A mutation in the propeptide of Factor IX leads to warfarin sensitivity by a novel mechanism

J Clin Invest. 1996 Oct 1;98(7):1619-25. doi: 10.1172/JCI118956.

Abstract

The propeptide sequences of the vitamin K-dependent clotting factors serve as a recognition site for the enzyme gamma-glutamylcarboxylase, which catalyzes the carboxylation of glutamic acid residues at the NH2 terminus of the mature protein. We describe a mutation in the propeptide of Factor IX that results in warfarin sensitivity because of reduced affinity of the carboxylase for the Factor IX precursor. The proband has a Factor IX activity level of > 100% off warfarin and < 1% on warfarin, at a point where other vitamin K-dependent factors were at 30-40% activity levels. Direct sequence analysis of amplified genomic DNA from all eight exons and exon-intron junctions showed a single guanosine-->adenosine transition at nucleotide 6346 resulting in an alanine to threonine change at residue -10 in the propeptide. To define the mechanism by which the mutation resulted in warfarin sensitivity, we analyzed wild-type and mutant recombinant peptides in an in vitro carboxylation reaction. The peptides that were analyzed included the wild-type sequence, the Ala-10-->Thr sequence, and Ala-10-->Gly, a substitution based on the sequence in bone gamma-carboxyglutamic acid protein. Measurement of C02 incorporation at a range of peptide concentrations yielded a Vmax of 343 cpm/min/reaction for the wild-type peptide, and Vmax values of 638 and 726 for A-10T and A-10G respectively, a difference of only twofold. The Km values, on the other hand, showed a 33-fold difference between wild-type and the variants, with a value of 0.29 microM for wild-type, and 10.9 and 9.50 microM, respectively, for A-10T and A-10G. Similar kinetic experiments showed no substantial differences between wild-type and mutant peptides in kinetic parameters of the carboxylase-peptide complexes for reduced vitamin K. We conclude that the major defect resulting from the Factor IX Ala-l0-->Thr mutation is a reduction in affinity of the carboxylase for the mutant propeptide. These studies delineate a novel mechanism for warfarin sensitivity. In addition, the data may also explain the observation that bone Gla protein is more sensitive to warfarin than the coagulation proteins.

Publication types

  • Case Reports
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Anticoagulants / pharmacology*
  • Anticoagulants / therapeutic use
  • Carbon-Carbon Ligases*
  • European Continental Ancestry Group
  • Exons / genetics
  • Factor IX / genetics*
  • Factor IX / metabolism
  • Humans
  • Kinetics
  • Ligases / metabolism
  • Male
  • Microsomes / metabolism
  • Middle Aged
  • Molecular Sequence Data
  • Mutation*
  • Protein Precursors / genetics*
  • Protein Precursors / metabolism
  • Protein Processing, Post-Translational
  • Sequence Alignment
  • Vitamin K / metabolism
  • Warfarin / pharmacology*
  • Warfarin / therapeutic use

Substances

  • Anticoagulants
  • Protein Precursors
  • factor IX Cambridge
  • Vitamin K
  • Warfarin
  • Factor IX
  • Ligases
  • Carbon-Carbon Ligases
  • glutamyl carboxylase