Factor IXHollywood: substitution of Pro55 by Ala in the first epidermal
growth factor-like domain
SG Spitzer, MN Kuppuswamy, R Saini, CK Kasper, JJ Birktoft and SP Bajaj
Department of Medicine, St Louis University School of Medicine, MO.
Factor IX is a multidomain protein essential for hemostasis. We describe a
mutation in a patient affecting the first epidermal growth factor
(EGF)-like domain of the protein. All exons and the promoter region of the
gene were amplified by the polymerase chain reaction method, and sequenced.
Only a single mutation (C----G), that predicts the substitution of Pro55 by
Ala in the first EGF domain was found in the patient's gene. This mutation
leads to new restriction sites for four enzymes. One new site (Nsi) was
tested in the amplified exon IV fragment and was shown to provide a rapid
and reliable marker for carrier detection and prenatal diagnosis in the
affected family. The factor IX protein, termed factor IXHollywood (IXHW),
was isolated to homogeneity from the patient's plasma. As compared with
normal factor IX (IXN), IXHW contained the same amount of gamma-carboxy
glutamic acid but twice the amount of beta-OH aspartic acid. Both IXHW and
IXN contained no detectable free -SH groups. Further, IXHW could be readily
cleaved to yield a factor IXa-like molecule by factor Xla/Ca2+. However,
IXaHW (compared with IXaN) activated factor X approximately twofold slower
in the presence of Ca2+ and phospholipid (PL), and 8- to 12-fold slower in
the presence of Ca2+, PL, and factor VIIIa. Additionally, IXaHW had only
approximately 10% of the activity of IXaN in an aPTT assay. In agreement
with the nuclear magnetic resonance- derived structure of EGF, the
Chou-Fasman algorithm strongly predicted a beta turn involving residues
Asn-Pro55-Cys-Leu in IXN. Replacement of Pro55 by Ala gave a fourfold
decrease in the beta turn probability for this peptide, suggesting a
change(s) in the secondary structure in the EGF domain of IXHW. Since this
domain of IXN is thought to have one high-affinity Ca2+ binding site and
may be involved in PL and/or factor VIIIa binding, the localized secondary
structural changes in IXHW could lead to distortion of the binding site(s)
for the cofactor(s) and, thus, a dysfunctional molecule.
Volume 76,
Issue 8,
pp. 1530-1537,
10/15/1990
Copyright © 1990 by The American Society of Hematology
