2003; baxter - Supplements - Haematologica

50S. Lacroix-Desmazes et al.bodies and that of FVIII inhibitory IgG. Nevertheless,patients were heterogeneous when ratesof FVIII hydrolysis were compared with inhibitoryactivity: thus, 11 patients presented with FVIIIinhibitory activity in plasma but with no FVIIIhydrolyzingactivity among purified IgG; otherspresented with high rates of FVIII hydrolysis andlow inhibitory activity in plasma; a few patientshad both high rates of FVIII hydrolysis and highinhibitory activity in plasma. A moderate correlationbetween the rate of FVIII hydrolysis andthe FVIII-neutralizing activity in plasma mayreflect the complex and multiple mechanismsthat participate simultaneously in inactivatingFVIII in vivo.The cleavage fragments generated among IgGof different patients ranged between 70 kD andless than 30 kD. Smaller digestion fragmentswere not detected under the experimental conditionsused (i.e., 10% SDS-PAGE). N-terminalsequencing of the generated digestion fragmentsof FVIII allowed the identification of the cleavagesites in the case of four patients with FVIIIhydrolyzingantibodies. Overall, the cleavage sitesare evenly spread throughout the A1, A2, B, A3and C1 domains of the FVIII molecule and arelocated after an arginine or a lysine in 85% of thecases (unpublished data). Identification of thecleavage sites on the available 3 dimensionalmodels of the A1, A2, A3 and C1 domains indicatesthat most cleavage sites are located on theouter core of the molecule and are readily accessibleby the antibodies. Using both radiolabeledFVIII and synthetic peptides coupled to a fluorescentdye as substrates, we have calculated thekinetic parameters of the anti-FVIII hydrolyticantibodies. Vmax and Km were between 6 and600 fmol/min and 10 and 1500 µM, respectively.The estimated catalytic efficiency was in therange of 0.6 or 60 M -1 .sec -1 (unpublished data). Incalculating the kinetic parameters, we overestimatedthe quantity of hydrolytic IgG, assumingthat all IgG molecules present in the assay arecatalytic. Paul et al. estimated the amount ofantibodies hydrolytic to the vasoactive intestinalpeptide within pools of purified IgG to be equalto 73.4 fmol/mg of IgG (Paul et al., 1989). Thecatalytic efficiency calculated in the case of FVIIIhydrolyzingantibodies may thus be underestimatedby several orders of magnitude.Taken together, our data demonstrate thathydrolytic antibodies are present among anti-FVIII IgG of inhibitor-positive patients withhemophilia A. Hemophilia thus appears as thefirst human disease in which hydrolysis of thetarget molecule by catalytic antibodies mayaccount for the clinical manifestations. We hopethat the identification of molecules that specificallyblock the catalytic activity of anti-FVIII antibodieswill provide novel therapeutic approachesfor patients with inhibitors in the near future.References1. 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Haemophilia A: mutationtype determines risk of inhibitor formation. ThrombHaemost 1995; 74:1402-6.6. Scandella D, Timmons L, Mattingly M, Trabold N, HoyerLW. A soluble recombinant factor VIII fragment containingthe A2 domain binds to some human anti-factorVIII antibodies that are not detected by immunoblotting.Thromb Haemost 1992; 67:665-71.7. Gilles JG, Desqueper B, Lenk H, Vermylen J, Saint-RemyJM. Neutralizing antiidiotypic antibodies to factor VIIIinhibitors after desensitization in patients with hemophiliaA. J Clin Invest 1996; 97:1382-8.8. Lollar P, Parker ET, Curtis JE, Helgerson SL, Hoyer LW,Scott ME, et al. Inhibition of human factor VIIIa by anti-A2 subunit antibodies. J Clin Invest 1994; 93:2497-504.9. Scandella D, Mattingly M, Prescott R. A recombinantfactor VIII A2 domain polypeptide quantitatively neutralizeshuman inhibitor antibodies that bind to A2.Blood 1993; 82:1767-75.10. Saenko EL, Shima M, Rajalakshmi KJ, Scandella D. 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Localization of the binding regionsof a murine monoclonal anti-factor VIII antibody anda human anti-factor VIII alloantibody, both of whichinhibit factor VIII procoagulant activity, to amino acidresidues threonine351-serine365 of the factor VIIIheavy chain. J Clin Invest 1988; 82:123-8.15. Lubahn BC, Ware J, Stafford DW, Reisner HM. Identificationof a F.VIII epitope recognized by a humanhemophilic inhibitor. Blood 1989; 73:497-9.16. Zhong D, Saenko EL, Shima M, Felch M, Scandella D.Some human inhibitor antibodies interfere with factorVIII binding to factor IX. Blood 1998; 92:136-42.17. Arai M, Scandella D, Hoyer LW. Molecular basis of factorVIII inhibition by human antibodies. Antibodies thatbind to the factor VIII light chain prevent the interactionof factor VIII with phospholipid. J Clin Invest 1989;83: 1978-84.18. Jacquemin MG, Desqueper BG, Benhida A, Vander ElstL, Hoylaerts MF, Bakkus M, et al. Mechanism and kinet-haematologica vol. 88(supplement n. 12):september 2003