y. Planque S. Mitsuda Y. Taguchi H. Karle S. Paul S. J. Biol. Chem. 2004; 279: 7877-7883Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) as follows. The resin with the 2303–2332 peptide protected at Lys-2320 side chain with the 4-methyltrityl group was treated with 1% trifluoroacetic acid in dichloromethane to remove this protecting group, and the Lys-2320 side chain amine was acylated with the N-hydroxysuccinimidyl ester of the phosphonate reagent used for E-FVIII preparation. Protecting groups and the solid support were removed with trifluoroacetic acid containing 2% phenol, 5% thioanisole, and 5% ethanedithiol, and E-(2303–2332) was purified by HPLC (m/z (ESI) 1529.5 (MH33+, calculated MH33+ 1529.5), 1147.3 (MH44+, 1147.4), 917.6 (MH55+, 918.1)]. The electrophilic analogs were stored at –80 °C as lyophilized powders. Protein concentrations were measured by the bicinchoninic acid assay.Patients and Antibodies—This study was approved by the University of Texas Institutional Review Board. Plasma was prepared from blood in 3.2% sodium citrate from 8 inhibitor-positive HA patients (our lab codes: HA1828, HA1834, HA1835, HA2084, HA2085, HA2222, HA2223, and HA3112; age range 5–59 years). The patients had a history of FVIII inhibitors for at least 5 years but had not received FVIII replacement therapy for at least 2 months prior to the blood draw). The plasma FVIII titers were determined by Bethesda assay (24Kasper C.K. Pool J.G. Thromb. Diath. Haemorrh. 1975; 34: 875-876PubMed Google Scholar) and are reported in Table 1. Control plasma was from a nonhemophiliac human subject without known coagulation or autoimmune disorders (code NH1941). Electrophoretically homogeneous IgG from the plasma samples was purified by affinity chromatography using immobilized protein G (25Paul S. Said S.I. Thompson A.B. Volle D.J. Agrawal D.K. Foda H. de la Rocha S. J. Neuroimmunol. 1989; 23: 133-142Abstract Full Text PDF PubMed Scopus (34) Google Scholar). Fab fragments were prepared by digestion with immobilized papain and chromatography using immobilized protein A (26Paul S. Mei S. Mody B. Eklund S.H. Beach C.M. Massey R.J. Hamel F. J. Biol. Chem. 1991; 266: 16128-16134Abstract Full Text PDF PubMed Google Scholar). Murine monoclonal anti-C2 IgG (clone ESH8) was from American Diagnostica. The isotype-matched control was monoclonal anti-VIP IgG (clone c23.5) (27Paul S. Sun M. Mody R. Tewary H.K. Stemmer P. Massey R.J. Gianferrara T. Mehrotra S. Dreyer T. Meldal M. J. Biol. Chem. 1992; 267: 13142-13145Abstract Full Text PDF PubMed Google Scholar).TABLE 1E-FVIII and E-C2 binding activity and FVIII inhibitor titer of antibodies from HA patientsPatient IDPlasma FVIII inhibitorE-FVIII binding, IgG0.25 in μg/mlE-C2 binding, IgG0.25 in μg/mlBU/mlHA18288002.72.6HA18341503.37.9HA18351494.55.6HA208410244.312.9HA20854919.217.4HA222210110.51.6HA22237029.956.1HA31121985.152.8NH1941<0.5>100>100 Open table in a new tab Hapten Phosphonate Binding—Synthesis of hapten electrophilic probe E-hapten-1, E-hapten-2, and E-hapten-3 was described (14Planque S. Taguchi H. Burr G. Bhatia G. Karle S. Zhou Y.X. Nishiyama Y. Paul S. J. Biol. Chem. 2003; 278: 20436-20443Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 23Planque S. Bangale Y. Song X.T. Karle S. Taguchi H. Poindexter B. Bick R. Edmundson A. Nishiyama Y. Paul S. J. Biol. Chem. 2004; 279: 14024-14032Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 28Paul S. Tramontano A. Gololobov G. Zhou Y.X. Taguchi H. Karle S. Nishiyama Y. Planque S. George S. J. Biol. Chem. 2001; 276: 28314-28320Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). FVIII (0.5 μm) was treated with the E-hapten probes (100 μm) for 4 h, and formation of irreversible adducts was measured by reducing SDS-electrophoresis as described (14Planque S. Taguchi H. Burr G. Bhatia G. Karle S. Zhou Y.X. Nishiyama Y. Paul S. J. Biol. Chem. 2003; 278: 20436-20443Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar).E-FVIII and FVIII Binding—Abs were incubated with electrophilic or control polypeptides devoid of electrophilic groups in 10 mm HEPES, pH 7.5, 150 mm NaCl, 0.025% Tween 20 (HEPES/Tween) at 37 °C. The reaction mixtures were boiled (5 min) in 2% SDS and 3.3% 2-mercaptoethanol and subjected to SDS-electrophoresis. Adducts were detected and quantified in gel blots with peroxidase-conjugated streptavidin or goat anti-human IgG (Fc and κ/λ chain-specific, 1:1000; Sigma (16Paul S. Planque S. Zhou Y.X. Taguchi H. Bhatia G. Karle S. Hanson C. Nishiyama Y. J. Biol. Chem. 2003; 278: 20429-20435Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar)). ELISAs were done using microtiter plates (Nunc) coated with 0.1 ml of FVIII, E-FVIII, C2, E-C2 (1 μg/ml), or synthetic FVIII peptides (4 μg/ml) in 100 mm NaHCO3, pH 9.5, and blocked with 5% skimmed milk in 10 mm sodium phosphate, pH 7.4, 137 mm NaCl, 2.7 mm KCl, 0.05% Tween 20 (PBS/Tween). Ab binding was measured using peroxidase-conjugated goat anti-human IgG as above or goat anti-human Fab (Sigma) followed by peroxidase-conjugated rabbit anti-goat IgG (Pierce) (16Paul S. Planque S. Zhou Y.X. Taguchi H. Bhatia G. Karle S. Hanson C. Nishiyama Y. J. Biol. Chem. 2003; 278: 20429-20435Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Binding to biotinylated E-(2303–2332) was measured similarly using streptavidin-coated plates (1 μg/ml). To measure irreversible binding, Abs were permitted to bind the immobilized antigens; the fluid was removed, and the wells were incubated for 30 min in PBS/Tween without (total binding) or with 2% SDS (SDS-refractory binding), followed by washing with PBS/Tween (16Paul S. Planque S. Zhou Y.X. Taguchi H. Bhatia G. Karle S. Hanson C. Nishiyama Y. J. Biol. Chem. 2003; 278: 20429-20435Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Percent residual binding in SDS-treated wells was (A490, SDS-treated wells) × 100/(A490, PBS/Tween-treated wells). Samples displaying A490 > mean ± 3 S.D. for control IgG (from subject NH1941) were considered positive. Binding rate data were fitted to the equation A490/A490,max = 1 – exp(– Kt), where K is the pseudo-first order rate constant (K = (k3 × [Ab])/Kd); k3 is the first order rate constant for covalent bonding; Kd is the equilibrium dissociation constant for noncovalent binding step; [Ab] is the Ab concentration). t½ was computed as ln2/K. In the immunoadsorption experiment, HA IgG (0.7 μm) was incubated in diluent or biotinylated E-FVIII (0.1 μm) for 20 h, and the reaction mixture (0.04 ml) was incubated for 1 h with immobilized streptavidin in spin columns (30 μl of settled gel, UltraLink Plus columns, Pierce). The unbound fraction and three washes (0.05 ml each) were pooled and assayed for binding to FVIII and E-FVIII by ELISA.Clotting Factor Assays—FVIII and E-FVIII cofactor activity was determined using the Diapharma Coamatic FVIII kit® as instructed by the manufacturer using 0.045-ml solutions of the proteins in HEPES/Tween. The method measures the ability of FVIIIa generated by thrombin cleavage to form the FIXa tenase complex responsible for FXa generation, which in turn hydrolyzes the chromogenic substrate N-α-Z-d-Arg-Gly-Arg-p-nitroanilide. To measure FVIII inhibitor activity of Abs, IgG preparations from patients HA1828 (0.2 mg/ml), HA2222 (0.1 mg/ml), and HA3112 (0.1 mg/ml) were incubated with diluent or the electrophilic FVIII analogs in HEPES/Tween (0.1 ml; 20 h, 37 °C). Unbound electrophilic analogs in the reaction mixtures (0.1 ml) were removed by chromatography on protein G-Sepharose (0.04 ml of settled gel packed in Micro Biospin columns, Bio-Rad). The columns were washed with 5 ml of 50 mm Tris-HCl, pH 7.4, 0.1 mm CHAPS. Bound IgG was eluted with 0.1 m glycine, pH 2.7, 0.1 mm CHAPS (0.2 ml) in tubes containing 0.01 ml of 1 m Tris base, pH 9. The FVIII inhibitory activity of eluates was determined using the Coamatic assay or the one-stage activated partial thromboplastin time (APTT) clotting assay using APTT-SP reagent (29Ten Boekel E. Bock M. Vrielink G.J. Liem R. Hendriks H. Kieviet W.D. Thromb. Res. 2007; 12: 361-367Abstract Full Text Full Text PDF Scopus (34) Google Scholar) (Instrumentation Laboratory) and an ACL300 plus coagulometer (Instrumentation Laboratory) according to the manufacturer's instructions. The standard curve was constructed from the clotting times of reference FVIII-containing plasma diluted in FVIII-depleted plasma (both from George King Bio-Medical, Inc.). Prior to the chromogenic FVIII inhibitor assay, FVIII (0.2 IU/ml, 0.025 ml) was incubated with the IgG eluates from protein G columns (0.025 ml; 1 h). Prior to the APTT clotting assay, pooled plasma from normal subjects (0.06 ml) was incubated with the IgG eluates (0.06 ml; 2 h). The concentrations of the FVIII inhibitory IgG in these assays yielded FVIII inhibition in the linear range of the inhibition curve (25–75% residual activity of reference FVIII-containing plasma).RESULTSE-FVIII and E-C2—Multiple electrophilic phosphonate groups were placed on FVIII and C2 Lys residues (E-FVIII, 52 mol/mol; E-C2, 7 mol/mol; total available Lys residues, respectively, 158 and 9; Fig. 1A), producing diverse electrophilic epitopes. Reducing SDS-electrophoresis and silver staining of E-C2 indicated a single band with mass similar to underivatized C2 (18.6 kDa; Fig. 1B, lane 3). SDS gels of E-FVIII revealed major 225- and 86-kDa bands (respectively, intact FVIII and FVIII light chain), minor ∼96–200-kDa bands corresponding to known proteolytic FVIII fragments (3Lacroix-Desmazes S. Moreau A. Sooryanarayana Bonnemain C. Stieltjes N. Pashov A. Sultan Y. Hoebeke J. Kazatchkine M.D. Kaveri S.V. Nat. Med. 1999; 5: 1044-1047Crossref PubMed Scopus (174) Google Scholar, 30Bhopale G.M. Nanda R.K. J. Biosci. 2003; 28: 783-789Crossref PubMed Scopus (29) Google Scholar), and smeared aggregate bands (nominal mass values ∼350 and 580 kDa close to the loading position; Fig. 1B, lane 1). Other than the aggregates, these bands were present in underivatized FVIII obtained from the supplier (Fig. 1B, lane 2). The aggregates constituted 21–32% of the total silver-stainable protein present in three preparations of E-FVIII examined.We reported recently the presence of nucleophilic sites in various nonenzymatic proteins, evident from their covalent reaction with small molecule phosphonate diester compounds (E-haptens) containing a positive charge neighboring the electrophilic phosphorus atom (31Nishiyama Y. Mitsuda Y. Taguchi H. Planque S. Hara M. Karle S. Hanson C.V. Uda T. Paul S. J. Mol. Recognit. 2005; 18: 295-306Crossref PubMed Scopus (16) Google Scholar). The presence of a naturally occurring nucleophilic site(s) in FVIII was suggested by the formation of a major 225-kDa adduct and a faint 86-kDa adduct of FVIII treated with a positively charged E-hapten-1 (Fig. 1C, lane 1). Only faint adduct bands were observed in reaction mixtures containing the poorly electrophilic control phosphonic acid (E-hapten-2) or the neutral phosphonate E-hapten-3. Control ovalbumin, a protein with minimal nucleophilic reactivity (31Nishiyama Y. Mitsuda Y. Taguchi H. Planque S. Hara M. Karle S. Hanson C.V. Uda T. Paul S. J. Mol. Recognit. 2005; 18: 295-306Crossref PubMed Scopus (16) Google Scholar), did not form detectable E-hapten-1 adducts. The E-FVIII aggregates may therefore be interpreted to derive from intermolecular covalent bonding between the electrophilic phosphonate and a naturally occurring nucleophilic site(s) of FVIII.To assess antigenic integrity, the binding of E-FVIII and E-C2 by Abs was determined by ELISA using affinity-purified IgG preparations from eight HA patients positive for FVIII inhibitory antibodies. All of the IgG preparations at 25 μg/ml displayed E-FVIII and E-C2 binding exceeding the mean ± 3 S.D. values for control IgG from the nonhemophilia subject (code NH1941; A490, respectively, 0.01 ± 0.01 and 0.07 ± 0.01). The IgG concentrations yielding an A490 value of 0.25 computed from dose-response curves are reported in Table 1. Fast protein liquid chromatography-gel filtration of E-FVIII permitted separation of an aggregate peak eluting close to the column void volume (retention time 18.3–21.6 min; 13% of protein loaded on the column) from unaggregated E-FVIII (retention time 43.3–46.0 min). The aggregates did not display reduced reactivity with IgG from an HA patient (HA1828) compared with unfractionated E-FVIII (A490 1.22 ± 0.02 and 0.85 ± 0.10, respectively; 30 μg/ml IgG, 93 ng/well E-FVIII aggregates or unfractionated FVIII), suggesting that the Ab-reactive epitopes aggregates are present in the aggregates. The ability of E-FVIII to consume anti-FVIII Abs was measured following solution phase reactions of HA1828 IgG with biotinylated E-FVIII. Immune complexes were removed using immobilized streptavidin, and free Abs in the unbound fraction were measured. This procedure resulted in es" @default.
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• W2073294017 title "Covalent Inactivation of Factor VIII Antibodies from Hemophilia A Patients by an Electrophilic FVIII Analog" @default.
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