title: Direct characterization of factor VIII in plasma: detection of a mutation altering a thrombin cleavage site (arginine-372----histidine).
abstract: 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.
authors.affiliation: Holland Laboratory, American Red Cross, Rockville, MD 20855.
authors.forename: M
authors.initials: M
authors.lastname: Arai
authors.forename: H
authors.initials: H
authors.lastname: Inaba
authors.forename: M
authors.initials: M
authors.lastname: Higuchi
authors.forename: S E
authors.initials: SE
authors.lastname: Antonarakis
authors.forename: H H
authors.initials: HH
authors.lastname: Kazazian
authors.forename: M
authors.initials: M
authors.lastname: Fujimaki
authors.forename: L W
authors.initials: LW
authors.lastname: Hoyer
mesh_terms: D001120:Arginine; D001483:Base Sequence; D005169:Factor VIII / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D005796:Genes; D006467:Hemophilia A / Q000097:blood* / Q000235:genetics; D006639:Histidine; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation; D013917:Thrombin / Q000378:metabolism
chemical_list: D006639:Histidine; D005169:Factor VIII; D001120:Arginine; D013917:Thrombin