Blood clotting factor IX BM Nagoya. Substitution of arginine 180 by tryptophan and its activation by alpha-chymotrypsin and rat mast cell chymase.
Factor IX BM Nagoya (IX Nagoya) is a natural mutant of factor IX responsible for severe hemophilia B. A patient with this mutant is characterized by a markedly prolonged ox brain prothrombin time. IX Nagoya was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that treatment of IX Nagoya with factor XIa/Ca2+ resulted in cleavage only at the Arg145-Ala146 bond. Reversed-phase high performance liquid chromatography of a trypsin digest of IX Nagoya showed an aberrant peptide, which was further digested with proteinase Asp-N. Primary structure analysis of one of the Asp-N peptides revealed that Arg180 is replaced by Trp. An essentially complete (99%) amino acid sequence of IX Nagoya was obtained by sequencing fragments derived from a lysyl endopeptidase digest in which no other substitutions in the catalytic triad or substrate binding site were found. We also found that IX Nagoya is activated by alpha-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VIII, phospholipids, and Ca2+. These results indicate that the substitution of Arg180 by Trp impairs the cleavage by factor XIa required for activation of this zymogen and that the substitution causes hemophilia BM.
Suehiro
K
K
First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Kawabata
S
S
Miyata
T
T
Takeya
H
H
Takamatsu
J
J
Ogata
K
K
Kamiya
T
T
Saito
H
H
Niho
Y
Y
Iwanaga
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D053818:Chymases; D002918:Chymotrypsin / Q000378:metabolism*; D004789:Enzyme Activation; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008407:Mast Cells / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D051381:Rats; D012016:Reference Values; D012697:Serine Endopeptidases / Q000378:metabolism*; D014364:Tryptophan
C062013:factor IX BM Nagoya; D014364:Tryptophan; D005164:Factor IX; D001120:Arginine; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D053818:Chymases