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Hemophilia A is genetically very heterogeneous because disease-causing mutations involving deletions, point mutations, insertions, and inversions are scattered throughout the factor VIII gene. Of these mutations, inversions, which are intrachromosomal recombinations between int22h-1 (intron 22 homologous region 1) and 1 of 2 other extragenic copies located 500 kilobases upstream, are the more frequently found defects, especially in patients with severe hemophilia A. Reportedly, approximately half of all severe hemophilia A patients have inversions in intron 22. A group of unrelated patients from the middle of Japan with severe hemophilia A were screened by Southern blot analysis for gene inversions. Forty-two of 100 severely affected patients presented factor VIII gene rearrangements. Of these patients, 36 exhibited the distal type of inversion, and 6 exhibited the proximal type. No other variant type of recombination was observed. In this study, neither the prevalence of inhibitor development against factor VIII nor the frequency of sporadic cases in the group presenting gene inversions was significantly different from that in the group without chromosomal inversions. Southern blot analysis successfully detected a carrier in a hemophilia family for which no patient was available. Genetic counseling of patients with severe hemophilia A and their families will be considerably improved, because the inversions occur in 42% of the Japanese patients with severe hemophilia.

症例は8歳,男性.腹痛,嘔吐を主訴に当院小児科を受診した.浣腸後の観便および腹部超音波検査により腸重積と診断し,バリウムによる高圧洗腸整復を行った.整復後の腹部単純X線および注腸X線検査上,大部分の大腸を左腹部に,回盲部を腹部正中に認め,腸回転異常症が疑われた.翌日,再度腹痛が出現し,腹部超音波検査にて腸重積の再発と診断された.手術加療目的にて当科紹介となり,同日緊急手術を施行した.結腸結腸型および回腸盲腸型の2カ所の腸重積を認め,ともに徒手整復した.整復後,上腸間膜動脈の右側に十二指腸空腸ループを,また盲腸結腸ループをその左側に認めた. 90°回転型の腸回転異常症と診断し,線維性癒着(Ladd靭帯)の切離と虫垂切除術を施行した.腸回転異常症の多くは新生児期に発見されるが,乳児期以降に繰り返す腹痛と嘔吐が認められた場合本症を念頭におき,検査を進めることが必要である.

Factor VIII binds to activated platelets and contributes to the tenase complex assembled on the platelet membrane surface. We have examined the role of platelet von Willebrand factor in the binding of factor VIII to platelets using a platelet captured enzyme-linked immunosorbent assay. Purified factor VIII bound to activated normal platelets in a dose dependent manner. Factor VIII also bound to platelets obtained from a patient with Type 2N von Willebrand disease, although in this case the binding was reduced to approximately 50% of that seen with control platelets. Furthermore, factor VIII bound to Type 3 von Willebrand disease platelets in the absence of detectable von Willebrand factor. In this instance the binding reaction appeared to be approximately 30% of that seen with the same number of normal platelets. An anti-A3 domain monoclonal antibody, NMC-VIII/10, which recognizes the amino-terminal acidic region of the factor VIII light chain, and an anti-C2 domain monoclonal antibody, NMC-VIII/5, which also moderates the binding of factor VIII to phosphatidylserine, inhibited the association between factor VIII and platelets. Inhibition was more remarkable with NMC-VIII/5 than with NMC-VIII/10 but not complete. The findings suggest that the binding of factor VIII to activated platelets is not based on a single ligand-receptor relationship, although a predominant role exists for the platelet von Willebrand factor. Furthermore, both the amino-terminal acidic region of the A3 domain and the C2 domain participate in the binding of factor VIII to activated platelets.

We report the successful treatment of a subepiglottic hematoma accompanied by progressive dyspnea and vocal difficulty in a 17-year-old hemophilia A patient with inhibitor. The inhibitor titer to human factor VIII was 16 Bethesda Units (B. U.)/ml, whereas that to porcine factor VIII was 5 B. U./ml (cross-reactivity: 31.3%). Then, he was treated by 19, 885u (310.7/kg) (15, 000u for inhibitor neutralizing dose plus about 5, 000u for factor VIII activity (FVIII: C) incremental dose up to 100%) of porcine factor VIII concentrate (MTI-9002; Hyate: C) in a single infusion. The vocal difficulty and respiratry obstruction started disappearing 2 hours later and completely disappeared 2 days later. The recovery of the FVIII: C at 30min and 60min after infusion was 130 and 110%, respectively. Although shivering and a transient and mild decrease in the platelet counts occurred at 30min after the end of the infusion, the symptom disappeared by using hydrocortisone infusion 30min later and the thrombocytopenia recovered within 8 hours. Anamnestic response appeared 7 days after infusion. The peak anti-human and anti-porcine factor VIII titers were 448 and 160 B. U./ml at week 4, respectively. Inhibitor titers decreased to 220 and 84 B. U./ml at week 8. We concluded that only a single but a large infusion more than the calculated neutralizing dose of porcine factor VIII concentrates is effective for lifethreatening hemorrhages in hemophillia A patients with low cross-reactivity.

SummaryWe report the development of a FVIII inhibitor in a patient with severe, cross reacting material reduced (CRMR) haemophilia A. The level of Factor VIII antigen (FVIII:Ag) measured by ELISA using anti- C2 monoclonal and alloantibodies was 1.9 U/dl. This baseline FVIII:Ag level was increased to 8.3 U/dl after administration of DDAVP. The anti-FVIII inhibitor titer was 2.9 Bethesda U/ml. DNA analysis showed a large deletion of the FVIII gene from exon 4 to 7, corresponding to amino acid residues 111-317 included within the A1 domain. The size of the gene deletion was approximately 28 kb. 5' and 3' breakpoints were identified by sequencing in intron 3 and intron 7, respectively. FVIII mRNA was detected in the patient’s peripheral lymphocytes and the deletion spanning exon 4 to 7 was confirmed at the RNA level. Immunoprecipitation experiments using 125I labeled A1, A2 and light chain demonstrated that the inhibitor reacted only with the 54 kDa A1 domain. The inhibitor activity was more than 95% neutralized by A1 domain polypeptide. Our findings suggest a close relationship between the inhibitor epitope and the specific gene deletion with regard to the pathogenesis of the inhibitor in this patient.

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.

Haemophilia A is the most common X-linked blood coagulation disorder; it is caused by deficiency of factor VIII activity (FVIII:C). Half of the affected patients do not have detectable levels of FVIII protein in their plasma, whereas about 5% have normal levels of the FVIII antigen (FVIII:Ag) (> 50 u/dl), and are called cross-reacting material (CRM) positive (CRM + or A + ). About 45% of patients have reduced levels of the FVIII:Ag (1-50u/dl), classified as CRM reduced (CRM R or A R ). We screened the FVIII gene of 13 Japanese patients (five CRM + and eight CRM R ) by single-strand conformation polymorphism, and identified 11 different mutations in 13 patients by analysing all 26 exons (Trp255Cys, Tyr473Cys, Gly479Arg, Arg531His, Thr667Arg, Argl689Cys, Argl941Gln, Arg2150His, Arg2159Cys, Thr2245Ala and Gly2285Val). Seven mutations were identified in the A domains (four in the A2 domain). All the mutations are point mutations resulting in missense codons. Four mutations (Trp255Cys, Thr667Arg, Thr2245Ala and Gly2285Val) have not been described previously.