(Received for publication, August 25, 1995)
From the
The contribution of the catalytic and noncatalytic domains of
factor IXa to the interaction with its cofactor, factor VIIIa, was
evaluated. Two proteolytic fragments of factor IXa, lacking some or all
of the serine protease domain, failed to mimic the ability of factor
IXa to enhance the reconstitution of factor VIIIa from isolated
A1/A3-C1-C2 dimer and A2 subunit. Both fragments, however, inhibited
this factor IXa-dependent activity. Selective thermal denaturation of
the factor IXa serine protease domain eliminated its effect on factor
VIIIa reconstitution. Modification of factor IXa with
dansyl-Glu-Gly-Arg chloromethyl ketone (DEGR-IXa) stabilized this
domain, and heat-treated DEGR-IXa retained its ability to enhance
factor VIIIa reconstitution. These results indicate the importance of
the serine protease domain as well as structures residing in the factor
IXa light chain (-carboxyglutamic acid and/or epidermal growth
factor domains) for cofactor stabilizing activity. In the presence of
phospholipid, the A1/A3-C1-C2 dimer produced a saturable increase in
the fluorescence anisotropy of fluorescein-Phe-Phe-Arg chloromethyl
ketone-modified factor IXa (Fl-FFR-IXa). This effect was inhibited by a
factor IXa fragment comprised of the
-carboxyglutamic acid and
epidermal growth factor domains. The difference in Fl-FFR-IXa
anisotropy in the presence of A1/A3-C1-C2 dimer (
r = 0.043) compared with factor VIIIa (
r = 0.069) represented the contribution of the A2 subunit. A
peptide corresponding to factor VIII A2 domain residues 558-565
decreased the factor VIIIa dependent-anisotropy of Fl-FFR-IXa to a
value similar to that observed with the A1/A3-C1-C2 dimer. These
results support a model of multiple interactive sites in the
association of the enzyme-cofactor complex and localize sites for the
A1/A3-C1-C2 dimer and the A2 subunit to the factor IXa light chain and
serine protease domain, respectively.
Factors VIII and IX are essential plasma glycoproteins, which
when absent or defective, result in hemophilia A and hemophilia B,
respectively. Factor VIII is synthesized as a 300-kDa
protein(1, 2) with domain structure A1-A2-B-A3-C1-C2 (3) that is subsequently processed and circulates in plasma as
a series of Me heterodimers(4, 5, 6) . Factor VIII is
converted to its active form, factor VIIIa, upon proteolytic cleavage
by thrombin(7) . Factor VIIIa is a heterotrimer composed of the
A1, A2, and A3-C1-C2 subunits. (
)The A1 and A3-C1-C2
subunits retain the Me
ion linkage and can be
isolated as a stable dimer(8, 9) . The A2 subunit is
weakly associated with the dimer primarily through electrostatic forces (10) and at physiologic pH readily dissociates resulting in the
loss of factor VIIIa activity(11, 12, 13) .
Under the appropriate conditions, factor VIIIa activity can be
reconstituted from the isolated A2 subunit and the A1/A3-C1-C2
dimer(10, 12, 14) .
Factor IX circulates
in blood as a single chain zymogen. It is composed of an NH terminus Gla (
)domain which is rich in
-carboxyglutamic acid, followed by two EGF-like domains, a
35-residue activation peptide and the serine protease domain (see (15) for review). In the intrinsic blood coagulation pathway,
factor IX is activated to factor IXa by factor XIa and
Ca
(15) . Upon activation, the 35-residue
activation peptide is released to yield factor IXa light chain,
comprised of the Gla and two EGF domains, linked via a disulfide bond
to the heavy chain which contains the serine protease
domain(16) .
Factor VIIIa functions as a cofactor for the
serine protease factor IXa, which in the presence of Ca and a membrane surface, converts factor X to factor Xa. Factor
VIIIa increases the k
for this reaction by
several orders of magnitude(17) . Association of factor VIIIa
with factor IXa in the presence of phospholipid stabilizes cofactor
activity(18) . While reconstitution of heterotrimer factor
VIIIa from the isolated A2 subunit and A1/A3-C1-C2 dimer is enhanced
severalfold in the presence of factor IXa and
phospholipid(14) , prolonged incubation of factor VIIIa with
factor IXa results in a loss of factor VIIIa activity due to
proteolytic cleavage within the A1 domain(19) . However, stable
enhancement of factor VIIIa reconstitution can be achieved in the
presence of active site-inhibited factor IXa(14) .
Sites of
interaction between factor IXa and its cofactor are not well defined.
In addition to its role in binding phospholipid (20) and
endothelial cell (21) surfaces, the isolated factor IX Gla
domain has also been shown to inhibit the factor VIIIa-dependent
conversion of factor X to Xa by factor IXa(22) . Furthermore, a
monoclonal antibody specific for an epitope within the heavy chain of
factor IXa was shown to interfere with the binding of factor VIIIa to
factor IXa suggesting the serine protease domain also interacts with
factor VIIIa(23) . Similarly, little information is known about
sites within factor VIII(a) involved in its interaction with factor
IXa. Recently it has been shown that the light chain of factor VIII
(likely within the A3 domain) contains a high affinity binding site for
factor IXa(24) . In addition, peptides corresponding to the
sequence surrounding the activated protein C cleavage site in the A2
subunit of factor VIIIa (Arg)(8) , inhibit the
factor IXa-dependent enhancement of factor VIIIa reconstitution (25) . Thus, interaction of factor IXa and factor VIIIa appears
to involve multiple sites on each protein.
In this study we demonstrate that the serine protease domain of factor IXa is essential for stabilizing the structurally labile, factor VIIIa heterotrimer and likely contributes to this association by interaction with the A2 subunit. Additional results obtained with proteolytic fragments of factor IX indicate that the Gla and/or EGF domains contain an interactive site for the A1/A3-C1-C2 dimer factor VIIIa. These results support a model for the factor VIIIa-factor IXa interaction consistent with proposed surface proximal and surface distal regions of the molecules.
Factor IXa was modified with active site specific reagent DEGR-CK as described elsewhere(31) . Fluorescein-Phe-Phe-Arg-factor IXa (Fl-FFR-IXa) was purchased from Molecular Innovations, Wayne MI.
Fluorescence measurements of the thermal
unfolding of factor IXa and DEGR-IXa were performed with a protein
concentration of 0.1 mg/ml by monitoring the ratio of intrinsic
fluorescence intensity at 350 nm to that at 320 nm with excitation at
280 nm in an SLM 8000-C fluorometer. The temperature was controlled
with a circulating water bath programmed to raise the temperature at
1 °C/min. Changes in the ratio parameter provide a sensitive
method for detecting melting transitions and determination of their
midpoints (T
).
where r is the anisotropy of Fl-FFR-IXa in
the absence of added A1/A3-C1-C2 dimer, r
is the
anisotropy of Fl-FFR-IXa in the presence of saturating levels of dimer. f
is the fraction of Fl-FFR-IXa binding sites that
are occupied by the dimer and is defined as,
where B is the concentration of bound A1/A3-C1-C2 dimer
and E is the total concentration of Fl-FFR-IXa.
The concentration of the bound dimer is given by the quadratic equation
where K is the dissociation constant and C
is the total concentration of A1/A3-C1-C2 dimer.
For experiments in which FPR-IXa or the 25-kDa factor IX fragment were added to displace Fl-FFR-IXa, reactions initially contained Fl-FFR-IXa (20 nM) in buffer A with 0.1 mg/ml PSPCPE. The change in Fl-FFR-IXa anisotropy upon addition of A1/A3-C1-C2 (50 nM) was determined. Subsequent anisotropy measurements were made following successive additions of either FPR-IXa or the 25-kDa factor IX fragment.
Figure 1:
Melting curves
of factor IXa and DEGR-IXa. Fluorescence detected thermal denaturation
of factor IXa and DEGR-IXa in 20 mM Hepes, pH 7.2, 100 mM NaCl, 5 mM CaCl and 0.01% Tween-20 was
determined as described under ``Materials and
Methods.''
To examine the role the serine protease domain of factor IXa serves in its interaction with factor VIIIa, we took advantage of the sensitivity of this domain to elevated temperature and monitored the capacity of factor IXa to enhance reconstitution of factor VIIIa from isolated subunits. Factor IXa or DEGR-IXa was incubated for 5 min at 60 °C prior to reaction with factor VIIIa subunits. This treatment destroyed greater than 98% of the proteolytic activity of factor IXa as determined by its inability to convert factor X to factor Xa in the presence of factor VIIIa and phospholipid (data not shown). Reconstitution of factor VIIIa activity from the isolated A1/A3-C1-C2 dimer and the A2 subunit was carried out in the presence of the various factor IXa forms (Fig. 2). Untreated, native factor IXa resulted in a transient enhancement of factor VIIIa reconstitution that decayed over the time course of the reaction, consistent with our previously reported observation(14) . The transient nature of this reaction reflects the subsequent inactivation of factor VIIIa following factor IXa-catalyzed cleavage within the A1 subunit(19, 35) . Modification of the factor IXa active site by DEGR-CK yields catalytically inactive enzyme that still retains its ability to enhance factor VIIIa reconstitution (14) (Fig. 2, diamonds). Furthermore, the enhancement activity observed with DEGR-IXa is stable and occurs to a greater extent since the protease activity is nonfunctional. However, heat-treated factor IXa was unable to enhance the reconstitution of factor VIIIa activity while heat treated-DEGR-IXa, whose serine protease domain was stabilized by the active site modification, retains its ability to enhance factor VIIIa reconstitution. These results suggest that the serine protease domain is necessary for the factor IXa-dependent enhancement of this factor VIIIa intersubunit interaction.
Figure 2:
Effect of heat treatment on factor IXa
enhancement of factor VIIIa reconstitution. A1/A3-C1-C2 (20
nM) and A2 (20 nM) were incubated in buffer A (20
mM Hepes, pH 7.2, 100 mM NaCl, 1 mM
CaCl, 0.01% Tween-20) containing 0.5 mg/ml bovine serum
albumin, and phospholipid (0.1 mg/ml inosithin) with the following: no
additions (
), 5 nM factor IXa (
), 5 nM heat-treated factor IXa (
), 5 nM DEGR-IXa
(
), 5 nM heat-treated DEGR-IXa (
). Heat-treated
factor IXa or heat-treated DEGR-IXa were incubated at 60 °C for 5
min prior to use in the reconstitution assay. Reactions were initiated
by the addition of A2 subunit and aliquots were assayed at the
indicated times for factor VIIIa activity in a one-stage clotting
assay.
To determine whether the Gla and/or EGF domains
participate in the interaction between factor IXa and factor VIIIa,
competition experiments were performed in which the ability of the
25-kDa factor IX fragment to inhibit the DEGR-IXa-dependent enhancement
of factor VIIIa reconstitution was examined. As shown in Fig. 3,
factor VIIIa reconstitution from 10 nM subunits was enhanced
approximately 8-fold in the presence of 10 nM DEGR-IXa.
However, the 25-kDa fragment was able to inhibit this
DEGR-IXa-dependent effect with 50% inhibition occurring at 4
µM. The 45-kDa fragment, which contained the Gla and EGF
domains as well as the amino-terminal portion of the serine protease
domain, also inhibited the DEGR-IXa-dependent enhancement of
reconstitution with 50% inhibition seen at
3 µM (data
not shown). This effect did not result from titration of the
phospholipid vesicles by the high levels of fragment in the reaction
since increasing the phospholipid concentration 10-fold failed to
reverse the inhibition (data not shown). This result suggests that the
Gla and/or EGF domains of factor IX interact with one or more factor
VIIIa subunits, and this interaction, like the interaction with the
serine protease domain, is necessary for the factor IXa-dependent
stabilization of factor VIIIa.
Figure 3: Inhibition of DEGR-IXa enhancement of factor VIIIa reconstitution by the 25-kDa factor IX fragment. A1/A3-C1-C2 (10 nM) was preincubated for 5 min in buffer A containing inosithin (0.4 mg/ml) with no additions or the indicated concentration of 25 kDa factor IX fragment. DEGR-IXa (10 nM) was then added and reconstitution was initiated by the addition of A2 (10 nM). Aliquots of each reaction were assayed after 60 min for factor VIIIa activity. The fold enhancement of factor VIIIa activity is plotted relative to a reaction in which no DEGR-IXa was present.
Figure 4:
Binding of A1/A3-C1-C2 to Fl-FFR -IXa.
Reactions contained Fl-FFR-IXa (20 nM), and the indicated
concentration of A1/A3-C1-C2 in buffer A with phospholipid (0.1 mg/ml
PSPCPE). The fluorescence anisotropy was measured as described under
``Materials and Methods.'' Values from the fitted constants
were K = 11.1 ± 6.7
nM, n = 5.5 ± 0.52, r
= 0.1988 ± 0.0012, and r
= 0.2668 ±
0.0033.
The ability of a nonfluorescent, active site modified factor IXa molecule, FPR-IXa to inhibit the binding of Fl-FFR-IXa to the A1/A3-C1-C2 dimer was examined. The fluorescence anisotropy of 20 nM Fl-FFR-IXa and 50 nM A1/A3-C1-C2 dimer in the presence of 0.1 mg/ml PSPCPE was determined. Addition of increasing concentrations of FPR-IXa produced a decrease in the anisotropy demonstrating that binding to the active site-modified factor IXa was reversible (Fig. 5).
Figure 5:
Inhibition of A1/A3-C1-C2 binding to
Fl-FFR-IXa. Reactions contained Fl-FFR-IXa (20 nM),
A1/A3-C1-C2 (50 nM) in buffer A with phospholipid (0.1 mg/ml
PSPCPE). FPR-IXa () or 25-kDa factor IX fragment (
) were
added as indicated and fluorescence anisotropy was measured.
Fluorescence anisotropy of Fl-FFR-IXa in the presence of A1/A3-C1-C2
and absence of inhibitor was 0.238. The anisotropy of Fl-FFR-IXa alone
was 0.205.
To determine the role of
the Gla and/or EGF domains in the interaction of factor IXa and the
A1/A3-C1-C2 dimer, the 25-kDa factor IX fragment was used as an
inhibitor of the dimer-dependent increase in anisotropy. Successive
additions of the factor IX fragment produced a decrease in the
fluorescence anisotropy in a reaction containing 20 nM Fl-FFR-IXa and 50 nM A1/A3-C1-C2 in the presence of 0.1
mg/ml PSPCPE vesicles. A 50% inhibition of the anisotropy increase
occurred at 1 µM. This fragment was observed to be
about 10-fold less potent of an inhibitor of Fl-FFR-IXa anisotropy
compared with the intact FPR-IXa molecule.
Recently, we observed that a peptide
corresponding to factor VIII residues 558-565 of the A2 subunit
noncompetitively inhibited factor Xase activity (K
100 µM) as well as eliminated the factor
IXa-dependent enhancement of factor VIIIa reconstitution from isolated
subunits(25) . This result indicated an interaction of factor
VIIIa A2 subunit with factor IXa. The ability FVIII
to affect the fluorescence anisotropy of Fl-FFR-IXa upon
association of factor VIIIa or the A1/A3-C1-C2 dimer was examined. As
shown in Table 1, in the presence of 300 µM peptide,
the anisotropy of Fl-FFR-IXa alone was unaffected, whereas the
magnitude of the increase in anisotropy observed upon association of
Fl-FFR-IXa and factor VIIIa was reduced to a value similar to that seen
when the A1/A3-C1-C2 dimer is used. Furthermore, inclusion of the
FVIII
peptide had no effect on the anisotropy
value measured in presence of the A1/A3-C1-C2 dimer. That
FVIII
blocks the A2-dependent incremental
increase in anisotropy rather than abolishing the total factor VIIIa
(or A1/A3-C1-C2)-dependent anisotropy increase suggests that the A2
subunit may interact within the serine protease domain, at or near the
active site of factor IXa.
The interaction of factor IXa with its cofactor, factor VIIIa, was investigated by evaluating the contribution of both the catalytic (serine protease) and noncatalytic (Gla and two EGF) domains of the molecule. In the present study, we demonstrate that a structurally intact serine protease domain is required for interaction with factor VIIIa, as judged by the factor IXa-dependent enhancement of factor VIIIa reconstitution. Two proteolytic fragments of factor IX in which all or part of the serine protease domain are deleted, failed to enhance factor VIIIa reconstitution. Furthermore, selective thermal denaturation of the serine protease domain abolished the ability of the molecule to enhance factor VIIIa reconstitution. Modification of the factor IXa active site with DEGR-CK stabilized this domain, preventing the irreversible denaturation at the elevated temperature. This heat-treated DEGR-IXa retained the ability to enhance factor VIIIa reconstitution.
The above results indicate the importance of the serine protease domain for the cofactor stabilizing activity and are consistent with previous studies that proposed a factor VIIIa-interactive site on the factor IXa heavy chain. Earlier studies described a monoclonal antibody that binds an epitope within factor IXa heavy chain (serine protease domain) residues 181 and 310 (36) and interferes with subsequent binding of factor VIIIa(23) . However, the exact mechanism of this inhibition is unknown since creation of recombinant factor IX molecules, in which surface residues in this region were individually replaced with factor X residues from the homologous position, eliminated or reduced affinity for the antibody yet retained normal clotting activity(37) .
A role for the noncatalytic domains of factor IXa in the interaction
with its cofactor was indicated by the capacity of the 25-kDa factor IX
fragment to inhibit the DEGR-IXa-dependent enhancement of factor VIIIa
reconstitution. Localization of factor VIIIa interactive sites within
these domains remains controversial. Lin et al.(38) reported that recombinant factor IX in which the two
EGF-like domains have been replaced by the corresponding domains of
factor X possessed only 4% normal biological activity, while
recombinant factor IX, in which only the first EGF domain was replaced
with the corresponding region from factor X, retained normal activity.
This suggests that the first EGF domain is not likely to be involved in
the interaction with factor VIIIa. In contrast, mutational analysis has
indicated that Tyr, located within the first EGF domain,
is essential for the factor VIIIa-dependent conversion of factor X by
factor IXa(39) . Evidence that the Gla domain may be required
for interaction with factor VIIIa is based on the observation that a
fragment of bovine factor IX consisting of the isolated Gla domain
weakly inhibited (K
10 µM) the
factor VIIIa-dependent conversion of factor X to Xa(22) .
Fluorescence anisotropy has been used to investigate the interaction
of factor IXa and factor VIIIa(33, 40) . In this
report, we demonstrate that the binding of the A1/A3-C1-C2 dimer to
Fl-FFR-IXa in the presence of phospholipid is a high affinity
interaction (K = 11.1 ± 6.7
nM) which is capable of altering the active site. Previous
work from our laboratory suggested that the presence of both the A2
subunit and the A1/A3-C1-C2 dimer were required for the observed
increase in the fluorescence polarization of DEGR-IXa (14) .
This disparity may be attributed to fluorophore sensitivity, since a
greater increase in factor VIIIa-dependent anisotropy was observed when
fluorescein (
r = 0.09) rather than dansyl
(
r = 0.03) was the reporter group (33) .
The similar affinity of the above interaction to interactions of factor
IXa with the free factor VIII light chain (K
14 nM) (24) and human factor VIIIa (K
23 nM) (27) suggests
little if any contribution of factor VIII heavy chain-derived subunits
(A1 and A2) to the binding energy. However, factor VIII light chain
produces a fractional increase the fluorescence anisotropy of
Fl-FFR-IXa (
)compared with the A1/A3-C1-C2 dimer, suggesting
that the A1 subunit may contribute to the change in conformation near
the active site. FPR-IXa eliminated the dimer-dependent anisotropy
increment by displacing Fl-FFR-IXa. The 25-kDa factor IX fragment was
also able to displace Fl-FFR-IXa from the complex demonstrating a
direct physical interaction of the Gla and/or EGF domains with the
A1/A3-C1-C2 subunit. The observed 10-fold reduction in inhibitor
potency of the fragment compared with the intact FPR-FIXa molecule
could result from partial denaturation of the fragment during isolation
and/or indicate that additional sites of dimer interaction are located
within the serine protease domain.
Heterotrimeric factor VIIIa
produces a greater increase in the anisotropy of Fl-FFR-IXa than the
A1/A3-C1-C2 dimer, thus suggesting a role for the A2 subunit in
modulating the factor IXa active site. Recently, Duffy et al.(33) showed that the fluorescence anisotropy of factor IXa
is increased in the presence of factor VIII and that a further
incremental increase is observed upon thrombin-catalyzed activation.
This incremental increase likely reflects contribution from cleavage of
both the light chain (Arg) and the heavy chain
(Arg
) of factor VIII. A hybrid factor VIII molecule
possessing a native heavy chain plus thrombin-cleaved light chain
increased the anisotropy of Fl-FFR-IXa to a value that was intermediate
to that of factor VIII and factor VIIIa(40) . Furthermore,
addition of thrombin to fully activate the hybrid was required to
observe the maximal anisotropy effect. Recently, we reported that
residues 558-565 of the A2 subunit contained a factor IXa
interactive site based upon the observation that a peptide
corresponding to this region was able inhibit the factor
VIIIa-dependent conversion of factor X to Xa as well as inhibit the
factor IXa-dependent enhancement of factor VIIIa reconstitution (25) . We now show that this peptide can block the interaction
of the A2 subunit with factor IXa based upon its capacity to eliminate
the A2-dependent contribution of the anisotropy increase of Fl-FFR-IXa
in the presence of factor VIIIa. The ability of A2 subunit to alter the
conformation around the active site is compatible with the localization
of this interaction to the serine protease domain of factor IXa.
Based upon the above results we suggest the following model for the
relative orientation of factors VIIIa and IXa and surface in the
intrinsic tenase complex. Factor IXa is an elongated molecule with its
light chain (Gla domain) involved in binding the phospholipid surface,
whereas the heavy chain contains the serine protease domain (see (41) for review). Recently, Mutucumarana et al.(42) determined that the active site of factor IXa is located
high above the membrane surface (>70 Å), and this distance is
unaffected by the presence of factor VIIIa. However, the conformation
around the active site of factor IXa is altered by the
cofactor(42) . The A3-C1-C2 subunit of factor VIIIa contains
the binding site for phospholipid (C2 domain) (43) as well as
the high affinity site for factor IXa (A3 domain)(24) . Since
this factor VIIIa subunit is also surface proximal, it is reasonable to
speculate an interaction between the A3 domain and the light chain of
factor IXa. This inference is supported by the inhibition of the
A1/A3-C1-C2 dimer-dependent anisotropy increase of Fl-FFR-IXa by the
25-kDa fragment. The factor VIIIa A1 subunit is linked by divalent
metal ion(s) to the A3-C1-C2 subunit and is likely oriented above the
surface since its presence is required for the anisotropy effect and it
contains a site (Arg) (19) proteolyzed by factor
IXa. We predict that the factor VIIIa A2 subunit is also localized
above the surface since it contains a primary cleavage site for
activated protein C (Arg
)(8) , an homologous
enzyme to factor IXa (see (41) for review) whose active site
is likely similarly positioned. Additional support for the surface
distal positioning of A2 subunit is inferred from its direct effect on
the magnitude of the factor VIIIa-dependent anisotropy of Fl-FFR-IXa
when the contribution of A2 is eliminated. Thus, we propose that factor
IXa contains at least two interactive sites for factor VIIIa and
include: (i) a surface-proximal site possessing a high affinity
interaction and involving the light chain of factor IXa and the factor
VIII A3-C1-C2 subunit and (ii) a weak affinity, surface-distal site(s)
involving participation of the serine protease domain of factor IXa and
the A2 (plus possibly A1) subunit(s) of factor VIIIa.