Direct Characterization of Factor VIII in Plasma: Detection of a Mutation Altering a Thrombin Cleavage Site (arginine-372 → histidine)

• Published in: Proceedings of the National Academy of Sciences - PNAS 1989-06, Vol.86 (11), p.4277-4281
• Main Authors: Arai, M., Inaba, H., Higuchi, M., Antonarakis, S. E., Kazazian, H. H., Fujimaki, M., Hoyer, L. W.
• Format: Article
• Language: English
• Subjects: Antigens; Arginine; Base Sequence; Biological and medical sciences; Blood plasma; DNA; Exons; Factor VIII - analysis; Factor VIII - genetics; Factor VIII - isolation & purification; Genes; Genetic mutation; Hematologic and hematopoietic diseases; Hemophilia; Hemophilia A; Hemophilia A - blood; Hemophilia A - genetics; Histidine; Humans; Immunoblotting; man; Medical sciences; Molecular Sequence Data; Monoclonal antibodies; Mutation; Platelet diseases and coagulopathies; Polymerase chain reaction; Sequencing; thrombin; Thrombin - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.11.4277

An immunoadsorbent method has been developed for the direct analysis of normal and variant plasma factor VIII. Using this method, the molecular defect responsible for mild hemophilia A has been identified for a patient whose plasma factor VIII activity is 0.05 unit/ml, even though the factor VIII antigen content is 3.25 units/ml. Although the variant factor VIII has an apparently normal molecular mass and chain composition, the 92-kDa heavy chain accumulates when the variant protein is incubated with thrombin and the 44-kDa heavy chain fragment cannot be detected. In contrast, thrombin cleavage of the 80-kDa light chain to the 72-kDa fragment is normal. As these data indicate a loss of factor VIII cleavage by thrombin at arginine-372, the genetic defect was determined by polymerase-chain-reaction amplification of exon 8 of the factor VIII gene and direct sequencing of the amplified product. A single-base substitution (guanine → adenine) was identified that produces an arginine to histidine substitution at amino acid residue 372. These data identify the molecular basis of an abnormal factor VIII, ``factor VIII-Kumamoto,'' that lacks procoagulant function because of impaired thrombin activation.