Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).

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Hemophilia A is an X chromosome-linked recessive disorder resulting in defective or deficient factor VIII (FVIII) molecules, which, in its severe form, is a life-threatening and crippling hemorrhagic disease. Infusion of homologous FVIII to patients with severe hemophilia A results, in 25% of patients, in the emergence of alloantibodies against FVIII (inhibitors)( ref. 1) that inhibit FVIII procoagulant activity by steric hindrance of the interaction of FVIII either with stabilizing molecules, with molecules essential for its activity or with activating molecules. Here, we report on the proteolysis of FVIII by alloantibodies of two patients with severe hemophilia A, demonstrating a previously unknown mechanism by which FVIII inhibitors may prevent the pro-coagulant function of FVIII. The kinetic parameters of FVIII hydrolysis indicate a functional role for the catalytic immune response in the inactivation of FVIII in vivo. The characterization of alloantibodies against FVIII as site-specific proteases may provide new approaches to the treatment of FVIII inhibitors.

Spontaneous inhibitors of factor VIII (FVIII) are pathogenic IgG autoantibodies of restricted isotypic heterogeneity found in the plasma ofpatients presenting with bleeding episodes and low levels of FVM. We now report the presence of a natural FVIII-neuralizing activity in 85 of500 plasma samples (17%) from healthy donors. FVIII-inhibitory activity was present in F(ab')2 fragments of purified IgG and was dosedependent. The titer of anti-FVIII antibodies in normal plasma ranged between 0.4 (threshold of detection) and 2.0 Bethesda units. Anti-FVIII IgG was also detected in normal plasma by using an ELISA. Anti-FVIH antibodies from healthy individuals did not exhibit restricted isotypic heterogeneity. Mean levels of FVIII activity did not differ significantly between individuals with and without detectable anti-FVIU antibodies in plasma. Natural anti-FVIII IgG inhibited FV1I activity in pools of normal plasma and in plasma of certain donors in the pool but did not inhibit FVII activity in autologous plasma. These observations demonstrate that polyclonal IgG antibodies against procoagulant FVHI are present in healthy individuals. The antibodies are natural IgG autoantibodies and/or antibodies directed against epitopes associated with a so far unidentified allotypic polymorphism of the human FVIII molecule.The presence of autoantibodies to factor VIII (FVIII) has so far exclusively been investigated in the plasma of patients with spontaneously occurring severe bleeding episodes in whom circulating autoantibodies are associated with low levels of FVIII. The inhibitors are IgG autoantibodies of restricted isotypic heterogeneity (1) that bind to selective sites on the FVIII molecule, resulting in inhibition of FVIII procoagulant activity (2-4).Natural IgM and IgG autoantibodies reacting with a wide array of serum proteins and hormones, and nuclear and cellular antigens are found in normal human serum (5). In the present study, we found that the plasma of 17% of 500 healthy blood donors contained FVIII-neutralizing activity. Anti-FVIII activity was present in F(ab')2 fragments from the IgG fraction of the plasma samples with inhibitor activity. FVIIIneutralizing activity of anti-FVIII IgG from a given donor differed with the individual source of FVIII used in the neutralization assay. Blood was collected in 0.13 M sodium citrate, 1:9 (vol/vol), and immediately centrifuged at 2500 x g for 15 min at room temperature. Plasma was divided and stored at -800C for no longer than 4 weeks.Pooled plasma and IgG from the 420 healthy donors lacking detectable anti-FVIII activity in plasma were used as negative controls.FVIII Assays. FVIII activity was measured in a one-stage assay by a manual activated partial thromboplastin reagent (APTT) method using human plasma depleted of FVIII (containing <1% FVIII and normal levels of factor V; coagulation time in APTT, >200 sec) (General Diagnostics, Morris Plains, NJ) as substrate and human brain partial thromboplastin and Kaolin (5 mg/ml) as activators. The clotting time of four ...

Plasma samples obtained from a patient 6 wk,