| [PMID]: | 26023895 |
| [Au] Autor: | Lu Q; Yang L; Manithody C; Wang X; Rezaie AR |
| [Ad] Endereço: | †Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China. |
| [Ti] Título: | Expression and Characterization of Gly-317 Variants of Factor IX Causing Variable Bleeding in Hemophilia B Patients. |
| [So] Source: | Biochemistry;54(24):3814-21, 2015 Jun 23. |
| [Is] ISSN: | 1520-4995 |
| [Cp] País de publicação: | United States |
| [La] Idioma: | eng |
| [Ab] Resumo: | 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. |
| [Mh] Termos MeSH primário: |
Precursores Enzimáticos/metabolismo
Fator IX/metabolismo
Glicina/química
Hemofilia B/genética
Hemorragia/etiologia
Proteínas Mutantes/metabolismo
Mutação
|
| [Mh] Termos MeSH secundário: |
Substituição de Aminoácidos
Cisteína Endopeptidases/metabolismo
Ativação Enzimática
Precursores Enzimáticos/genética
Fator IX/genética
Fator VIIIa/metabolismo
Fator X/metabolismo
Fator XIa/metabolismo
Células HEK293
Hemofilia B/metabolismo
Hemofilia B/fisiopatologia
Seres Humanos
Cinética
Masculino
Mutagênese Sítio-Dirigida
Proteínas de Neoplasias/metabolismo
Proteínas Recombinantes/metabolismo
Índice de Gravidade de Doença
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| [Pt] Tipo de publicação: | JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL |
[Nm] Nome de substância:
| 0 (Enzyme Precursors); 0 (Mutant Proteins); 0 (Neoplasm Proteins); 0 (Recombinant Proteins); 72175-66-7 (Factor VIIIa); 9001-28-9 (Factor IX); 9001-29-0 (Factor X); EC 3.4.21.27 (Factor XIa); EC 3.4.22.- (Cysteine Endopeptidases); EC 3.4.22.26 (cancer procoagulant); TE7660XO1C (Glycine) |
| [Em] Mês de entrada: | 1509 |
| [Cu] Atualização por classe: | 161019 |
[Lr] Data última revisão:
| 161019 |
| [Sb] Subgrupo de revista: | IM |
| [Da] Data de entrada para processamento: | 150530 |
| [St] Status: | MEDLINE |
| [do] DOI: | 10.1021/acs.biochem.5b00270 |
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