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Characterization of a factor IX variant with a glycine207 to glutamic acid
mutation
SW Lin, CN Lin, N Hamaguchi, KJ Smith and MC Shen
Graduate Institute of Medical Technology, National Taiwan University,
School of Medicine, Taipei, Republic of China.
Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with
reduced levels of circulating protein molecules (cross-reacting material
reduced, CRM). This variant contained a glycine (Gly) to glutamic acid
(Glu) substitution at the 207th codon of mature factor IX. The functional
consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E)
were characterized in this study. Plasma-derived IXG207E exhibited a
mobility similar to that of normal factor IX on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Its specific activity was
estimated to be 3.5% that of the purified normal factor IX in a one-stage
partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by
factor XIa or factor VIIa-tissue factor complex appeared to be normal. When
the calcium-dependent conformational change was examined by monitoring
quenching of intrinsic fluorescence, both normal factor IX and IXG207E
exhibited equivalent intrinsic fluorescence quenching. Activated factor
IXG207E (IXaG207E) also binds antithrombin III equally as well as normal
factor IXa. However, aberrant binding of the active site probe
p-aminobenzamidine was observed for factor XIa-activated factor IXG207E,
indicating that the active site pocket of the heavy chain of factor
IXaG207E was abnormal. Moreover, the rate of activation of factor X by
factor IXaG207E, as measured in a purified system using chromogenic
substrates, was estimated to be 1/40 of that of normal factor IXa. A
computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic
environment surrounding Gly-207 and this Gly forms a hydrogen bound to the
active site serine-365. The molecular mechanism of the Gly-->Glu
mutation in factor IXTaipei9 might result in the alteration of the
microenvironment of the active site pocket which renders the active site
serine-365 inaccessible to its substrate.
Volume 84,
Issue 6,
pp. 1866-1873,
09/15/1994
Copyright © 1994 by The American Society of Hematology
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