(Received for publication, January 17, 1996; and in revised form, February 15, 1996)
From the
We recently showed that not only Ca ions but
also Mg
ions play a crucial role in stabilizing the
native conformation of coagulation factor IX. We here report that
Mg
ions at physiological concentrations greatly
augment the biological activities of factor IX. In clotting assays with
dialyzed plasma, addition of Mg
ions enhanced the
apparent coagulant activity of factor IXa, while that of factor Xa was
scarcely affected. Activation of factor X by factor IXa in the presence
of factor VIIIa, phospholipids, and Ca
ions was
accelerated by Mg
ions. It appeared that the cation
increased the affinity between factor IXa and factor VIIIa, thereby
increasing the apparent catalytic efficacy of the enzyme. We also
evaluated the effect of Mg
ions in the coagulation
pathway initiated by tissue factor and found that activation of factor
IX by factor VIIa
tissue factor was accelerated by the cation.
Consequently, clotting of normal plasma induced by factor
VIIa
tissue factor was shortened by the cation, while no such
effect was observed in plasma deficient in factor IX or VIII. These
results indicate that the previously unrecognized plasma component,
Mg
ions, plays crucial roles in blood coagulation
and, moreover, that contributions of factors IX and VIII in the
coagulation cascade have been seriously underestimated in previous
investigations.
The coagulation proenzyme factor IX plays a central role in
hemostasis in concert with its essential procofactor factor VIII, as is
apparent from the fact that genetic defects in these factors cause a
life-threatening tendency to bleed, hemophilia(1) . Once
activated, factor IX forms a complex with the activated form of factor
VIII (IXaVIIIa) (
)on phospholipid membranes in the
presence of Ca
ions. The enzyme complex catalyzes
efficient activation of factor X, and factor Xa in turn converts
prothrombin to thrombin, which ultimately leads to successful formation
of hemostatic plugs. In the classical cascade model of coagulation,
factors VIII and IX are classified as components of the
``intrinsic pathway'' and are placed downstream of factor XI
( (2) and (3) ; for recent review, see (4) and (5) ). However, it is now considered that the intrinsic pathway
is insignificant in normal hemostasis, since individuals who lack
initiators of this mechanism, i.e. factor XII(6) ,
high molecular weight kininogen(7) , and prekallikrein (8) , are all asymptomatic. To today's best knowledge,
the single mechanism that solely governs blood coagulation is the
so-called ``extrinsic pathway,'' which starts with the
exposure of the extravascular component, tissue factor, to the
bloodstream and formation of the active factor VIIa
tissue factor
complex (VIIa
TF). The complex is able to activate factor X
without a requirement for factors VIII and IX. Why, then, do
hemophiliacs experience such severe hemorrhagic diatheses? This
question was partly answered by the finding that VIIa
TF can
activate not only factor X but also factor IX(9) . Since then,
evidence has accumulated for the indispensable roles of factors VIII
and IX in the coagulation pathway initiated by tissue factor. Komiyama et al.(10) showed that factor IX is preferred to
factor X as a substrate for VIIa
TF. More recently, Mann and
co-workers (11, 12) demonstrated that the
VIIa
TF-induced thrombin generation is strongly dependent on
factor VIII. These studies clearly demonstrated the significance of
factors VIII and IX. With these observations in mind, we attempted to
validate the importance of these proteins considering a previously
unrecognized factor, Mg
ions.
Calcium ions
(concentration in plasma: total, 2.2-2.6 mM; free,
1.1-1.3 mM) are the essential constituents of the
coagulation cascade. However, participation of Mg ions (total, 0.8-1.2 mM; free, 0.4-0.6
mM) in this mechanism has been ignored.
Nonetheless, we
found recently that folding of the native tertiary structure of factor
IX requires not only Ca but also Mg
ions(13) . The Ca
-dependent binding of
various conformation-specific probes was greatly enhanced by
Mg
ions at physiological concentrations even in the
presence of excess Ca
ions. This result suggests that
factor IX has a specific binding site(s) for Mg
ions
that does not interact with Ca
ions, and, moreover,
that the binding of Mg
ions promotes additional
changes in the Ca
-bound conformation of the molecule.
We failed to detect such Mg
-induced additional
conformational changes in other vitamin K-dependent coagulation
factors, such as factor X. Moreover, the Ca
-dependent
activation of factor IX by the intrinsic pathway activator factor XIa
was accelerated by Mg
ions with a dramatic reduction
in the apparent K
(13) . Thus, the
Mg
-induced additional conformational change appears
to enhance the protein's ability to function. The activation by
factor XIa is, however, not essential for physiological
coagulation(11) . Therefore, we examined the effect of
Mg
ions on the function of factor IX in the
coagulation pathway initiated by VIIa
TF. Our results shed light
on the indispensable roles of factors VIII and IX in vivo.
We examined the effect of Mg ions on
clotting. Pooled normal plasma was dialyzed to remove endogenous metal
ions and the exogenously added anticoagulant, sodium citrate. It was
then incubated with activated forms of coagulation factors and
phospholipids. Clotting was initiated by addition of Ca
ions (plus Mg
ions) at concentrations close to
physiological. When factor XIa was used as initiator, clotting times
were reduced by Mg
ions. Approximately 10-fold lower
concentrations of factor XIa were sufficient to yield the same clotting
times as seen in the absence of Mg
ions (Fig. 1A). This result was reasonable because
Mg
ions potentiate factor IX activation by factor XIa (13) . Next, we tested the effect of Mg
ions
on factor IXa-induced clotting. Shorter clotting times were again
obtained with Mg
ions (Fig. 1B). The
apparent clotting activity of factor IXa was increased approximately
3-fold by Mg
ions. By contrast, the cation had a
minimal effect on factor Xa-induced clotting (Fig. 1C).
It was thus clear that Mg
ions accelerated not only
factor IX activation by factor XIa but also factor X activation by
factor IXa, while all the processes after the generation of factor Xa
were unmodified. This result is consistent with our previous result
that, among various vitamin K-dependent coagulation enzymes, only
factor IX is responsive to Mg
ions(13) .
Figure 1:
Effects of Mg ions on
clotting of normal plasma induced by factors XIa, IXa, and Xa. Dialyzed
normal plasma was incubated with indicated concentrations of factor XIa (A), factor IXa (B), or factor Xa (C) plus
200 µM phospholipids (75% PC, 25% PS; w/w). Clotting was
initiated by the addition of metal ions. Detailed methods are given
under ``Experimental Procedures.'' Closed circles,
2.5 mM Ca
alone; open circles, 2.5
mM Ca
+ 1 mM Mg
.
We next investigated the activation of factor X by factor IXa in the
presence of factor VIIIa and phospholipids. Factor X at a physiological
concentration (0.2 µM) was activated in the presence of
various concentrations of Ca ions (Fig. 2A). The activation rate at any Ca
concentrations was increased by Mg
ions, while
Mg
ions alone were ineffective. When a 10-fold higher
concentration of factor X was used, the amount of factor Xa generated
and the effect of Mg
ions were essentially the same
(data not shown). These results indicated that the physiological level
of factor X was considerably higher than the K
value so that the reaction could proceed at maximum velocity (V
), and that Mg
ions
increased the catalytic efficacy (k
) of the
enzyme. Indeed, the reported K
values of the
IXa
VIIIa complex (approximately 0.06 µM) are well
below the plasma concentration of factor X(19, 20) .
When we varied the concentration of factor VIIIa, a pronounced leftward
shift of the required cofactor concentration was observed in the
presence of Mg
ions (Fig. 2B). No
such shift was observed upon varying the concentration of phospholipids (Fig. 2C). It appeared that Mg
ions
augmented the coagulant activity of factor IXa by increasing the
affinity of the enzyme for factor VIIIa rather than for phospholipids.
This result reflects the observed effect on catalytic efficacy, since
it is known that factor VIIIa increases the apparent k
while phospholipids decrease the apparent K
(19, 20) .
Figure 2:
Effects of Mg ions on
the activation of factor X by factor IXa. A, dose-response to
Ca
ions. Factor X (0.2 µM) was incubated
with 1 nM factor IXa in the presence of 2 units/ml
thrombin-activated factor VIIIa, 10 µM phospholipids (75%
PC, 25% PS), and various concentrations of Ca
(plus 1
mM Mg
) for 5 min. B, dose-response
to factor VIIIa. Factor X was activated as in A in the
presence of the indicated concentrations of factor VIIIa.
Concentrations of Ca
, Mg
, and
phospholipids were fixed at 1 mM, 0.5 mM, and 30
µM, respectively. C, dose-response to
phospholipids. Factor X was activated as in A in the presence
of indicated concentrations of phospholipids. Concentrations of
Ca
, Mg
, and factor VIIIa were fixed
at 1 mM, 0.5 mM, and 10 units/ml, respectively.
Detailed methods are given under ``Experimental Procedures.'' Closed circles, Ca
alone; open
circles, Ca
+
Mg
.
We also evaluated
the effect of Mg ions on VIIa
TF-initiated
coagulation, the main physiological pathway. We tested the effect on
factor IX activation in a purified system. Factor IX at a physiological
concentration (0.1 µM) was incubated with VIIa
TF and
the factor IXa generated was quantified. The cation increased the rate
of activation, and the velocity increased approximately 2-fold by
Mg
ions under the experimental condition (Fig. 3). This effect of Mg
ions seemed rather
small and, in plasma, the effect was more striking (see below). We are
presently uncertain of the reason of this quantitative discrepancy but,
nevertheless, it appeared that the cation also has a positive effect on
factor IX activation.
Figure 3:
Effects of Mg ions on
the activation of factor IX by VIIa
TF. Factor IX (0.1
µM) was incubated with 1 nM factor VIIa plus 0.1
nM tissue factor reconstituted on 30 µM phospholipid vesicles (75% PC, 25% PS) for indicated periods. The
amount of factor IXa generated was quantified as described under
``Experimental Procedures.'' Closed circles, 1
mM Ca
alone; open circles, 1 mM Ca
+ 0.5 mM Mg
.
We next investigated the effect of
Mg ions on VIIa
TF-induced clotting. Tissue
factor, reconstituted on vesicles of 75% PC, 25% PS, and factor VIIa
were added to dialyzed plasma. Clotting times were again decreased by
Mg
ions in normal plasma and approximately 5 times
lower concentrations of tissue factor was sufficient to yield the same
clotting time (Fig. 4A). However, the cation was
ineffective in factor IX- or VIII-deficient plasma (data not shown).
These results suggest that the cation is indeed involved in the
mechanism of physiologic coagulation through its action on factor IX.
We also examined the effect of Mg
ions with
phospholipids of different composition. Gilbert and Arena (21) recently showed that PE, a constituent of natural
procoagulant membranes (activated platelets), provides high affinity
binding sites for factor VIII in membranes with low PS content and
thereby facilitates efficient assembly of the IXa
VIIIa complex.
We employed a preparation of phospholipids with a composition (75% PC,
20% PE, 5% PS) much closer to physiological than frequently used, and
re-evaluated VIIa
TF-induced clotting. This preparation was
reported to be almost equipotent to the preparation of 75% PC, 25% PS
with respect to the assembly of the active IXa
VIIIa
complex(21) . We expected that the contribution of factors VIII
and IX would be emphasized and thus the effect of Mg
ions would be more apparent.
Figure 4:
Effects of Mg ions on
clotting of plasma induced by VIIa
TF. A, dialyzed plasma
was challenged by indicated concentrations of tissues factor
reconstituted on vesicles of 75% PC, 25% PS (250 µM) plus
1 nM factor VIIa. In B and C, phospholipids
consisting of 75% PC, 20% PE, 5% PS (250 µM) were used,
and other conditions were as in A. A and B,
normal plasma; C, factor IX-deficient plasma. Closed
circles, 2.5 mM Ca
alone; open
circles, 2.5 mM Ca
+ 1 mM Mg
.
Indeed, the effect of
Mg ions was very striking with this preparation (Fig. 4B), and the cation again had minimal effects in
plasma deficient in factor IX (Fig. 4C) or factor VIII
(data not shown). Raising the concentration of tissue factor hardly
decreased the clotting time without Mg
ions (i.e. under conditions wherein the apparent coagulant activity of factor
IX was attenuated), suggesting that the rate of generation of factor Xa
should be reduced considerably. By contrast, factor Xa should be
produced very rapidly in the presence of Mg
ions. It
appeared that VIIa
TF-induced factor Xa generation is very
strongly dependent on Mg
ions and this action is
solely mediated through the effects on factor IX/IXa. It is thus
strongly suggested that most factor Xa is generated by IXa
VIIIa,
not by the direct action of VIIa
TF, in blood plasma where both
Ca
and Mg
ions are present.
Magnesium ions potentiated all the factor IX-dependent processes in
coagulation, and the cation appeared to be a crucial constituent of the
coagulation cascade. Our data also underline the physiological
significance of factors VIII and IX. The effect of Mg ions on the activation by factor IXa of factor X is especially
noteworthy. The cation augmented the apparent catalytic efficacy of
factor IXa by increasing the affinity of the protease for the cofactor.
The plasma concentration of factor VIII is quite low (0.7 nM)
as compared with those of factor IX (90 nM) and other
components(11) . Moreover, the active form, factor VIIIa,
readily loses its activity either through spontaneous inactivation or
through degradation by activated protein C(22, 23) .
Therefore, only minute amounts of factor VIIIa should be available
during coagulation, and its availability seems to represent a critical
bottleneck in the cascade. Our present observation that Mg
ions facilitated efficient factor Xa generation with much lower
amounts of factor VIIIa than those needed with Ca
alone (Fig. 2B) effectively explains how the
hemostatic process in vivo is guaranteed.
Although the cascade (or waterfall) theory (2, 3) constitutes a fundamental biochemical basis for the mechanism of coagulation and, in principle, it remains a viable theory after several revisions, erroneous descriptions in early cascade models still seriously influence the current models, found in many textbooks (e.g.(24) ). Because the original model was based on observations in test tubes rather than in vivo, it explains the mechanism of widely used diagnostic tests in vitro, i.e. APTT (activated partial thromboplastin time) and PT (prothrombin time). However, it does not reflect the actual hemostatic process. The most obvious discrepancy between the model and events in vivo is the role of factors VIII and IX, which have been classified as components of the intrinsic pathway. The intrinsic pathway, whose importance was stressed in the original hypothesis, now seems likely to be a mere in vitro artifact. Only the extrinsic (or tissue factor) pathway is operative in vivo.
Since the discovery of the mechanism of factor IX activation by
VIIaTF(9) , many investigations have been undertaken to
re-evaluate the roles of factors VIII and IX in the cascade. Kinetic
parameters of activation of factors IX and X by VIIa
TF (10) provide a solid biochemical basis for the hypothesis that
factor IX is the preferred substrate of the enzyme complex. Mann and
co-workers(11, 12) definitively demonstrated the
importance of factors VIII and IX in the tissue factor pathway through
elucidation of the detailed activation kinetics of each constituent in
a reconstitution system containing plasma concentrations of factors V,
VIII, IX, and X and prothrombin. The effects of Mg
ions were, however, not examined in these earlier studies. Since
Mg
ions enhanced the coagulant activity of factor IX
in the tissue factor pathway, the contributions of factors VIII and IX
in this pathway seem to be much greater than previously assumed. In
addition, the differences in lipid composition of procoagulant
membranes between those used experiments in vitro (containing
large amounts of PS; 20-30%) and those available in vivo (containing rather small amounts of PS;
5%) also must be
considered as pointed by Gilbert and Arena (21) . Thus, the
relative significance of each reaction step that involves
protein/phospholipid interactions might be different from that
predicted from investigations with artificial membranes.
The
significance of factors VIII and IX has clearly been seriously
underestimated in previous laboratory investigations. We propose here a
revised cascade theory of coagulation, which includes a novel
component, the Mg ion, and is much simpler than
previous models (Fig. 5). Although several important reactions (e.g. conversion of procofactors to active cofactors) are
omitted from the figure for simplicity, our model illustrates the main
elements of the principal chain reaction, in which an active protease
activates another downstream protease zymogen. We have also omitted the
mechanism of factor X activation by VIIa
TF for clarity. The
evidence shows unequivocally that most, if not all, factor Xa is
generated by the action of IXa
VIIIa. However, the significance of
direct activation cannot be ignored. As suggested by Hoffman et al.(25) , the amount of factor Xa generated directly by
VIIa
TF at the very early phase of coagulation is, by itself, very
limited but it does produce thrombin, perhaps in a minuscule amount but
perhaps sufficient for activation of platelets in the vicinity and,
thus, for promotion of the expression of procoagulant surfaces. This
small amount of factor Xa at the early phase should be immediately
neutralized by plasma protease inhibitors and is unlikely to
participate in the wholesale generation of thrombin at the later
propagation phase. However, it might play a role in the initiation of
coagulation. Our hypothesis must be examined by detailed analyses and
is now open to discussion. We believe, however, that this simple model
can explain the events in our bodies much better than previous models,
and it also provides a clue to the answer of the long-standing
question: why do hemophiliacs hemorrhage so severely?
Figure 5:
The main stream of the blood coagulation
cascade: a revised hypothesis. This model focuses on the main pathway
of the chain reaction of protease activation and stresses the action of
Mg ions. Some important accessory reactions have been
omitted for clarity. To reproduce the precise events that occur in
vivo, we must consider the following: activation of factor X by
VIIa
TF (see text), conversion of procofactors (V and VIII) to
active cofactors, inactivation of factors Va and VIIIa via the
thrombomodulin/protein C pathway(23) , feedback activation of
factor XI by thrombin(26, 27) , neutralization of
proteases by serpins, in particular by antithrombin III and tissue
factor pathway inhibitor (27) , and so on. We must also
consider the effects of certain cellular mechanisms (in particular
participation of platelets) and blood flow for an understanding of the
dynamic hemostatic system. PL,
phospholipids.