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The A2 domain (residues 373-740) of human blood coagulation factor VIII (fVIII) contains a major epitope for inhibitory alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine fVIII and mapped a major determinant of the A2 epitope by using a series of active recombinant hybrid human/porcine fVIII molecules. Hybrids containing a substitution of porcine sequence at segment 410-508, 445-508, or 484-508 of the human A2 domain were not inhibited by a murine monoclonal antibody A2 inhibitory, mAb 413, whereas hybrids containing substitutions at 387-403, 387-444, and 387-468 were inhibited by mAb 413. This indicates that the segment bounded by Arg484 and Ile508 contains a major determinant of the A2 epitope. mAb 413 did not inhibit two more hybrids that contained porcine substitutions at residues 484-488 and 489-508, indicating that amino acid side chains on both sides of the Ser488-Arg489 bond within the Arg484-Ile508 segment contribute to the A2 epitope. The 484-508, 484-488, and 489-508 porcine substitution hybrids displayed decreased inhibition by A2 inhibitors from four patient plasmas, suggesting that there is little variation in the structure of the A2 epitope in the inhibitor population.

We have established an ELISA for detecting thrombin cleavage of the FVIII light chain at Arg1689. The method used a coating alloantibody which recognized amino acid residues 2248-2312 in the C2 domain, together with a second monoclonal antibody, NMC-VIII/10, which recognized residues 1675-1684 in the amino-terminal region of the light chain. FVIII antigen (FVIII:Ag) was measured after treatment of plasma with various concentrations of thrombin. The FVIII:Ag of normal plasma was reduced in a dose-dependent manner by the thrombin, falling to 28% in the presence of 100 U/ml enzyme. The concentration of thrombin that achieved 50% reduction (IC50) was approximately 1.0 U/ml. The plasma of four haemophilia A positive (A+) and two haemophilia A reduced (AR) patients were analysed. The IC50 of all patients was more than 1.0 U/ml, indicating that thrombin cleavage of the FVIII light chain was defective. One haemophilia A+ plasma did not respond to thrombin in this ELISA system. The patient (TI) was a haemophiliac with FVIII coagulant activity of 0.04 U/ml and FVIII:Ag of 1.78 U/ml. In addition, immunoblotting of the purified FVIII from TI showed that thrombin cleavage of the 80 kilodalton (kD) light chain was impaired. The patient's DNA was amplified using the polymerase chain reaction with a set of synthetic oligonucleotide primers spanning amino acid residues 1646-1714. Sequence analysis of the amplified DNA fragments revealed a cytosine to thymine transition, converting an arginine 1689 to cysteine. This abnormal FVIII was designated as FVIII Hiroshima. Our ELISA system is a simple and useful method of evaluating the proteolytic cleavage by thrombin at Arg1689.

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

Anti-factor VIII (FVIII) inhibitor alloantibodies from 11 patients with hemophilia A, along with five anti-FVIII neutralizing monoclonal antibodies, were examined for differences in their reactivities with the A2 and C2 domains of human and porcine FVIII. None of the patients had been previously treated with porcine FVIII. Six inhibitors which specifically recognized the human FVIII C2 domain bound to both the 76-kDa porcine FVIII light chain and its 69-kDa proteolyzed fragments, showing cross-reactivity against porcine FVIII between 33 and 100%. Two A2-specific inhibitors did not react with porcine FVIII. The cross-reactivity was low (0-0.5%). The inhibitors recognizing both C2 and A2 reacted with the 76- and 69-kDa bands of porcine FVIII light chain, with cross-reactivity of between 11 and 33%. Monoclonal antibodies recognizing A1 (C-5) and A2 (JR8) did not react with the porcine FVIII. No anti-porcine FVIII neutralizing activity was detected in these antibodies. Monoclonal antibodies to the amino-terminal portion of A3 (NMC-VIII/10 and C-2) poorly reacted with the 76-kDa band, the cross-reactivities being 0 and 0.5%, respectively. NMC-VIII/5 recognizing C2 which competes with the C2-specific inhibitor, reacted with both the 76- and 69-kDa fragments showing cross-reactivity of 13%. These findings suggest that porcine A2 is antigenically different from human A2. The A2-specific inhibitor is a useful indicator for therapy with porcine FVIII.

Using enzyme-linked immunosorbent assay (ELISA), we measured anti-factor VIII immunoglobulin G (IgG), IgG4 and IgM in serum samples obtained from hemophilia A patients treated with a recombinant factor VIII (rFVIII) preparation (Kogenate). Twelve pre- and post-treatment serum samples from 3 previously untreated patients who developed an inhibitor (alloantibody) were evaluated. Both rFVIII and plasma-derived factor VIII (pdFVIII) were used as antigen. Serum samples were diluted 37.5-fold. For the measurement of IgM antibody, protein A Sephadex suspension was added to the patients' serum samples and the supernatant was assayed. Anti-human IgG, IgG4, and IgM monoclonal antibodies labelled with peroxidase were added and absorbance at 450nm was determined. The reactivity of the IgG antibody with rFVIII and pdFVIII was extremely low. Samples containing the IgG4 inhibitor with a neutralizing activity of approximately 7.5 Bethesda units (BU)/ml resulted in absorbance values of 0.451-0.551, thus demonstrating a considerably high degree of sensitivity. The correlation between the neutralizing activity and reactions of the IgG4 antibody with rFVIII and pdFVIII antigens was high, with correlation coefficients of 0.912 and 0.966, respectively. Furthermore, the correlation coefficient between the measured absorbance values for the antibody reacted with pdFVIII and rFVIII was 0.961. No correlation was found between the reactivity of IgM to rFVIII and pdFVIII.

The authors propose a new model on cleavage fracture toughness of steels. Fracture toughness of steels is controlled by the weakest link mechanism, so it has intrinsic scatter. Beremin proposed Weibull stress through probabilistic fracture initiation model based on the weakest link theory, and it has been widely used for describing fracture toughness scatter. In his model, only propagation of micro crack is considered. However, micro crack nucleation should also be incorporated in order to estimate fracture toughness distribution more accurately. Bordet introduced micro crack initiation to the Beremin model, in which probability of micro crack nucleation is assumed proportional to plastic strain. Some previous studies have shown, however, that micro crack nucleation probability increases non-linearly with plastic strain. Then, we developed a new probabilistic model by introducing micro crack nucleation probability as a non-linear function of plastic strain. Furthermore, the authors developed a new method for obtaining Weibull parameters, in which not only distribution of fracture toughness values but also location of fracture initiation sites are considered through a newly developed likelihood function. We conducted fracture toughness tests with different specimen configurations and carried out convergence calculation for determining Weibull parameters by applying the likelihood function mentioned above. As a result of the calculation, the authors confirmed that the present model can obtain the Weibull parameters less sensitive to the specimen configurations and simulate distribution of fracture toughness more accurately than the previous models.

We isolated a highly transformable thermophile, Bacillus stearothermophilus SIC1, which exhibited the following features. The growth temperature ranged from 45 to 65°C in L broth. The maximum cell concentration in 2L broth (2% tryptone, 1% yeast extract, 0.5% NaCl, pH 7.2) was determined as an optical density at 660 nm of 7.8, and the generation time was 11 min at 60°C. Strain SIC1 was a prototroph and was transformed by the protoplast procedure not only with repB plasmids (high-copy-number plasmids such as pTB913 and pUB110) but also with repA plasmids (low-copy-number plasmids such as pTB53). Transformation efficiencies with repB and repA plasmids were about 2 × 10 6 to 5 × 10 6 and 5 × 10 4 transformants per μg of DNA, respectively. The transformant carrying plasmid pTB913Y/K could grow at 63°C in the presence of kanamycin. The regeneration frequency of protoplasts was 60%, and only 1 day was needed for regeneration at 55°C.

Four monoclonal antibodies against factor VIII, NMC-VIII/1, NMC-VIII/2, NMC-VIII/3 and NMC-VIII/4, were produced. The first three antibodies were of the IgG1 immunoglobulin subclass, while the fourth was IgM. The affinity of each antibody for factor VIII was high and antifactor VIII clotting activity was detected in NMC-VIII/2, NMC-VIII/3 and NMC-VIII/4. NMC-VIII/1 had no inhibitory effect on factor VIII clotting activity. Immunoblotting using purified intact and thrombin-treated factor VIII identified the antibodies’factor VIII polypeptide specificities. With thrombin-treated factor VIII the factor VIII fragments for NMC-VIII/1 and NMC-VIII/2 were 80 kDa and 54 kDa, respectively, while both NMC-VIII/3 and NMC-VIII/4 recognised a 44 kDa fragment. With intact factor VIII, antibodies NMC-VIII/2-4 bound to polypeptides larger than 90 kDa, especially NMC-VIII/2, which reacted with a chain of 330 kDa thought to be a mature form of factor VIII protein. We also detected a mature form of factor VIII in hepatic sinusoidal endothelial cells by immunoperoxidase staining. Identification of the factor VIII polypeptides bearing these fragments enabled us to clarify the localization of the factor VIII thrombin cleavage site, and suggested the existence of factor VIII in hepatic sinusoidal endothelial cells.

In order to clarify human factor VIII inhibitor epitopes to factor VIII (F. VIII), we analyzed the inhibitor IgG developed in the patients with hemophilia A and autoimmune disease using immunoblotting of purified F. VIII and thrombin-degraded F. VIII. IgG fractions were obtained from 6 cases of severe hemophilia A and one autoimmune disease. The titer of the inhibitor plasma ranged from 50 to 3,000 Bethesda units/ml. Purification of F. VIII from commercial F. VIII concentrate was performed by immunoadsorbent column chromatography using anti-von Willebrand factor monoclonal antibody and anti-fibrinogen, anti-fibronectin and anti-IgM goat antibodies and subsequently by Aminohexyl Sepharose column chromatography essentially according to the method of Fulcher et al.. The specific activity of the purified F. VIII was 2,700 units/mg. On sodium-dodecylsulfate (SDS) 5 to 10% gradient polyacrylamide gel electrophoresis (PAGE), 80 kDa of a main fragment and a series of 90 to 210 kDa fragments were visualyzed by Coomassie blue staining. Immunoblotting of 5-10 ug of each of unreduced purified F. VIII and thrombin-degraded F. VIII. followed by reaction with inhibitor IgG samples, monoclonal antihuman IgG-3 and IgG-4 antibodies and radiolabeled rabbit antimouse IgG.All inhibitor IgG samples reacted with both purified F. VIII and thrombin-degraded F. VIII. The pattern of reactivity of inhibitor antibodies was divided into three groups; 1) inhibitor IgG which reacted with 80 and 70 kDa derived from carboxy-terminus of the F. VIII molecule, 2) inhibitor IgG which reacted with 90 to 210 kDa and 54 and/or 44 kDa derived from amino-terminus of the F. VIII, and 3) inhibitor IgG which reacted with both of N- and C-terminus of the F. VIII. One inhibitor IgG belonged to group 3 strongly reacted with a series of higher molecular polypeptides of F. VIII ranged from 180 .to 210 kDa. One autoantibody IgG from the patient with autoimmune disease was also belonged to group 3. There was no relationship between the titer and the pattern of reactivity of each inhibitor plasma. In summary, there is a heterogeneity of F. VIII inhibitor epitopes to F. VIII molecules.