(Received for publication, July 18, 1995; and in revised form, November 10, 1995)
From the
In previous studies we have shown that the interaction between
factors IXa and VIII involves the light chain of factor VIII and that
this interaction is inhibited by the monoclonal antibody CLB-CAg A
against the factor VIII region Gln-Asp
(Lenting, P. J., Donath, M. J. S. H., van Mourik, J. A., and
Mertens, K.(1994) J. Biol. Chem. 269, 7150-7155).
Employing distinct recombinant factor VIII fragments, we now have
localized the epitope of this antibody more precisely between the A3
domain residues Glu
and Met
. Hydropathy
analysis indicated that this region is part of a major hydrophilic
exosite within the A3 domain. The interaction of factor IXa with this
exosite was studied by employing overlapping synthetic peptides
encompassing the factor VIII region
Tyr
-Ala
. Factor IXa binding was
found to be particularly efficient to peptides corresponding to the
factor VIII sequences Lys
-Lys
and
Glu
-Gln
. The same peptides proved
effective in binding antibody CLB-CAg A. Further analysis revealed that
peptides Lys
-Lys
and
Glu
-Gln
interfere with binding of factor
IXa to immobilized factor VIII light chain (K
0.2 mM and 0.3 mM, respectively).
Moreover, these peptides inhibit factor X activation by factor IXa in
the presence of factor VIIIa (K
0.2
mM and 0.3 mM, respectively) but not in its absence.
Equilibrium binding studies revealed that these two peptides bind to
the factor IX zymogen and its activated form, factor IXa, with the same
affinity (apparent K
0.2
mM), whereas the complete factor VIII light chain displays
preferential binding to factor IXa. In conclusion, our results
demonstrate that peptides consisting of the factor VIII light chain
residues Lys
-Lys
and
Glu
-Gln
share a factor IXa binding
site that is essential for the assembly of the factor X-activating
factor IXa-factor VIIIa complex. We propose that the overlapping
sequence Glu
-Lys
comprises the
minimal requirements for binding to activated factor IX.
Human blood coagulation factor VIII (FVIII) ()is an
essential protein of the hemostatic system, which is evident from the
severe bleeding disorder hemophilia A that is associated with FVIII
deficiency or dysfunction (1) . FVIII is synthesized as a
single chain polypeptide containing a number of discrete domains
arranged in the sequence A1-A2-B-A3-C1-C2 (2, 3) .
Examination of its primary structure reveals that FVIII shares
considerable homology with the plasma proteins factor V (FV) and
ceruloplasmin(4, 5, 6) . Whereas
ceruloplasmin comprises a triple A domain structure (A1-A2-A3), FV
displays the same domain structure(7, 8) . In contrast
to FV, FVIII predominantly circulates as a heterodimeric protein,
consisting of a Me
-linked light and heavy chain (9, 10, 11) . The heavy chain contains the
A1-A2-B domains and is heterogeneous (M
90,000-200,000) due to limited proteolysis at a number of
positions within the B domain. The light chain of FVIII (M
80,000) comprises the domains
A3-C1-C2(10, 12) .
In the intrinsic pathway of
blood coagulation, FVIII functions as a nonenzymatic cofactor in the
factor X (FX)-activating complex(13) . Within this complex, the
serine protease factor IXa (FIXa) activates FX in the presence of
calcium ions, phospholipids, and activated FVIII. In order to play its
role in the generation of FXa, FVIII has to be
activated(14, 15) . Activation is achieved by limited
proteolysis in both the FVIII heavy and light chain by FXa or
thrombin(12) , which results in the formation of a
heterotrimeric product, FVIIIa(16, 17) . The
relatively labile FVIIIa heterotrimer is known to be stabilized by the
enzyme FIXa in the presence of phospholipids(18) . In addition,
it has been reported that the phospholipid-FIXa complex enhances the
reassociation of isolated FVIIIa subunits into the FVIIIa heterotrimer (19) , indicating that FVIIIa is capable of directly
interacting with FIXa.
Several studies have been performed in order
to characterize the assembly of the FIXFVIII complex in more
detail(19, 20, 21, 22) . The FVIII
heavy chain regions Ser
-Gln
and
Arg
-Ser
have been recognized to
represent FIXa interactive sites(22, 23) . Previously,
we have shown that FVIII light chain comprises an exosite that binds
FIXa with high affinity(21) . In the same study, we found that
the FIXa-FVIII light chain interaction was inhibited by the anti-FVIII
antibody CLB-CAg A, which is known to bind to the FVIII A3 domain
region Gln
-Asp
(24) . In the
present study, we addressed the possibility that this region is
involved in the assembly of the enzyme-cofactor complex. Therefore, we
first located the binding site of antibody CLB-CAg A in more detail.
Subsequently, a series of synthetic peptides was employed in order to
define the FVIII region involved in FIXa binding. This approach allowed
us to identify the FVIII light chain region
Glu
-Lys
as being involved in FIXa
binding and in the assembly of the FX-activating FIXa
FVIIIa
complex.
Figure 1:
Immunoprecipitation of FVIII fragments
with monoclonal antibodies. Recombinant FVIII fragments were obtained
as outlined under ``Experimental Procedures.'' The fragments
were labeled by in vitro translation employing
[S]methionine(24) . Immunocomplexes were
analyzed by 12% (w/v) SDS-polyacrylamide gel electrophoresis and
subsequent autoradiography. Lanes represent immunocomplexes of
the radiolabeled FVIII fragments from Asp
to
Ser
(lanes 1 and 5), Glu
(lanes 2 and 6), Glu
(lanes
3 and 7) and Met
(lanes 4 and 8) with antibodies CLB-CAg 69 and CLB-CAg A, respectively. The
residues Lys
-Arg
encompass the
previously defined epitope for antibody CLB-CAg
69(24) .
Figure 2:
Synthetic peptides of the FVIII A3 domain.
Kyte-Doolittle hydropathy analysis (32) of the FVIII A3 domain
sequence using a sliding window of 19 amino acids is shown on top. The most hydrophilic region comprises the residues
Arg-Asp
. Peptides overlapping the
FVIII region Tyr
-Ala
that are used
in this study are shown below. The primary sequence of the
FVIII region Tyr
-Ala
is represented
using the single-letter code. The lines below the amino acid
sequence denote the inclusive amino acid residues of the peptides
synthesized.
Figure 3:
Binding of CLB-CAg A and FIXa to
immobilized synthetic peptides. Antibody CLB-CAg A (625 nM) or
FIXa (50 nM) were incubated with immobilized synthetic
peptides (0.8 nmol/well added) in 1% (w/v) HSA, 0.1% (v/v) Tween 20, 10
mM CaCl, 0.1 M NaCl, 25 mM Tris
(pH 7.4) for 1 h at 37 °C. After washing the microtiter plate with
the same buffer without HSA, bound CLB-CAg A (solid bars) and
FIXa (shaded bars) were quantified by incubating for 15 min at
room temperature with peroxidase-labeled goat anti-mouse antibodies and
peroxidase-labeled anti-FIX antibody CLB-FIX 14, respectively, as
described under ``Experimental Procedures.'' Binding is
expressed as percentage of the maximum response. Data represent the
mean ± S.D. of at least three experiments. Peptides are
indicated by the positions of the amino- and carboxyl-terminal amino
acid in the corresponding FVIII sequence (cf.Fig. 2).
Figure 4:
Effect of synthetic peptides on FIXa-FVIII
light chain interaction and FX activation. A, Glu-Gly-Arg
chloromethyl ketone-treated FIXa (30 nM) was incubated with
immobilized FVIII light chain (0.7 pmol/well) in 0.15 M NaCl,
1% (w/v) HSA, 0.1% (v/v) Tween 20, 5 mM CaCl, 25
mM histidine (pH 6.2) for 4 h at 37 °C in the presence of
various concentrations of peptide Gly
-Lys
(
), Lys
-Lys
(
), or
Glu
-Gln
(
). FIXa binding
and calculation of binding parameters was performed as described
previously (21) . Binding parameters revealed a K
of 0.19 ± 0.01 mM and
0.27 ± 0.02 mM for peptides
Lys
-Lys
and
Glu
-Gln
, respectively. B,
activation of FX (0.2 µM) by FIXa (0.7 nM) in the
presence of phospholipids (0.1 µM), FVIIIa (0.4
nM), and various concentrations of peptide
Gly
-Lys
(
),
Lys
-Lys
(
), or
Glu
-Gln
(
) in 3 mM CaCl
, 0.1 M NaCl, 0.2 mg/ml HSA, 0.05 M Tris (pH 7.4) was assayed as described(31) . FXa formation
was quantified as described under ``Experimental
Procedures.'' The data represent the mean ± S.D. of three
experiments.
Figure 5:
Kinetic analysis of FX activation in the
presence of peptide Lys-Lys
. FXa
generation experiments were performed in the absence (
) or
presence of 0.2 mM (
), 0.4 mM (
), or 0.6
mM (
) of the peptide
Lys
-Lys
as described under
``Experimental Procedures,'' except that variable FX
concentrations (2.5-50 nM) were used. Initial rates of
FXa formation are plotted as a function of the substrate concentration.
Data represent the mean ± S.D. of three experiments. The curves were obtained by fitting the data employing the
Michaelis-Menten equation. The calculated apparent V
values in the absence or presence of 0.2 mM, 0.4
mM, or 0.6 mM peptide were 6.0 ± 0.3, 4.5
± 0.2, 3.8 ± 0.2, and 2.8 ± 0.1 nM FXa/min, respectively (mean ± S.D.). The apparent K
values were 5.3 ± 1.0, 5.0
± 0.7, 6.1 ± 0.9, and 4.6 ± 0.8 nM FX,
respectively.
Figure 6:
Binding of FIX or FIXa to immobilized
peptide Lys-Lys
or FVIII light
chain. Various concentrations of Glu-Gly-Arg chloromethyl
ketone-treated FIXa (
) or FIX (
) were incubated with
immobilized FVIII light chain (0.7 pmol/well) as described under
``Experimental Procedures.'' Association between FVIII light
chain and FIX or FIXa was assessed as described(21) . Inset, various concentrations of Glu-Gly-Arg chloromethyl
ketone-treated FIXa (
) or FIX (
) were incubated with
immobilized peptide Lys
-Lys
(0.8
nmol/well added) as described under ``Experimental
Procedures.'' Binding was detected employing the
peroxidase-labeled anti-FIX antibody CLB-FIX 14. Absorbance was
measured at 450 nm using 540 nm as reference. Plotted is the absorbance versus the concentration of FIX or FIXa. Data represent mean
values ± S.D. of three to six
experiments.
Figure 7:
Effect of FVIII light chain on binding of
FIX or FIXa to peptide Lys-Lys
. FIX
(
) or FIXa (
) (both 50 nM) were incubated with
immobilized peptide Lys
-Lys
(0.8
nmol/well added) in the presence of various concentrations of FVIII
light chain as described under ``Experimental Procedures.''
Binding is presented as percentage of binding in the absence of FVIII
light chain. The data represent the mean ± S.D. of three
experiments.
During the process of FX activation, the enzyme FIXa
assembles with the nonenzymatic cofactor FVIIIa into a lipid-bound
complex. In previous studies we have shown that FVIII light chain
contains a site that binds FIXa with high affinity (K
15 nM) and that FIXa binding is inhibited by the
FVIII light chain-directed antibody CLB-CAg A(21) . Here we
show that this antibody is directed against an extensive hydrophilic
exosite within the A3 domain ( Fig. 2and Fig. 3). Because
such hydrophilic regions are likely to be exposed at the exterior of
the protein(32) , we addressed the possibility that this
exosite comprises a FIXa binding site. Indeed, FIXa binds to synthetic
peptides that consist of FVIII sequences that are part of the
hydrophilic exosite Arg
-Asp
(Fig. 3). Competition studies demonstrated that peptides
corresponding to the exosite regions
Lys
-Lys
and
Glu
-Gln
effectively inhibit binding
of FIXa to immobilized FVIII light chain (Fig. 4A). The
same peptides also interfere with FVIII-dependent activation of FX by
FIXa (Fig. 4B). Inhibition of FX activation is
noncompetitive (Fig. 5), which strongly suggests that the
peptides inhibit the enzyme FIXa by binding at a site distinct from the
substrate binding pocket. Collectively, our data demonstrate that
peptides consisting of the FVIII amino acid residues
Lys
-Lys
and
Glu
-Gln
represent a FIXa binding
site. It is of importance to note that the K
for
the binary FIXa-FVIII light chain interaction is similar to the K
found for FX activation in the complete FX
activating complex, thus including the entire FVIIIa heterotrimer ( Fig. 4and Fig. 5). Assembly of the functional
FIXa
FVIIIa complex apparently is directly related to binding of
FIXa to the FVIII A3 domain exosite. In this respect it should be
mentioned that FIXa binding is not an exclusive property of the FVIII
A3 domain. FIXa recognition sites have been identified within the FVIII
A2 domain regions Ser
-Gln
(22) and
Arg
-Ser
(23) . As synthetic
peptides corresponding to these A2 domain regions also interfere with
FVIIIa cofactor function, it seems reasonable to assume that both FVIII
heavy chain and light chain regions participate in FIXa-FVIIIa complex
formation.
As peptides Lys-Lys
and Glu
-Gln
proved more
efficient in their interaction with FIXa than the other peptides tested
( Fig. 3and Fig. 4), we propose that the minimal
requirements for FIXa binding are met by the overlapping residues
Glu
-Lys
. This region, including its
direct environment (residues
Gly
-Gln
), is strikingly rich in
basic Lys residues, which are located at positions 1804, 1808, 1813,
and 1818 (Fig. 8). These Lys residues appear to be unique for
the FVIII A3 domain, as they are not only lacking in the FVIII A1- and
A2 domains but also in the A3 domains of the structurally related
proteins FV and ceruloplasmin (Fig. 8). The same Lys residues
are conserved in the FVIII A3 domain of a rodent species (Fig. 8), which would be compatible with the involvement of
these residues in a FVIII A3 domain-specific event such as FIXa
binding. However, peptide Gly
-Lys
with Lys residues at 1804, 1808, and 1813 proved considerably
less efficient in its interaction with FIXa than peptide
Glu
-Gln
with Lys residues at 1813
and 1818 ( Fig. 3and Fig. 4). Apparently, the presence of
the Lys residues alone is not sufficient for FIXa binding. It should be
mentioned that peptide Glu
-Gln
contains a triplet of aromatic residues
(Tyr
-Phe
-Trp
), which is
also present in the inhibitory peptide
Lys
-Lys
but lacking in the
noninhibitory peptide Gly
-Lys
.
Because part of this sequence is conserved in other A domains (Fig. 8), it is unclear how these residues may be involved in a
FVIII A3 domain-specific function. To what extent individual amino
acids in the FIXa binding region contribute to FIXa
FVIII light
chain complex formation, therefore, remains to be investigated. It
seems of interest to note that mutations at positions
Ser
, Leu
, Met
,
Pro
, and Thr
have been determined to be
associated with moderately severe hemophilia A(33) . As these
mutations are in close proximity to the FIXa binding region, it is
tempting to speculate that the bleeding tendency that is associated
with these mutations is due to a suboptimal assembly of the
FIXa
FVIII light chain complex.
Figure 8:
Comparison of the FVIII A3 domain region
Gly-Gln
with corresponding portions of
other A domains. The primary structure of the A3 domain of human FVIII (2, 3) is compared with the corresponding regions of
the A3 domains of murine FVIII (mFVIII)(36) , human FV (hFV)(4, 5) , and ceruloplasmin (hCer.) (6) and with both A1 and A2 domains of human
FVIII (hFVIII)(2, 3) . The sequences are
aligned as described(36, 37) . Amino acids that are
identical to the corresponding residues in the FVIII A3 domain are boxed.
Recently, we demonstrated that
uncleaved FVIII light chain is similar to FVIII light chain derivatives
that have been cleaved by the activators thrombin or FXa in that they
display similar affinity for FIXa(31) . Apparently, the FIXa
recognition site is fully exposed in the intact FVIII light chain. In
agreement with previous observations, we have found that FVIII light
chain is more efficient in binding the fully activated FIXa than the
uncleaved FIX zymogen ( Fig. 6and Fig. 7; (27) ).
These data indicate that FVIII light chain displays preferential
binding to the enzyme FIXa rather than to the nonactivated FIX zymogen.
In this regard, FVIII light chain seems similar to FVa and
thrombomodulin, because these cofactors are more efficient in binding
to their respective enzymes than to the uncleaved
proenzymes(34, 35) . Surprisingly, this seems to be
untrue for the FIXa-binding peptides
Lys-Lys
and
Glu
-Gln
, because these peptides do
not distinguish between the enzyme FIXa and the FIX zymogen ( Fig. 6and 7). Several possibilities may be considered that may
explain these observations. First, it is possible that the relative
size of the FVIII light chain prevents binding to the intact FIX
zymogen, while this restriction is overcome by limited proteolysis of
the zymogen at its activation sites Arg
or
Arg
(27) . It should be noted here that cleavage
at Arg
is sufficient for full exposure of the FVIII light
chain binding site, whereas cleavage at Arg
results in a
suboptimal exposure(27) . Alternatively, the conformation of
the FIXa-binding motif in synthetic peptides may differ from its
conformation in the complete FVIII light chain. This may be due to
other portions of the light chain that provide the region
Glu
-Lys
its specificity for binding
to the activated form of FIX. In this respect it is of importance to
note that the Asn residue at position 1810 is a potential site for N-linked glycosylation in FVIII(2, 3) . As
this site is located adjacent to the FIX-binding motif
Glu
-Lys
, it seems conceivable that
glycosylation of this site contributes to the specificity for binding
of FIXa to its binding sequence
Glu
-Lys
.