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

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).