Expression and Characterization of Gly-317 Variants of Factor IX Causing Variable Bleeding in Hemophilia B Patients

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• 作者：Qiuya Lu ; Likui Yang ; Chandrashekhara Manithody ; Xuefeng Wang ; Alireza R. Rezaie
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：June 23, 2015
• 年：2015
• 卷：54
• 期：24
• 页码：3814-3821
• 全文大小：332K
• ISSN：1520-4995

文摘

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鈥搕issue factor), activated both zymogen variants with an 鈭?.5-fold elevated K_m; however, extrinsic Tenase activated FIX-G317E with an 鈭?-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.