A mutation in the propeptide of Factor IX leads to warfarin sensitivity by a novel mechanism.
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.
Chu
K
K
Department of Pediatrics and Pathology, University of Pennslyvania and Children's Hospital of Philadelphia, 19104, USA.
Wu
S M
SM
Stanley
T
T
Stafford
D W
DW
High
K A
KA
D000595:Amino Acid Sequence; D000925:Anticoagulants / Q000494:pharmacology* / Q000627:therapeutic use; D019735:Carbon-Carbon Ligases; D005091:Exons / Q000235:genetics; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000378:metabolism; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D014812:Vitamin K / Q000378:metabolism; D014859:Warfarin / Q000494:pharmacology* / Q000627:therapeutic use; D044465:White People
D000925:Anticoagulants; D011498:Protein Precursors; C049838:factor IX Cambridge; D014812:Vitamin K; D014859:Warfarin; D005164:Factor IX; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase