Blood clotting factor IX B(M) Nagoya. Substitution of arginine 180 by tryptophan and its activation by α-chymotrypsin and rat mast cell chymase

K. Suehiro, S. Kawabata, T. Miyata, H. Takeya, J. Takamatsu, K. Ogata, T. Kamiya, H. Saito, Y. Niho, S. Iwanaga

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Abstract

Factor IX B(M) 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 α-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VII, 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 B(M).

Original languageEnglish
Pages (from-to)21257-21265
Number of pages9
JournalJournal of Biological Chemistry
Volume264
Issue number35
Publication statusPublished - Dec 1 1989

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Suehiro, K., Kawabata, S., Miyata, T., Takeya, H., Takamatsu, J., Ogata, K., Kamiya, T., Saito, H., Niho, Y., & Iwanaga, S. (1989). Blood clotting factor IX B(M) Nagoya. Substitution of arginine 180 by tryptophan and its activation by α-chymotrypsin and rat mast cell chymase. Journal of Biological Chemistry, 264(35), 21257-21265.