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the A1, A2, B, A3and C1 domains of the FVIII molecule and arelocated after an arginine or a lysine in 85% of thecases (unpublished data).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fmol/min and 10 and 1500 µM, respectively.The estimated catalytic efficiency was in therange of 0.6 or 60 M -1 .sec -1 (unpublished data).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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