title: Expression and Characterization of Gly-317 Variants of Factor IX Causing Variable Bleeding in Hemophilia B Patients.
abstract: We recently identified two hemophilia B patients who carried Gly-317 to Arg (FIX-G317R) or Gly-317 to Glu (FIX-G317E) substitutions in their FIX gene. The former mutation caused severe and the latter moderate bleeding in afflicted patients. To understand the molecular basis for the variable clinical manifestation of Gly-317 mutations, we prepared recombinant G317R and G317E derivatives of FIX and compared their kinetic properties to those of recombinant wild-type FIX in appropriate assay systems. Both physiological activators, factor XIa and extrinsic Tenase (factor VIIa-tissue factor), activated both zymogen variants with an ∼1.5-fold elevated K(m); however, extrinsic Tenase activated FIX-G317E with an ∼2-fold improved k(cat). By contrast to zymogen activation, the catalytic activities of both FIXa-G317R and FIXa-G317E enzymes toward the natural substrate, factor X, were dramatically (>4 orders of magnitude) impaired, but their apparent affinity for interaction with factor VIIIa was only slightly (<2-fold) decreased. Further studies revealed that the reactivity of FIXa-G317R and FIXa-G317E with antithrombin has been impaired 10- and 13-fold, respectively, in the absence and 166- and 500-fold, respectively, in the presence of pentasaccharide. As expected, the clotting activities of FIX variants could not be measured by the aPTT assay. These results implicate a critical role for Gly-317 in maintaining normal catalytic function for FIX/FIXa in the clotting cascade. The results further suggest that improved k(cat) of FIX-G317E activation in the extrinsic pathway together with dramatically impaired reactivity of FIXa-G317E with antithrombin may account for the less severe bleeding phenotype of a hemophilia B patient carrying the FIX-G317E mutation.
authors.affiliation: †Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China.
authors.forename: Qiuya
authors.initials: Q
authors.lastname: Lu
authors.affiliation: ‡Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, United States.
authors.forename: Likui
authors.initials: L
authors.lastname: Yang
authors.affiliation: ‡Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, United States.
authors.forename: Chandrashekhara
authors.initials: C
authors.lastname: Manithody
authors.affiliation: †Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China.
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Wang
authors.affiliation: ‡Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, United States.
authors.forename: Alireza R
authors.initials: AR
authors.lastname: Rezaie
mesh_terms: D019943:Amino Acid Substitution; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D005164:Factor IX / Q000235:genetics / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D015945:Factor XIa / Q000378:metabolism; D005998:Glycine / Q000737:chemistry*; D057809:HEK293 Cells; D002836:Hemophilia B / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D006470:Hemorrhage / Q000209:etiology*; D006801:Humans; D007700:Kinetics; D008297:Male; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism*; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012720:Severity of Illness Index
chemical_list: D004792:Enzyme Precursors; D050505:Mutant Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D015944:Factor VIIIa; D005164:Factor IX; D005170:Factor X; D015945:Factor XIa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant; D005998:Glycine