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 s erve as a recognition site for the enzyme gamma-glutamylcarboxylase, w hich catalyzes the carboxylation of glutamic acid residues at the NH2 terminus of the mature protein. We describe a mutation in the propepti de of Factor IX that results in warfarin sensitivity because of reduce d affinity of the carboxylase for the Factor IX precursor. The proband has a Factor IX activity level of >100% off warfarin and <1% on warfa rin, at a point where other vitamin K-dependent factors were at 30-40% activity levels. Direct sequence analysis of amplified genomic DNA fr om 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 mecha nism by which the mutation resulted in warfarin sensitivity, we analyz ed wild-type and mutant recombinant peptides in an in vitro carboxylat ion reaction. The peptides that were analyzed included the wild-type s equence, the Ala-10-->Thr sequence, and Ala-10-->Gly, a substitution b ased on the sequence in bone gamma-carboxyglutamic acid protein. Measu rement of CO2 incorporation at a range of peptide concentrations yield ed a V-max of 343 cpm/min/reaction for the wild-type peptide, and V-ma x values of 638 and 726 for A-10T and A-10G respectively, a difference of only twofold. The K-m values, on the other hand, showed a 33-fold difference between wild-type and the variants, with a value of 0.29 mu M for wild-type, and 10.9 and 9.50 mu M, respectively, for A-10T and A-10G. Similar kinetic experiments showed no substantial differences b etween wild-type and mutant peptides in kinetic parameters of the carb oxylase-peptide complexes for reduced vitamin K. We conclude that the major defect resulting from the Factor IX Ala-10-->Thr mutation is a r eduction in affinity of the carboxylase for the mutant propeptide. The se studies delineate a novel mechanism for warfarin sensitivity. In ad dition, the data may also explain the observation that bone Gla protei n is more sensitive to warfarin than the coagulation proteins.