(Received for publication, September 6, 1995; and in revised form, December 1, 1995)
From the
This report describes the analysis of a novel mutant human
factor IX protein from a patient with hemophilia B (factor IX activity
<1%; factor IX antigen 45%). Enzymatic amplification of all eight
exons of the factor IX gene followed by direct sequence analysis
reveals a single nucleotide change (a guanine adenine
transition) in exon 2 at nucleotide 6409 which results in a glycine
arginine substitution at amino acid 12 in the
-carboxyglutamic acid rich (Gla) domain of the mature protein.
Factor IX was isolated by immunoaffinity chromatography from plasma
obtained from the proband. The purified protein is indistinguishable
from normal factor IX by polyacrylamide gel electrophoresis.
Characterization of the variant in purified component assays reveals
that it is activated normally by its physiologic activator factor XIa,
but its phospholipid-dependent activation by the factor VIIa-tissue
factor complex is diminished. In the presence of phospholipid and 5
mM Ca
, the activities of variant and normal
plasma-derived factor IX are similar; however, in the presence of
activated factor VIIIa (intrinsic tenase complex), the normal
augmentation of the cleavage of the specific substrate of factor IX,
factor X, is not observed. The determination of the association
constants for normal and variant factor IXa with factor VIIIa shows
that the affinity of the activated variant factor IX for the cofactor
factor VIIIa is 172-fold lower than normal. Competition studies using
active site-inactivated factor IXas in the intrinsic tenase complex
confirm that the defect in the variant protein is in its binding to
factor VIIIa. We conclude that the structural integrity of the Gla
domain of human factor IX is critical for the normal binding of factor
IXa to factor VIIIa in the intrinsic tenase complex. In addition, a
glycine at amino acid 12 is necessary for normal activation of factor
IX by the factor VIIa-tissue factor complex.
Factor IX (F.IX) ()is a vitamin K-dependent
trypsin-like serine protease which functions in the middle phase of
blood coagulation. F.IX is activated by either factor VIIa-tissue
factor (F.VIIa-TF) or factor XIa (F.XIa) in
Ca
-dependent reactions. Once activated, factor IXa
(F.IXa), with its cofactor factor VIIIa (F.VIIIa) in the presence of
Ca
and phospholipid, forms the intrinsic tenase
complex and is responsible for the cleavage of the
Arg
-Ile
bond in the heavy chain of its
specific substrate, factor X (F.X), generating activated F.X (F.Xa)
(F.X numbering according to Leytus et al.(1) ).
Inherited deficiency of F.IX results in a bleeding disorder, hemophilia
B.
Factor IX circulates as a 415-amino acid single chain zymogen
with a molecular mass of 55,000 daltons and is present in normal plasma
at approximately 5 µg/ml. Factor IX is synthesized in the
hepatocyte as a precursor containing an amino-terminal signal sequence
and a propeptide. The signal sequence and propeptide are cleaved off in
separate reactions prior to secretion, and failure to remove these
sequences results in a nonfunctional protein(2) . The signal
sequence directs the protein to the endoplasmic reticulum (ER). The
propeptide contains elements which are important for recognition of
F.IX by the vitamin K-dependent -glutamyl carboxylase which is
associated with the inner surface of the ER(3) . The
amino-terminal 38 residues of the mature protein comprise a functional
region known as the Gla domain, so named because its first 12 glutamic
acid residues undergo a post-translational vitamin K-dependent
carboxylation at the
-carbon (Gla residues). The Gla domain
functions in calcium-dependent lipid binding and in binding to
endothelial cells (residues 3-11)(4) .
The gene encoding F.IX is present on the X chromosome at Xq27.1 near the F.VIII locus; it spans 33.5 kilobases and is composed of eight exons which correspond to the functional domains of F.IX(5) . Exon 1 encodes the signal peptide, exon 2 the propeptide and Gla domain, exon 3 a small aromatic amino acid-rich domain, exons 4 and 5 the 2 epidermal growth factor-like domains, exon 6 the activation peptide, and exons 7 and 8 the trypsin-like catalytic domain. Point mutations in the Gla domain of F.IX (and other vitamin K-dependent clotting factors) result in mild to severe bleeding disorders (6, 7, 8) .
This report describes a point
mutation in exon 2 of the factor IX gene which results in a glycine to
arginine substitution at amino acid 12 in the Gla domain of mature
F.IX. ()Analysis reveals that the major defect associated
with the variant protein is a markedly diminished cleavage of the
substrate F.X by F.IXa when the variant F.IXa is associated with its
cofactor F.VIIIa in the membrane-bound intrinsic tenase complex. The
data suggest that the binding of the variant F.IXa to F.VIIIa is
diminished 172-fold, and that this defect accounts for the decreased
activity of F.IXa in the intrinsic tenase complex. In addition, the
functional characterization of this variant protein provides evidence
that this substitution affects its normal activation by the
membrane-bound F.VIIa-TF complex.
where k is the rate of F.IX activation(19) .
This equation assumes that the rate is normalized to 1 as the maximum,
and that at t there is no F.IXa. Raw data can be
accommodated with the equation:
thus, for normal F.IX, k = 0.050; for variant F.IX, k = 0.013.
where a gives the amount of cleaved
substrate at time 0; a
gives the amount of F.Xa
present in the zymogen F.X, and a
reflects the
rate at which F.Xa cleaves the chromogenic substrate, the rate at which
F.X is cleaved, and the amount of enzyme (F.IXa/PSPC) or
(F.IXa-F.VIIIa/PSPC) present in the reaction(20) .
(subscript t indicates the total concentration) and
Since [F.IXa] = [F.IXa total] - [F.IXa in the intrinsic tenase complex], then:
Solving for [F.IXa-F.VIIIa] gives:
which can be substituted into . Curves were derived from a least-squares fit of the data to substituted .
In the presence of DEGR-F.IXa:
Since [F.IXa-F.VIIIa] is small compared to
[F.VIIIa], this equation simplifies to:
Since the rate of F.Xa generation is a direct function of the amount of F.IXa-F.VIIIa, the data can be expressed as:
The concentration of DEGR-F.IXa-F.VIIIa can be determined as described for the F.IXa-F.VIIIa complex in above assuming that the concentration of the F.IXa-F.VIIIa complex is small compared to the total amount of F.VIIIa and is given by the equation:
which can be substituted into . Curves were derived from a least-squares fit of the data to substituted . Curve fits were performed using the SlideWrite program (Advanced Graphics Software Inc., Carlsbad, CA).
Direct sequence analysis of amplified genomic DNA from all
eight exons and the exon-intron junctions of the variant F.IX gene
demonstrated a single G A transition at nucleotide 6409 in exon
2 (Fig. 1). The mutation was confirmed by sequence analysis of
the opposite strand. This single nucleotide change results in the
substitution of arginine for glycine at amino acid 12 in the Gla domain
of the mature F.IX protein. The mutation neither creates nor destroys
an endonuclease cleavage site useful for confirming the mutation by
restriction analysis.
Figure 1:
Sequence analysis of
genomic DNA from the proband. A G A transition at nucleotide
6409 (denoted by arrow) results in the substitution of
arginine for glycine at amino acid 12 of the mature
protein.
Screening coagulation studies on patient plasma showed a prothrombin time of 10.4 s (control = 10.6) and a partial thromboplastin time of 64.9 s (control = 37). F.IX activity was <1%. F.IX antigen level was 41% by crossed immunoelectrophoresis and 45% by enzyme-linked immunosorbent assay (normal range 60-150%).
Immunoaffinity-purified F.IX from patient plasma comigrated with normal F.IX on electrophoresis in an 8-25% gradient SDS-polyacrylamide gel (Fig. 2). Immunoblotting confirmed this material to be human F.IX (data not shown). In order to confirm the mutation, purified variant F.IX was subjected to amino-terminal amino acid analysis. Edman degradation demonstrated a single amino acid species, arginine, at position 12. Gla analysis demonstrated 9.1 mol of Gla/mol of variant F.IX and 8.7 mol of Gla/mol of normal plasma-derived F.IX.
Figure 2: 8-25% gradient SDS-polyacrylamide gel electrophoresis of purified F.IX. Protein is visualized by silver staining. Lane M, molecular weight markers. Lane A, immunoaffinity-purified normal plasma-derived F.IX. Lane B, immunoaffinity-purified variant F.IX.
F.IXa activities in the
following purified component assays were determined by the calculation
of initial rates of F.IXa-mediated activation of F.X. The optimal
concentration of PSPC vesicles (40 µM) was determined by
varying the concentration of PSPC from 5 µM to 1 mM and determining the rate of F.Xa generation using normal F.IXa (10
nM) and normal F.X (300 nM) (data not shown). The
rate of activation of purified variant F.IX by F.XIa was identical with
that of normal F.IXa (Fig. 3). However, activation of the
variant F.IX by the F.VIIa-TF complex was slower (Fig. 4),
approximately 25% of normal based on kinetic analysis of time courses
of activation (rate for normal F.IX is 0.050 ± 0.002
min, for variant F.IX is 0.013 ± 0.001
min
; see data analysis).
Figure 3:
Time course of F.IX activation by F.XIa.
Variant () and normal (
) F.IX (250 nM) are
activated by F.XIa (1 nM) at 5 mM Ca
. Activation is complete at 20 min. F.IXa
activity is expressed as the concentration (nM) of F.Xa
generated/min.
Figure 4:
Time course of F.IX activation by F.VIIa
and tissue factor. Variant () and normal (
) F.IX (100
nM) are activated by F.VIIa (1 nM) and TF (1
nM) at 3 mM Ca
. F.IXa activity is
expressed as the concentration (nM) of F.Xa generated/min. The
rate of activation of variant F.IX by F.VIIa-TF is 25% that of normal
(0.050 ± 0.002 min
for normal F.IX and 0.013
± 0.001 min
for the variant). Rates were
determined by a least-squares fit of the data to (see
``Experimental Procedures'').
Varying F.IXa between 0
and 10 nM showed the expected linear increase in the rate of
F.Xa generation in the presence of 40 µM PSPC vesicles at
5 mM Ca (Fig. 5). As shown in the inset to Fig. 5, there was no difference in F.Xa
generation between normal F.IXa and variant F.IXa at subnanomolar
concentrations (between 40 and 700 pM), indicating no defect
in phospholipid binding by the variant F.IXa. As shown in Fig. 6A, the kinetics of F.X activation by purified
variant F.IXa and normal F.IXa in the absence of F.VIIIa (in the
presence of 40 µM PSPC vesicles at 5 mM Ca
) were not significantly different (K
= 341 ± 21.7 nM, k
= 9.0
10
M F.Xa s
M
F.IXa for normal F.IXa; K
= 217
± 8.3 nM, k
= 9.2
10
M F.Xa s
M
F.IXa for variant F.IXa). These
data are summarized in Table 1. When the cofactor F.VIIIa was
added to the reaction to form the intrinsic tenase complex, however,
the variant F.IXa did not show the normal augmentation in the rate of
cleavage of F.X (Fig. 6B). This was due to a minor
extent to a difference in K
values (45 nM for normal F.IXa-F.VIIIa complex, 142 nM for the variant
complex), but principally to a 26-fold difference in V
between normal and variant F.IXa (Fig. 6B). This
defect is the likely explanation for the patient's severe
bleeding diathesis.
Figure 5:
F.IXa activation of F.X in the presence of
phospholipid. Variant () and normal (
) F.IXa (between 0 and
7.5 nM) were used to activate 100 nM F.X in the
presence of 40 µM PSPC vesicles. The inset shows
rates of activation at subnanomolar concentrations of F.IXa (between 0
and 1 nM).
Figure 6:
F.IXa activation of F.X. A, F.IXa
(10 nM normal () or variant (
)) are used to
activate F.X in the presence of 40 µM PSPC vesicles and 5
mM Ca
and in the absence of F.VIIIa. The K
and V
values of
variant F.IXa are 217 ± 8.3 nM and 0.546 ± 0.009
nM F.Xa/min, respectively. For normal F.IXa, the K
is 341 ± 21.7 nM and
the V
is 0.536 ± 0.017 nM F.Xa/min. B, F.IXa (0.1 nM normal (
) or
variant (
)) are used to activate F.X in the presence of 40
µM PSPC vesicles and 5 mM Ca
with 0.25 nM F.VIIIa (intrinsic tenase complex). The K
for F.X of the intrinsic tenase complex
containing variant F.IXa is 142 ± 10.0 nM, and the V
is 0.067 ± 0.003 nM F.Xa/min.
The K
for F.X of the normal intrinsic
tenase complex is 45 nM ± 4.6, and the V
is 1.834 ± 0.071 nM F.Xa/min
(see Table 1). Curves from which the constants were derived were
determined based on a least-squares fit of the data to the
Michaelis-Menten equation.
Failure of the variant F.IXa to show enhanced
cleavage of F.X in the intrinsic tenase complex may be due either to
altered affinity of variant F.IXa for the cofactor, F.VIIIa, or failure
of the variant F.IXa to undergo a required conformational change
following its binding to F.VIIIa on the phospholipid
surface(23) . To determine whether diminished binding of F.IXa
to F.VIIIa was responsible for the defect in the activated variant
F.IX, we first determined K values for the variant
and normal F.IXas (Fig. 7). The association of F.IXa with
F.VIIIa was determined by the addition of F.VIIIa and PSPC vesicles to
varying concentrations of F.IXa and measured by the increased
activation of F.X by generated intrinsic tenase (F.IXa-F.VIIIa-PSPC
vesicles-Ca
). Both the variant and normal F.IXa
showed a similar V
(V
= 25.7 ± 0.5 for normal; V
= 27.5 ± 4.9 for variant); however, the K
of the variant F.IXa for F.VIIIa-PSPC vesicles
was 172-fold higher than that of normal F.IXa (K
= 30.9 ± 7.27 nM; K
= 0.18 nM ± 0.02, respectively). Since the
kinetic parameters describing the interaction of variant F.IXa are
extrapolated values, we sought to corroborate the estimated K
using a second method. We determined K
values for the intrinsic tenase using normal and
variant F.IXas that had been blocked at the active site as competitive
inhibitors of normal F.IXa in the intrinsic tenase complex. In this
inhibition assay, activated species of normal and variant F.IX were
inhibited by the covalent linkage of the small peptide,
dansyl-Glu-Gly-Arg (DEGR), at the active site triad. If the defect in
the variant enzyme were due to abnormal binding to F.VIIIa, the variant
would be expected to exhibit diminished or no displacement of normal
F.IXa from intrinsic tenase. If, however, the defect were due to a
failure of the variant to undergo a conformational change (upon binding
of F.VIIIa) necessary for normal activity in the intrinsic tenase
complex, inactivated variant protein would bind F.VIIIa normally and
displace normal F.IXa from the intrinsic tenase complex in a fashion
similar to inactivated normal F.IXa. Varying amounts of these active
site-inactivated F.IXas were added to the intrinsic tenase complex
consisting of F.IXa, F.VIIIa, and PSPC vesicles (Fig. 8). K
values determined from this intrinsic tenase
inhibition assay were consistent with results obtained in the binding
experiment and showed a 45-fold difference between normal F.IXa (K
= 0.17 ± 0.011 nM) and
variant F.IXa (K
= 7.71 ± 0.50
nM).
Figure 7:
Determination of K
for normal or variant F.IXa and F.VIIIa. Thrombin-activated F.VIIIa
(0.5 nM) is incubated with variant (
) or normal (
)
F.IXa with 40 µM PSPC vesicles and 5 mM Ca
. The binding of F.IXa to F.VIIIa was
determined by measuring intrinsic tenase activity and detected by the
generation of F.Xa. The V
for normal F.IXa is
25.7 ± 0.5 nM F.Xa/min and for variant is 27.5 ±
4.9 nM F.Xa/min. The K
describing the binding of normal F.IXa to PSPC-F.VIIIa is
0.18 ± 0.02 nM; for variant F.IXa, the K
is 30.9 ± 7.3 nM as
determined from curves derived from least-squares fit of the data to (see ``Experimental
Procedures'').
Figure 8:
Inhibition of the normal intrinsic tenase
complex by dansyl-Glu-Gly-Arg (DEGR) inactivated F.IXas. Increasing
concentrations of DEGR-inactivated F.IXa normal () and variant
(
) are used as competitors for normal F.IXa in the intrinsic
tenase complex (50 pM F.IXa, 0.55 nM F.VIIIa, 40
µM PSPC vesicles, 5 mM Ca
).
Binding of the inactivated species is measured by the inhibition of
F.Xa generation by the normal intrinsic tenase complex. K
for DEGR-inactivated F.IXa normal is
0.17 ± 0.01 nM and for DEGR-inactivated F.IXa variant
is 7.71 ± 0.50 nM as determined from curves derived
from least-squares fit of the data to (see
``Experimental Procedures'').
We describe a novel mutation in the gene for human blood coagulation factor IX which results in the substitution of the large basic amino acid, arginine, for the normal glycine at position 12 in the Gla domain of the mature protein. This protein is of special interest because it provides evidence that the structural integrity of the Gla domain is critical for binding of F.IXa to the cofactor F.VIIIa. The variant protein is secreted into the circulation, but has no activity in coagulation. We have characterized the effects of the substitution on the function of the variant protein in coagulation by using immunoaffinity-purified plasma-derived F.IX in a series of functional assays. Other F.IX mutants reported in the Gla domain have not been studied in purified component assays(6) .
On
functional analysis, the major defect of the variant F.IXa is the
absence of the normal augmentation of the F.IXa-mediated activation of
F.X in the presence of phospholipid, Ca, and the
cofactor F.VIIIa (intrinsic tenase) (Fig. 6B).
Phospholipid binding by the variant is normal at concentrations of
F.IXa between 40 pM and 10 nM as demonstrated by
F.IXa-mediated generation of F.Xa (Fig. 5). Given the normal
function of the variant in the presence of Ca
and
phospholipid alone (Fig. 6A), the diminished activity
observed in the intrinsic tenase complex is due to a F.VIIIa-dependent
interaction of F.IXa within the intrinsic tenase complex. This reduced
activity could be due either to a defect in variant F.IXa binding to
F.VIIIa or a failure of the variant to undergo a conformational change
upon binding F.VIIIa that is necessary for normal activity in the
intrinsic tenase complex. Reduced binding of the variant F.IXa to
F.VIIIa is demonstrated by both the binding and competitive inhibition
assays. In the binding experiment, the K
of
variant F.IXa (30.9 nM) was 172-fold higher than that of
normal F.IXa (K
= 0.18 nM) (Fig. 7). Under the conditions used in this binding experiment,
the K
determined for F.VIIIa binding by variant
F.IXa exceeded the highest concentration of variant F.IXa used in the
experiment. To reasonably approach saturating conditions with variant
F.IXa in this experiment would require concentrations of F.IXa in the
range (>100 nM) where background F.X activation by F.IXa
alone would affect the analysis of the results. In addition, under such
conditions, the concentration of F.IXa would approach the concentration
of F.X used in the assay resulting in competition between F.IXa and F.X
for phospholipid binding. The use of higher concentrations of F.X to
overcome this limitation would result in unmeasurable reaction rates.
The intrinsic tenase inhibition assay was performed to overcome these
limitations and to corroborate the estimated K
using a second method. Normal F.IXa was 45 times more effective
as an inhibitor in the intrinsic tenase inhibition assay (K
= 0.17 nM for normal F.IXa and
7.71 nM for variant F.IXa) (Fig. 8). Several groups
have published constants between 0.02 and 16 nM describing the
interaction of F.VIIIa with F.IXa under conditions different from those
used in the experiments described
here(22, 24, 25, 26, 27, 28) .
Our values are at the lower limit of other reported ranges for binding
constants but are not inconsistent with previous studies which
evaluated human factors (25, 27) . In the inhibition
assay, similar K
values (similar inhibition
pattern) would have been expected for both the normal and variant if
the defect observed in the intrinsic tenase (Fig. 6B)
were due solely to the variant's failure to undergo the necessary
conformational change. If one assumes that there is no effect of the
small peptide blocking the active site of F.IXa on the binding of
inactivated F.IXa (normal or variant) to F.VIIIa, then the intrinsic
tenase inhibition assay, in which F.X activation is accomplished only
by normal F.IXa, measures only the difference in binding of F.VIIIa
between normal and variant F.IXa. Factor X activation in the binding
assay, however, relies on the measurement of F.X activation by either
normal F.IXa or variant F.IXa and, therefore, measures both binding and conformational effects. Thus, the inhibition assay
confirms that the major effect is due to abnormal binding of variant
F.IXa to F.VIIIa. The 4-fold discrepancy between the relative
differences in the normal and variant K
values
(45-fold) and the normal and variant K
values
(172-fold) ascertained in these two experiments is likely to be within
the range of experimental error. Alternatively, there may be a small
contribution to the defect from differences in the conformational
change undergone by wild-type versus variant F.IXa upon
binding to F.VIIIa. (A conformational difference would be reflected in
the binding assay shown in Fig. 7, but not in the competitive
inhibition assay shown in Fig. 8.) In any case, it is clear that
the major factor accounting for the reduced activity in the intrinsic
tenase complex is altered binding of the variant F.IXa to F.VIIIa.
Catalytic efficiencies (k/K
) were calculated from
experimentally determined K
and V
(Table 1). In the experiments characterizing F.IXa activity
in the intrinsic tenase complex, the true enzyme is the F.IXa-F.VIIIa
complex. The concentration of the F.IXa-F.VIIIa complex was determined
using observed K
values (Fig. 7) and (see ``Experimental Procedures,'' Data
Analysis). Note that the 172-fold difference between the K
values for normal and variant F.IXa results in a
marked difference in the concentration of the true enzyme complex
(normal or variant F.IXa-F.VIIIa) at identical concentrations of F.IXa
and F.VIIIa. In contrast to the difference in rates of F.X activation
at a given concentration of F.IXa between the normal and variant
intrinsic tenase (Fig. 6B), catalytic efficiencies of
the normal and variant complexes are similar, since they are determined
using concentrations of the true enzyme complex (Table 1).
In
the solution phase activation of F.IX by F.XIa in which two bonds in
the catalytic domain of F.IX (Arg-Ala
and
Arg
-Val
) are cleaved by F.XIa, variant F.IX
is activated normally. However, in the phospholipid-dependent
activation of F.IX by the F.VIIa-TF complex, in which the same two
bonds are cleaved, the variant is activated at a rate which is 25% of
normal. These data suggest that amino acid 12 of F.IX is either
involved in the binding site for F.VIIa-TF or has distal effects on the
conformation of the F.VIIa-TF binding site on phospholipid-bound F.IX.
The substitution of arginine, a bulky negatively charged residue,
for the small neutral glycine at position 12 would be predicted to
affect Ca binding by perturbing the surrounding Gla
residues. Such an effect might be expected to result in altered
phospholipid binding since Ca
binding in the Gla
domain is responsible for a conformational change which is necessary
for coagulant activity (29, 30, 31, 32) , and this activity
is correlated to phospholipid binding(33) . However, when the
kinetic activation of F.X by F.IXa was studied with phospholipid and
Ca
, there was little difference between the variant
and normal F.IXa, implying that binding of the variant to F.X and
phospholipid is normal and that the effect of the amino acid
substitution has minimal, if any, effect on calcium binding.
The
data indicate that the variant F.IX undergoes -carboxylation to
the same degree as the normal protein. Based on extensive work with
recombinant proteins (3, 34, 35, 36) and synthetic
peptides(37, 38) , the
-glutamyl carboxylase
recognition site is known to include residues at the amino-terminal
portion of the propeptide in vitamin K-dependent proteins. Observations
from naturally occurring F.IX variants with mutations affecting
residues -1 and -4 at the carboxyl terminus of the
propeptide, are not consistent; some have reported normal
-carboxylation (39, 40) and others, reduced
-carboxylation(2, 41) . Peptide substrates which
contain these same changes, however, have no effect on
carboxylation(38, 42) . Although a contribution to the
carboxylase binding site from the Gla domain has been suggested based
on sequence homology between vitamin K-dependent coagulation factors
and matrix Gla protein (43) , peptide substrates which include
the propeptide and this purported binding site in the Gla domain have
not been shown to affect carboxylase binding when compared to the
propeptide substrate alone (44) . Zhang et al.(45) have observed that only 10-20% of recombinant
protein C containing substitutions of aspartic acid for Gla residues
16, 20, and 26 is fully carboxylated. These data have led to the
hypothesis that amino acid substitutions which perturb the structure of
the Gla domain or the intervening portion of the propeptide may affect
carboxylation. Normal carboxylation of the variant F.IX described here
indicates that glycine 12 is not involved in carboxylase recognition,
and that a nonconservative amino acid substitution at this position in
F.IX does not affect
-carboxylation of the appropriate glutamic
acid residues by the carboxylase.
Alignment of the amino acid
sequences of the human vitamin K-dependent factors (F.IX, prothrombin,
F.VII, F.X, protein C, and protein S) reveals that the residue
corresponding to glycine 12 of mature factor IX is conserved in these
proteins, with the exception of protein C in which another small
neutral polar amino acid (serine) is present. This suggests a critical
role for glycine (or serine) at this position. Two other mutations have
been reported in F.IX affecting amino acid 12(6, 46) .
In F.IX, an alanine substitution results in a
molecule with 3% normal activity. F.IX
, in which the
charged residue, glutamic acid, is substituted for glycine 12, has
<1% normal activity. The mechanism for defective activity has not
been characterized for either of these variants.
The data presented
here demonstrate that a glycine to arginine substitution at position 12
in human F.IX prevents the normal augmentation of activity of activated
F.IX that occurs in the presence of the cofactor F.VIIIa. Competition
studies with active site-inactivated F.IXa provide strong evidence that
variant F.IXa binding to F.VIIIa is diminished and accounts for the
severe defect in the activity of the variant F.IXa in the intrinsic
tenase complex. These data thus indicate a critical role for the
structural integrity of the Gla domain in the binding of F.VIIIa by
F.IXa. Although it is possible that the Gla domain of F.IXa contacts
the F.VIIIa molecule directly, it is more likely that the alignment of
proper intermolecular contact points is critically dependent on residue
12 of F.IXa, and that the Gly Arg mutation exerts its effect on
F.VIIIa binding through this mechanism.