From the
The indispensable role of Ca
Calcium is an element that is essential in the regulation of a
great variety of biological processes. The metal is abundant in
extracellular fluids. In blood plasma, calcium is present at
concentrations of 2.2-2.6 mM (1.1-1.3 mM as free ions)(1) , and it plays an obligatory role in
hemostasis. Calcium ions bind to a number of coagulation proteins and
modulate their functions. Almost all the steps in blood coagulation
require Ca
Another alkaline-earth
metal, magnesium, is also abundant in blood plasma (0.4-0.6
mM as free ions)(1) . While we know much about the role
of Ca
We previously isolated a novel type of
anticoagulant protein that binds to the Gla domains of factors IX and X
in a Ca
Human factor XIa
was prepared using immunoaffinity chromatography by a modification of
the method of isolation of the zymogen(18) . In brief, factor XI
was activated by incubating 1 liter of plasma, anticoagulated with
acid-citrate dextrose, with 25 g of Celite (Hyflo-super-cel; Wako Pure
Chemicals, Osaka, Japan) at 37 °C for 10 min. Factor XIa absorbed
to the Celite was eluted by stirring the filter cake in 100 ml of 20
mM Tris-HCl, 1.5 M NaCl, pH 7.5, for 18 h at ambient
temperature. The eluate was passed through a column of soybean trypsin
inhibitor-Sepharose to remove factor XIIa, after dialysis against 20
mM Tris-HCl, 140 mM NaCl, pH 7.5 (TBS).
The snake venom anticoagulant IX/X-bp recognizes
Ca
We next examined the effects of Mg
Factor IX plays a central role in blood coagulation, as is
apparent from the fact that deficiency of this factor causes a severe
bleeding disorder, hemophilia B(25) . It has been established
that factor IX, similar to other vitamin K-dependent coagulation
factors, has multiple binding sites for polyvalent metal ions, and the
natural ligands are believed to be primarily Ca
The
importance of Ca
It is particularly
noteworthy that the effect of Mg
Furie and co-workers (10, 11) postulated
previously that there is a common mechanism for the metal-induced
conformational transitions of vitamin K-dependent coagulation factors.
Their hypothesis is based on observations with conformation-specific
antibodies. They developed a method for fractionating antibodies
according to the dependence on metal ions of the antigen recognition
(as employed here to prepare anti-factor IX:Ca(II) antibodies). Two
classes of metal-dependent antibodies have been described; one is
specific for Ca
The
effects of Mg
In conclusion, we have revealed a novel
physiological function of Mg
The steady-state kinetics of activation of factor IX were
analyzed as described under ``Experimental Procedures.'' The
data are means ± S.E. of three independent determinations. The k
We thank Tomohiro Nakagaki of The Chemo-Sero
Therapeutic Research Institute for providing factor IX-deficient
plasma. We also acknowledge the technical assistance of Shun Igarashi,
Hiromi Nakahata, and Daisuke Yamada.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
ions in the
maintenance of the functional tertiary structures of vitamin
K-dependent coagulation factors has been definitively established but
the participation of Mg
ions, another alkaline-earth
metal that is present abundantly in blood plasma, in such a process is
not yet understood. We show here that the
Ca
-stabilized conformation of coagulation factor IX
undergoes a further conformational change upon binding of
Mg
ions using three independent structural probes.
The probes we used were (i) IX/X-bp, a snake venom anticoagulant that
recognizes the Gla domains in coagulation factors IX and X, (ii)
conformation-specific polyclonal antibodies against bovine factor IX,
and (iii) monoclonal antibodies against the Gla domain of human factor
IX. The binding of all these probes had an absolute requirement for
Ca
ions, and Mg
ions alone were
ineffective. However, when added together with Ca
ions, Mg
ions at physiological concentrations
greatly augmented the binding of these probes to factor IX; the
required concentration of Ca
ions was much reduced,
and the affinity of each probe for factor IX was increased even in the
presence of an excess of Ca
ions. These results
suggest the presence of a Mg
-specific binding site
that does not interact with Ca
ions in factor IX.
Furthermore, Mg
ions potentiated the susceptibility
of factor IX to activation by factor XIa, concomitant with their effect
on the conformation. Similarly, the required Ca
concentration was reduced by Mg
ions, and the
rate of conversion to factor IXa was increased by Mg
ions in the presence of an excess of Ca
ions.
At a saturating concentration of Ca
ions (5
mM), addition of 1 mM Mg
reduced
the apparent K
value for factor IX from
0.31 to 0.18 µM, and in the presence of a physiological
concentration of Ca
ions (1 mM), the
reduction in K
by Mg
ions was far more striking (from 0.91 to 0.24 µM).
The apparent V
values were hardly affected by
Mg
ions. Our present data reveal a hitherto novel
physiological role of the Mg
ions in plasma. Not only
Ca
ions but also Mg
ions are
important regulators of the stabilization of the native conformation of
factor IX as well as of its efficient activation.
ions, and chelating agents such as EDTA
and citrate are very effective anticoagulants. Among the various
proteins that participate in coagulation, vitamin K-dependent
coagulation factors show strong dependence on Ca
ions
for maintenance of their functional conformation (for reviews, see
Refs. 2 and 3). Each factor binds about 10 Ca
ions at
the N-terminal Gla domain, and Gla-independent
Ca
-binding sites have also been identified in some of
these factors. These various proteins, namely prothrombin and factors
VII, IX and X, and also the anticoagulant protein C, are synthesized as
zymogens and are converted to active proteases via limited proteolysis
catalyzed by specific activators. Each activated protease in turn
activates another factor, constituting the cascade of coagulation. The
protein conformations with bound Ca
ions are
essential for recognition by the respective activators. Moreover, the
binding of these factors to procoagulant membranes rich in anionic
phospholipids (e.g. the surface of activated platelets), which
allows condensation of both activator and substrate at one site and
thereby facilitates efficient activation, is also a
Ca
-dependent process.
ions, the involvement of Mg
ions in hemostasis has been poorly understood. Various polyvalent
metal ions including Mg
ions have been shown to
interact with the Ca
-binding sites in Gla domains of
vitamin K-dependent coagulation factors (e.g. Refs.
4-7). According to previous studies, however, the affinity of
Mg
ions for these proteins is relatively weak and, so
far as is known to date, Mg
ions cannot be replaced
Ca
ions for the expression of biological activity. In
1980, Byrne et al.(8) reported that activation of
factor IX by factor XIa was accelerated to some extent by the addition
of Mg
ions in the presence of a suboptimal
concentration of Ca
ions (1.25 mM). However,
since only limited information about the structure of factor IX was
available, the underlying molecular mechanism of this phenomenon was
hard to explain at that time. Furthermore, the concentration of factor
IX employed in that report was far beyond the physiological range and,
therefore, it was unclear whether the observed phenomenon was
physiologically meaningful. In the mid-1980s, Furie and co-workers
(9-12) proposed the hypothesis that vitamin K-dependent
coagulation factors undergo two sequential conformational changes upon
binding to metal ions on the basis of observations with
conformation-specific antibodies as structural probes. Their scheme can
be summarized as follows: P
P`
P*. In this model, metal
ions other than Ca
ions (e.g. Mg
ions) can elicit only a partial transition, from the metal-free
form of the protein (P) to an intermediate form (P`) that is
insufficient for the expression of biological activity. Transition to a
final functional form (P*) is accomplished only by Ca
ions at physiological concentrations. The consequences of this
model are simple: only Ca
ions are essential, and
other cations are unnecessary for the regulation of the conformation
and function of the coagulation factors. By contrast, we show in the
present report that the Mg
ion is another regulator
of both the structure and the function of factor IX, which is crucial
for normal hemostasis.
-dependent fashion from snake
venom(13, 14) , and we have recently found that this
protein, designated IX/X-bp, recognizes the Ca
-bound
conformation of the Gla domains.
(
)In this
report, we now show that binding of IX/X-bp to factor IX, but not to
factor X, is greatly augmented by physiological concentrations of
Mg
ions when added together with Ca
ions. Furthermore, we demonstrate that not only Ca
ions but also Mg
ions are important for
stabilization of the conformation as well as for expression of the
biological activity of factor IX. Our discoveries highlight a novel
physiological role of Mg
ions in hemostasis.
Proteins
The following proteins were prepared by
published methods; IX/X-bp(13) , bovine factors IX and
X(15) , human factor IX (16), and human factor
IXa(17) . All of the preparations were homogeneous as
judged by SDS-polyacrylamide gel electrophoresis.
(
)The unbound fraction was then applied to a column of
immobilized monoclonal antibody against human factor XI (KMXI-1; Ref.
19). Factor XIa was eluted with 0.02% NH
OH, 0.15 M NaCl, pH 11. Trace amounts of high molecular weight contaminants
were removed by gel filtration.
Binding of
IX/X-bp was labeled with
NaI-IX/X-bp
I (Du Pont NEN) by use of IODOBEADS (Pierce) in
accordance with the manufacturer's instructions. The specific
activity of the labeled protein was in the range of 1.0-2.0
10
cpm/µg. Binding assays were conducted as
follows. Wells of breakable microtiter plates (Labsystems, Finland)
were coated with a solution of 10 µg/ml factor IX or X in 50 µl
of TBS at 4 °C overnight, and the remaining nonspecific binding
sites were blocked by incubation with 1% bovine serum albumin (BSA,
essentially fatty acid-free; Sigma) in TBS for 1 h. Each well was
incubated with approximately 50,000 cpm of
I-IX/X-bp
(approximately 0.5 µg/ml) in 50 µl of TBS containing 1 mg/ml
BSA and appropriate concentrations of Ca
and
Mg
ions for 30 min at 37 °C. Wells were then
washed twice with 200 µl of TBS plus metal ions at the same
concentrations as those used for the incubation, cut into pieces, and
counted for bound radioactivity in a
counter. Magnesium ions were
added as magnesium acetate, and acetate ions added as sodium acetate
had no effect on the binding.
Equilibrium Dialysis
This was conducted at ambient
temperature in a microvolume dialyzer with 250-µl cells (Hoffer
Scientific Instrument, San Francisco, CA). Dialysis membranes were
pretreated with a boiled solution of 0.1 M NaHCO,
2% EDTA and washed extensively with metal-free water prior to use. A
200-µl aliquot of a TBS solution of CaCl
containing
CaCl
as a tracer (1,000,000 dpm/cell; Du Pont
NEN) was dialyzed against 200 µl of 40 µM IX/X-bp in
TBS for 20 h with constant rotation. Protein-bound Ca
was quantified by liquid scintillation counting. In order to
negate effects of possible contamination by metal ions, the buffer used
was passed through a column of Chelex 100 (Bio-Rad) and the protein
solution was dialyzed against a suspension of Chelex 100 (1 g/liter) in
the buffer.
Polyclonal Antibodies
Antisera against bovine
factor IX were raised in rabbits by immunizing them with the pure
protein. Conformation-specific antibodies were prepared essentially by
the method of Liebman et al.(11) . In brief, a pool of
polyclonal antibodies was loaded onto an affinity gel of immobilized
bovine factor IX which had been equilibrated with a buffer containing
Ca ions. Anti-factor IX:Ca(II) antibodies,
(
)which recognized factor IX with an absolute
requirement for Ca
ions, were eluted by changing the
buffer to that containing Mg
ions and no
Ca
ions. Anti-factor IX:Mg(II) antibodies, which
recognized factor IX in the presence of various polyvalent metal ions
including Mg
, and metal-independent antibodies were
obtained by elution with buffers containing EDTA and guanidine
hydrochloride, respectively. Conformation-specific antibodies against
other coagulation factors were also prepared by the same method. The
nature of each antibody was confirmed by an enzyme-linked immunosorbent
assay (ELISA) as described below.
Monoclonal Antibodies
A panel of monoclonal
antibodies against human factor IX was prepared by the standard
hybridoma technique. Clones producing antibodies reactive to the
antigen were selected by ELISA. Characterization of the positive clones
obtained was also performed by ELISA. The metal dependence of the
binding was examined and preliminary epitope mapping utilizing various
factor IX derivatives were performed. Among 119 positive clones, 46
clones exhibited Ca dependence for the binding to the
antigen. Two clones for which binding was absolutely dependent on
Ca
ions, C1 (subclass IgG
,
) and 2A2
(IgG
,
), were selected for this study. The antibodies
reacted with the isolated Gla domain fragment of factor IX (17) in the presence of Ca
ions at millimolar
concentrations.
(
)
ELISA
Wells of microtiter plates were
coated with 50 µl of protein solutions (1 µg/ml in TBS) for 2 h
at ambient temperature with subsequent blocking by 1% BSA for 30 min.
Coated wells were then incubated with 50 µl of a diluted solution
of antibody in TBS containing 0.1% Tween-20 and various concentrations
of Ca and Mg
ions for 1 h, and the
same buffers were used throughout the subsequent washing and incubation
procedures. After two washes, the wells were reacted with 50 µl of
peroxidase-conjugated goat antibodies against either rabbit IgG (for
polyclonal antibodies) or mouse IgG (for monoclonal antibodies)
(diluted 1:100; Kirkegaard and Perry Laboratories, Gaithersburg, MD)
for 1 h. After extensive washing (five times), reaction with the
antigen was visualized by incubation with 1 mg/ml o-phenylenediamine and 0.06% H
O
in 100
µl of 0.1 M citrate buffer, pH 5.5, and absorbance at 492
nm was recorded.
Quantification of Factor IXa
A two-stage clotting
assay system was designed for the sensitive and selective
quantification of factor IXa. A typical protocol was as follows. Human
factor IX (10 µg/ml in 120 µl of TBS containing 1 mg/ml BSA and
various concentrations of Ca and Mg
ions) was incubated with 2.5 ng/ml factor XIa at 37 °C for 30
min. The reaction was stopped by the addition of 15 µl of a
solution of EDTA/Ca
/Mg
;
Ca
and Mg
ions were included so
that all the samples in a given set of experiments would include the
same amounts of metal ions at this step, and the concentration of EDTA
was adjusted to neutralize both the Ca
and
Mg
ions in the sample. Factor IXa in the samples was
then quantified; one part of the sample (50 µl) was mixed with one
part of plasma deficient in factor IX and one part of a suspension of
phospholipids (phosphatidylcholine/phosphatidylserine (3:1, w/w; both
from Sigma); 1 mg/ml in TBS) and equilibrated at 37 °C for 2 min.
Fifty µl of 20 mM CaCl
were then added, and
the time required for clotting was measured in an Amelung Coagulometer
KC 4A. The amount of factor IXa was determined by use of a standard
curve that had been prepared with pure human factor IXa
that
contained the same amounts of EDTA/Ca
/Mg
as the tested samples; the logarithm of the clotting time was
plotted against the logarithm of the concentration of standard factor
IXa. The low concentrations of factor XIa and the rather long initial
incubation period rendered the secondary activation of factor IX during
the subsequent clotting assay negligible and allowed the accurate
quantification of factor IXa. Note that sensitivity of the
quantification is strongly dependent on the preparation of factor
IX-deficient plasma, and the use of plasma with the lowest possible
level of factor IX is necessary for the determination of extremely low
levels of factor IXa. The preparations that we used were congenitally
deficient human plasma (Sigma) or human plasma from which factor IX had
been immunodepleted with monoclonal antibody (a gift from the
Chemo-Sero Therapeutic Research Institute, Kumamoto, Japan). For the
determination of kinetic parameters, concentrations of substrate
ranging from 0.2 to 1.2 µM were utilized and the
incubation period was set to ensure the linearity of the reaction. The
data obtained were analyzed by construction of Eadie-Hofstee plots.
-bound conformations of Gla domains in factors IX
and X.
Approximately 1 mM Ca
ions is sufficient for binding to both factors(14) ,
consistent with the affinities of the Gla domains for Ca
ions. By contrast, Mg
ions alone were without
effect (see Fig. 2). When Mg
ions were added
together with Ca
ions, however, the extent of binding
of IX/X-bp to factor IX was much increased (Fig. 1). In this
experiment, the effect of the addition of Mg
ions on
the binding of
I-IX/X-bp to solid-phase factor IX or X
was investigated in the presence of 1 mM Ca
ions. Upon the addition of Mg
ions, the binding
to factor IX was greatly enhanced whereas the binding to factor X was
unchanged. The effect of further addition of Ca
ions
was modest for both proteins (data not shown). Since the same amount of
Ca
ions had little effect and since the effect of
Mg
ions was specific for factor IX, the observed
phenomena appeared not to be due to a mere increase in the ionic
strength of the incubation medium. Increase in the ionic strength of
the medium tended, in fact, to inhibit the binding. In subsequent
experiments, we focused our attention on the effects of Mg
ions on factor IX. As is shown in Fig. 2, the
Ca
requirement curve for the binding to factor IX was
shifted leftward, implying an enhancement of sensitivity to
Ca
ions by Mg
ions. Furthermore,
the augmentation was clearly seen even at saturating concentrations of
Ca
ions. The affinity of IX/X-bp for factor IX was
increased by the addition of Mg
ions, and the
apparent K
value for the binding of these
proteins was decreased from 3.7 ± 1.6
10
M (3 mM Ca
alone; mean
± S.E., n = 4) to 1.3 ± 0.1
10
M (3 mM Ca
+ 3 mM Mg
), whereas that for
factor X was unchanged (3.5 ± 0.6
10
M and 3.5 ± 1.3
10
M in the absence and presence of Mg
ions, respectively). Half-maximal and maximal augmentation
occurred at 0.3 and 3 mM Mg
, respectively.
It was revealed recently that IX/X-bp is capable of binding
Ca
ions and other polyvalent metal ions, and
occupation of the two Ca
-binding sites in IX/X-bp is
a prerequisite for the recognition of Gla domains.
It is
unlikely, however, that the effect of Mg
ions
involves IX/X-bp directly, because the binding to factor X was
unaffected by Mg
ions. It seems that IX/X-bp does not
interact with Mg
ions; Ca
binding
to IX/X-bp was not altered at all by high concentrations of
Mg
ions (Fig. 3). These observations strongly
suggest that factor IX is the molecule that interacts directly with
Mg
ions. The result that relatively low
concentrations of Mg
ions at saturating
concentrations of Ca
ions were still effective
suggests that Mg
ion(s) interact at specific site(s)
that are separate from the putative Ca
-binding sites
in factor IX. The binding of Mg
ion(s) to this
specific site(s) appears to promote a further conformational change of
factor IX, yielding a form distinct from the form that is achieved by
binding of Ca
ions alone.
Figure 2:
Augmentation of binding of IX/X-bp to
factor IX by Mg ions. Binding of
I-IX/X-bp to solid-phase bovine factor IX was examined in
the presence of Ca
ions at indicated concentrations
with (open circles) or without (closed circles) 3
mM Mg
ions. Total radioactivity was 25,000
cpm. Other conditions were as in Fig. 1.
Figure 1:
Effects of
Mg ions on the binding of
I-IX/X-bp to
factors IX and X. Wells of microtiter plates coated with bovine factor
IX (left) or factor X (right) were incubated with
I-IX/X-bp in the presence of 1 mM Ca
plus Mg
ions at the indicated concentrations,
as described under ``Experimental Procedures.'' Bound
radioactivity is expressed as the percentage of the control value (1
mM Ca
alone). Total radioactivities and
control values for factors IX and X were 44,000 (total) and 2,200 and
10,500 (controls) cpm, respectively.
Figure 3:
Absence of an effect of Mg ions on the binding of Ca
ions to IX/X-bp.
Binding of Ca
ions to IX/X-bp was determined by
equilibrium dialysis with
Ca
as the
tracer as described under ``Experimental Procedures.'' Moles
of bound Ca
/mol of IX/X-bp (r) were plotted
as a function of the concentration of Ca
ions. Open circles indicate the results in the presence of 10 mM magnesium acetate. In the controls (closed circles), 30
mM sodium acetate was included to normalize the ionic
strength.
To confirm our
hypothesis we employed different probes, in an effort to exclude any
possible effects of Mg ions on IX/X-bp. A pool of
polyclonal antibodies raised against bovine factor IX was fractionated
by affinity chromatography on immobilized factor IX according to the
dependence on metal ions of the recognition of the antigen by the
method of Liebman et al.(11) . Anti-factor IX:Ca(II)
antibodies obtained in this way showed absolute Ca
dependence for the binding, while Mg
ions were
ineffective, a feature identical to that originally described. The
Ca
-dependent binding of the antibodies to bovine
factor IX was also potentiated by the addition of Mg
ions (Fig. 4). Low concentrations of Mg
ions augmented the binding in the presence of very high
concentrations of Ca
ions. Similar results were again
obtained with Ca
-dependent monoclonal antibodies
against human factor IX (Fig. 5). The epitopes of these
monoclonal antibodies were located within the Gla domain. Taking this
result together with the results obtained with IX/X-bp, we can see that
the tertiary structure of the Gla domain is greatly influenced by
Mg
ions. The conformation adopted by the Gla domain
in the presence of both cations is apparently different from that
adopted with either cation by itself.
Figure 4:
Augmentation of the binding of anti-factor
IX:Ca(II) antibodies to factor IX by Mg ions. Binding
of isolated anti-factor IX:Ca(II) antibodies to bovine factor IX was
determined by ELISA in the presence of Ca
ions at the
indicated concentrations, as detailed under ``Experimental
Procedures.'' Closed circles, Ca
alone; open squares, Ca
plus 0.3 mM Mg
; open circles, Ca
plus 3 mM Mg
.
Figure 5:
Augmentation of the binding of
Gla-domain-directed monoclonal antibodies to factor IX by
Mg ions. Binding of two different monoclonal
antibodies (right, C1; left, 2A2) to human factor IX
was determined by ELISA in the presence of Ca
ions at
the indicated concentrations as in Fig. 4. Closed circles,
Ca
alone; open squares, Ca
plus 0.3 mM Mg
; open circles,
Ca
plus 3 mM Mg
.
We also prepared
conformation-specific polyclonal antibodies against human prothrombin,
bovine factors VII and X, and bovine protein C by the same method as
that employed for the preparation of anti-factor IX:Ca(II) antibodies,
and we investigated the effects of Mg ions on the
binding of the respective antibodies to these proteins. The antibodies
again had absolute Ca
dependence, and Mg
ions alone were again ineffective. However, the
Ca
-dependent binding of these antibodies was barely
affected by the addition of Mg
ions, in contrast to
the case with factor IX. Augmentation of the binding was not seen in
the presence of excess Ca
ions (5-10
mM) in each case. In the presence of suboptimal concentrations
of Ca
ions (of the order of 0.1 mM), slight
leftward shifts in the Ca
titration curves by
Mg
ions were seen in some cases, but these effects
were negligible (data not shown). These results strongly suggest but do
not prove that all of the Gla-containing coagulation proteases other
than factor IX do not respond to Mg
ions. It seems
thus that factor IX is unique among various vitamin K-dependent
coagulation factors in such a way that its conformation is stabilized
not only by Ca
ions but also by Mg
ions.
ions on function of factor IX. The inactive zymogen factor IX is
converted to the active protease factor IXa by physiological activators
factor XIa or factor VIIa/tissue factor/phospholipid
complex(20) , or by the snake venom coagulant protease
RVV-X(21) . The Ca
-bound conformation of
factor IX is considered to be essential for efficient activation by
each of these activators. However, factor VIIa per se also
requires Ca
ions for the formation of the active
complex with tissue factor and phospholipids. Such a requirement is
also known for RVV-X, which is yet another metal-binding
protein(22) . These features would clearly hinder evaluation of
the effects of metal ions on the activation by these activators. By
contrast, factor XIa itself does not need Ca
ions,
and the effects of Ca
ions can be considered to be
solely those exerted on factor IX(23) . We therefore employed
factor XIa to examine whether the Mg
-induced further
conformational change in factor IX might affect its activation
kinetics. Human factor IX was incubated with human factor XIa in the
presence of Ca
and Mg
ions, and the
amount of factor IXa generated was quantified as described under
``Experimental Procedures.'' Addition of Mg
ions increased the rate of activation of factor IX in the
presence of Ca
ions, while Mg
ions
alone were ineffective (Fig. 6). In the presence of 1 mM Mg
, the necessary Ca
concentration was lowered by approximately 1 order of magnitude.
The rate of activation in the presence of excess Ca
ions, which reached a plateau value, was also increased by
Mg
ions. The shapes of curves and the range of
effective Ca
concentrations obtained here closely
resemble those obtained with the conformation-specific probes (compare Fig. 6with Fig. 2, 4, and 5). It appears that the
additional conformational change in factor IX induced by Mg
ions renders the molecule susceptible to factor XIa. To gain
further insight, we determined the kinetic parameters for the
activation of factor IX in the absence and presence of 1 mM
Mg
ions; the results are summarized in .
The parameters that we obtained are consistent with those in the
literature, which were determined by a different method (23, 24). In
the presence of a saturating concentration of Ca
ions
(5 mM), the K
value was 0.31
µM, which is still higher significantly than the plasma
concentration of factor IX (
0.1 µM). Addition of
Mg
ions reduced the apparent K
by a factor of 1.7, while the apparent V
value was not altered. In the presence of a physiological
concentration of Ca
ions (1 mM), the effect
of Mg
ions was far more striking; the K
was reduced from 0.91 to 0.24
µM (Fig. 7, ). Under these conditions, k
was slightly (
20%) reduced by
Mg
ions. These results mean that, if we assume that
concentrations of free Ca
ions and factor IX in the
plasma are 1 mM and 0.1 µM, respectively, the
initial velocity of factor IX activation under physiological conditions
in the presence of Mg
ions can be calculated to be
2.5 times higher than that in the absence of Mg
ions,
according to the Michaelis-Menten equation. With the results of this
calculation in mind, we verified the significance of Mg
ions by conducting an experiment in which physiological
conditions were simulated. Factor IX at 10 µg/ml (close to the
concentration in plasma) was activated in the presence of both cations
at approximately physiological concentrations (1 mM
Ca
+ 1 mM Mg
), and
the time course of activation was monitored. The activation proceeded
efficiently, but when Mg
ions were omitted the rate
of activation was much lower (Fig. 8). These data strongly
suggest that Mg
ions, present in blood plasma at
millimolar concentrations, are indeed involved in the activation of
factor IX in vivo.
Figure 6:
Acceleration of factor XIa-induced
activation of factor IX by Mg ions. Human factor IX
(10 µg/ml) was incubated with human factor XIa (2.5 ng/ml) for 30
min at 37 °C in the presence of Ca
ions at the
indicated concentrations, and then the amounts of factor IXa formed
were measured as described under ``Experimental Procedures.'' Closed circles, Ca
alone; open
circles, Ca
plus 1 mM Mg
.
Figure 7:
Effects of Mg ions on
the kinetics of activation of factor IX. Human factor IX at various
concentrations was activated by 2.5 ng/ml human factor XIa for 30 min
at 37 °C in the presence of 1 mM Ca
alone (closed circles) or 1 mM Ca
plus 1 mM Mg
(open circles).
The data were analyzed by construction of Eadie-Hofstee plots (v = -K(v/s) + V
).
Figure 8:
Time course of the activation of factor IX
under physiological conditions. Human factor IX (10 µg/ml) was
incubated with human factor XIa (2.5 ng/ml) at 37 °C in the
presence of either 1 mM Ca alone (closed
circles) or 1 mM Ca
plus 1 mM Mg
(open circles) for indicated
periods, and then the amounts of factor IXa formed were
measured.
ions.
The N-terminal Gla domain is the major Ca
-binding
site, and a dozen or so of Ca
ions bind to this small
segment with affinities comparable to the plasma concentration of
Ca
ions (K
1
mM)(3, 26, 27) . Binding of
Ca
ions to the Gla domain is essential for
maintenance of the native functional conformation of factor IX, which
is crucial for recognition and subsequent activation by specific
activators. The active form (factor IXa) produced in this way also
exhibits biological activity only when the Gla domain is associated
with the full complement of Ca
ions.
ions has been definitively proven,
as described above, but it is not yet understood whether Mg
ions, another physiological constituent of blood plasma, also
participate in coagulation. Various biochemical studies have been
undertaken to demonstrate interactions between various metal ions and
vitamin K-dependent coagulation factors, which include factor
IX(4, 5, 6, 7, 8, 9, 10, 11, 12, 26, 27) .
However, most studies have focused mainly on interactions with
Ca
ions, and little attention has been paid to the
action of Mg
ions. We have shown here that
Mg
ions also influence the tertiary structure of
factor IX using three independent structural probes. The first probe
was IX/X-bp, which recognizes the Gla domains of both factors IX and X
from any species(13, 14) ; the second one was a
preparation of conformation-specific polyclonal antibodies against
bovine factor IX (anti-factor IX:Ca(II) antibodies; Ref. 11); and the
third one was a preparation of monoclonal antibodies directed to the
Gla domain of human factor IX. All of these probes recognized factor IX
in the presence of millimolar concentrations of Ca
ions, while Mg
ions alone were ineffective.
Since the Gla domain binds Ca
ions with a K
value of
1 mM and
exhibits a dramatic conformational change upon the binding of
Ca
ions, we can conclude that these probes recognized
the Ca
-bound conformation of this module. Magnesium
ions at relatively low concentrations augmented binding of these
Gla-domain-directed probes even in the presence of excess
Ca
ions (Fig. 2, 4, and 5). These observations
indicate that factor IX has specific binding site(s) for Mg
ion(s) that do not interact with Ca
ion(s) and,
moreover, that the Gla domain that has already been filled with
Ca
ions undergoes a further conformational transition
upon binding of Mg
ion(s). Note that this conclusion
does not necessarily imply that the Mg
-specific
site(s) is within the Gla domain or that Mg
ions do
not affect the shape and/or function of other modules that form the
factor IX molecule (e.g. epidermal growth factor-like
modules). The Mg
-specific binding site(s) has not yet
been identified, and we are currently attempting to localize it using
protease-digested fragments of factor IX(28) . It is of interest
to note that Amphlett et al.(26) reported previously
that factor IX has a unique binding site for a Mn
ion
that cannot be filled by a Ca
ion even at extremely
high concentrations of Ca
ions. It is possible that
the natural ligand for this metal-binding site is a Mg
ion. This issue awaits clarification.
ions seems to be
specific for factor IX, since other coagulation factors, i.e. prothrombin, factors VII and X, and protein C, were not responsive
to Mg
ions. As is seen in Fig. 1, the binding
of IX/X-bp to factor X was not affected by Mg
ions.
The Ca
-dependent binding of conformation- specific
polyclonal antibodies directed to these proteins was barely affected by
Mg
ions. Prendergast and Mann (29) reported
previously that activation of prothrombin by prothrombinase complex
(factor Xa plus factor Va, phospholipids, and Ca
ions) was potentiated by Mg
ions at millimolar
concentrations when the concentration of Ca
ions was
low. We also examined the effect of Mg
ions on the
activation of prothrombin and obtained a similar result, but when the
activation was monitored in the presence of physiological
concentrations of Ca
ions (>1 mM), the
augmentation by Mg
ions no longer occurred (data not
shown). We conclude, therefore, that factor IX is the only molecule
among various vitamin K-dependent coagulation factors whose
conformation and function are regulated by Mg
ions
under physiological conditions. The conformation of (and probably the
mechanism of the metal-ion-induced conformational change in) factor IX
may differ somewhat from that of other Gla-containing coagulation
factors.
-bound conformers (Ca(II) antibodies),
and the other has lower specificity and recognizes the antigen in the
presence of various polyvalent metal ions, such as Mg
(Mg(II) antibodies)(10, 11) . The existence of two
different classes of metal-dependent antibodies clearly indicates that
these proteins can adopt at least three different conformations,
depending on the presence of metal ions. Moreover, the full biological
activities of the proteins are seen only in the presence of
physiological concentrations of Ca
ions, and other
cations cannot be substituted. Hence, the conclusions of Furie and
co-workers can be summarized as shown in Fig. 9(upper
panel). In this model, the metal-free form of the protein (IX) is
first converted to an intermediate, dysfunctional form (IX`) upon the
binding of Mg
ions (or other polyvalent cations
including Ca
ions when they are present at low
concentrations). Only Ca
ions at millimolar
concentrations elicit the transition to final functional form (IX*). By
contrast, our data show unequivocally that Mg
ions
actually play a crucial role in the regulation of the conformation and
function of factor IX. From our present observations, we present an
advanced model (Fig. 9, lower panel). In our model,
Ca
ions are essential but are insufficient to bring
about a full conformational change, and both Ca
and
Mg
ions are necessary. Since Mg
ions are a normal constituent of plasma, the conformer IX should
represent the physiological form. Furthermore, since the conformer IX
is the preferred substrate for factor XIa as compared to the conformer
IX*, Mg
ions are also crucial for the function of
factor IX. In addition, the function of Mg
ions is
cooperative with respect of Ca
ions rather than
additive. The leftward shifts in Ca
titration curves
( Fig. 2and Fig. 4-6) clearly imply an increase in
the affinity for Ca
ions of this protein. The
conformational rearrangement of factor IX induced by Mg
ions appears to make the molecule better able to bind
Ca
ions. This point is extremely important if we
consider the physiological level of the cations. The physiological
level of free Ca
ions (1-2 mM) is
insufficient to bring about the full conformational change and
concomitant expression of biological activity. When Mg
ions are present, however, the folding of the factor IX molecule
can be completed at this relatively low concentration of Ca
ions, allowing factor IX to function with maximum potency.
Figure 9:
The
role of Ca and Mg
ions in the
regulation of the conformation of factor IX. Upper panel, the
original hypothesis proposed by Liebman et al. The scheme
presented in Ref. 11 has been modified to stress the action of
Mg
ions. Only Ca
ions at
physiological concentrations induce the conformational transition to
the functional form (IX*), whereas Mg
ions or any
other polyvalent cations (or Ca
ions at low
concentrations) can induce a partial transition to a dysfunctional form
(IX`). Lower panel, a modified hypothesis, based on the
present results. The maximally active, functional form of factor IX
(IX**), which is equivalent to the physiologically active conformer, is
generated only when both Ca
and Mg
ions are present, and either cation alone can induce only a
partial transition (to IX` or IX*).
Factor IX is converted to the active protease either by factor XIa
(the intrinsic pathway) or by factor VIIa/tissue factor (the extrinsic
pathway). Factor IXa then forms a complex with factor VIIIa and anionic
phospholipids in the presence of Ca ions and, in
turn, activates factor X. Although it has long been known that factor
VIIa/tissue factor can directly activate factor X, it was shown
recently and unequivocally that factor IX is the preferred substrate of
this activator; the value of k
/K
for factor IX
is approximately 10 times higher than that for factor X(30) . It
now seems conceivable that the primary substrate of factor VIIa/tissue
factor in vivo is factor IX, and that the activation of factor
X is a secondary event, which, in large part, is mediated by factor
IXa/factor VIIIa subsequent to the exposure of tissue factor to the
bloodstream. On the other hand, the contribution of factor XI (and its
activator factor XII) to the initiation of coagulation is considered to
be relatively small, at least under normal physiological conditions
(31). This notion is supported by a number of clinical investigations
that indicate that abnormalities in factor VIII or IX result in a
severe tendency to hemorrhage, hemophilia, whereas persons who have
defects in the initiators of the intrinsic pathway (i.e. activators of factor XI, factor XII(32) , and high
molecular weight kininogen (33, 34)) are asymptomatic. Defects
in factor XI do, however, cause mild but significant hemorrhagic
tendencies (35, 36). These contradictory observations can be
explained by the recently identified positive feedback system for
factor XI activation that is mediated by thrombin (37, 38).
Factor XI may not be involved in the initiation of coagulation but
should play an important role in the amplification phase subsequent to
initiation via the mechanism associated with the extrinsic pathway.
Lawson et al. (39) recently reported that tissue
factor-induced thrombin generation in a reconstituted system that
included purified coagulation factors was indeed potentiated in the
presence of factor XI. However, they did not include Mg
ions in the reaction mixture. The significance of factor XI might
thus have been underestimated as can be deduced from our present
observations. In this regard, our finding that Mg
ions facilitate efficient activation contribute to a much better
understanding of the physiological significance of this process.
ions on the activation of factor IX by
factor VIIa/tissue factor and on the activation of factor X by factor
IXa remain to be determined. It seems likely that both of these
processes are positively modulated by Mg
ions since
these two processes are also strongly dependent on the correct folding
of the Gla domain of factor IX/IXa, and since Mg
ions
do affect the tertiary structure of this module. We are currently
investigating these issues. Our preliminary results indicate that the
time required for factor IXa-induced clotting is considerably decreased
by the addition of Mg
ions, while factor Xa-induced
clotting is not affected.
(
)These results imply
that the activation of factor X by factor IXa is also accelerated by
Mg
ions.
ions. Magnesium ions
appear to be another important regulator of hemostasis. At present,
there is a vast literature about the role of Ca
ions
in blood coagulation but we believe that most studies (in particular,
those with reconstituted systems composed of purified components) have
to be re-examined in view of the demonstrated effects of Mg
ions. Our findings should contribute to a further understanding
of the mechanism of coagulation, the process that is one of the major
subjects of today's medical sciences.
Table: Kinetic parameters of activation of factor IX by
factor XIa
values were calculated on the assumption that
all the active sites of factor XIa were intact. The data of Sinha et al. (23) were obtained by determination of the release of
H-labeled activation peptide from factor IX.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.