From the
The transition of the factor IX zymogen into the enzyme factor
IXa
The blood coagulation pathway comprises a cascade of sequential
steps in which proenzymes are converted into active serine proteases
(1). The serine protease precursor factor IX (FIX)
In contrast to most serine proteases, FIX requires
two cleavages to yield full enzymatic
activity(2, 12, 13, 14, 15) .
Dependent on the sequence of cleavage, FIX activation can follow two
distinct pathways. Physiological activators first cleave the
Arg
Within the blood coagulation
cascade, FIXa
The aim of the present study was to elucidate the role of cleavage
of the Arg
FIXa
FIX
The enzymatic properties of FIXa
In the proteolytic activation of human FIX, cleavage of the
Arg
Because conversion of FIX into FIX
With respect to the interaction with the
serine protease inhibitor (``serpin'') ATIII, FIX
Proteolytic activation of
the FIX zymogen is associated with the exposure of the cofactor binding
site, because the activation intermediates FIX
In an attempt to
identify the region involved in binding the FVIII light chain, we
employed monoclonal antibodies that inhibit FIX activity. One antibody,
designated CLB-FIX 11, was observed to interfere with the FIX-FVIII
light chain interaction (Fig. 5). It is of interest to note that
Bajaj and co-workers (55) have also described an anti-FIX
monoclonal antibody that interferes with FVIII-dependent FX activation
by FIXa
Hydrolysis of
CH
We thank F. Meijer-Huizinga, J. Rentenaar, and A. Blok
for preparing and characterizing the anti-FIX antibodies and Dr. J.
Voorberg for critically reading the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
was investigated. For this purpose, the activation
intermediate factors IX
and IXa
were purified after cleavage
of the Arg
-Ala
and
Arg
-Val
bonds, respectively. These
intermediates were compared for a number of functional properties with
factor IXa
, which is cleaved at both positions. Factor IXa
was equal to factor IXa
in hydrolyzing the synthetic substrate
CH
SO
-Leu-Gly-Arg-p-nitroanilide (k
/K
120
s
M
) but was less
efficient in factor X activation. Factor IX
was incapable of
generating factor Xa but displayed reactivity toward p-nitrophenol p-guanidinobenzoate and the peptide
substrate. The catalytic efficiency, however, was 4-fold lower compared
with factor IXa
and factor IXa
. Factor IX
and factor
IXa
had similar affinity for the inhibitor benzamidine (K
2.5 mM), and amidolytic
activity of both species was inhibited by Glu-Gly-Arg-chloromethyl
ketone and antithrombin III. Unlike factor IXa
, factor IX
was
unable to form SDS stable complexes with antithrombin III. Moreover,
inhibition of factor IXa
and factor IX
by
Glu-Gly-Arg-chloromethyl ketone followed distinct pathways, because
factor IX
was inhibited in a nonirreversible manner and displayed
only minor incorporation of the dansylated inhibitor into its catalytic
site. These data demonstrate that the catalytic site of factor IX
differs from that of the fully activated factor IXa
. Factor IX and
its derivatives were also compared with regard to complex assembly with
factor VIII in direct binding studies employing the immobilized factor
VIII light chain. Factor IX
and factor IXa
displayed a
30-fold higher affinity for the factor VIII light chain (K
12 nM) than the factor
IX zymogen. Factor IXa
showed lower affinity (K
50 nM) than factor IX
and factor
IXa
, which may explain the lower efficiency of factor X activation
by factor IXa
. Collectively, our data indicate that cleavage of
the Arg
-Val
bond develops full
amidolytic activity but results in suboptimal binding to the factor
VIII light chain. With regard to cleavage of the
Arg
-Ala
bond, we have demonstrated
that this results in the transition of the factor IX zymogen into an
enzyme that lacks proteolytic activity. Moreover, the same cleavage
fully exposes the binding site for the factor VIII light chain,
suggesting that cleavage of the Arg
-Ala
bond serves a previously unrecognized role in the assembly of the
factor IX-factor VIII complex.
(
)circulates in plasma as a single-chain polypeptide (M
= 57,000) (2) that comprises a
number of discrete domains(3) . At the amino-terminal site of
the molecule the so-called ``Gla domain'' is located. This
domain contains several glutamic acid residues that have been
carboxylated to yield Gla(4) . The presence of Gla residues
allows this region to bind metal ions (5) and is essential for
surface binding at platelets and endothelial
cells(6, 7) . Adjacent to the Gla domain a region is
located that shares homology with the epidermal growth factor (EGF)
(8). This region consists of two distinct EGF-like domains, which are
important for FIX function. The first EGF-like domain contains a single
calcium binding site(8) . The second EGF-like domain is
connected to the activation peptide, a segment that is liberated during
zymogen activation(9) . Finally, the carboxyl-terminal portion
comprises the trypsin-like serine protease domain, which contains a
single metal ion binding site (10) and the catalytic centre of
FIX(11) .
-Ala
bond, resulting in the
transient activation intermediate FIX
. So far, FIX
has been
characterized as being enzymatically inactive(16) , which is
underscored by the notion that FIX
lacks clotting
activity(12, 15) . Subsequent cleavage at the amino
terminus of the protease domain (i.e. position
Arg
) then results in the active enzyme FIXa
. The
nonphysiological activator isolated from Russell's viper venom
first cleaves at position Arg
(12) . The resulting
intermediate FIXa
displays proteolytic activity, although its
clotting activity is just 20-50% of that of the enzyme
FIXa
(12, 17) . Subsequent cleavage of the
Arg
-Ala
bond then converts FIXa
into the fully active FIXa
.
activates the zymogen factor X (FX) in a process
that requires the presence of phospholipids, calcium ions, and factor
VIIIa (FVIIIa)(18, 19) . During FX activation, FIXa
is in complex with its protein cofactor FVIIIa(20, 21) .
This interaction involves the FVIII light chain, which contains a high
affinity binding site for FIXa
(22) . Complex formation with
FVIIIa results in structural changes within the active site of
FIXa
(20) . Maximal response requires the presence of the
carboxyl-terminal portion of the FVIII heavy chain(20) ,
indicating that the FVIII heavy chain is involved in complex formation
with FIXa
as well. Optimal FX activation requires the two-step
cleavage of FIX into FIXa
, because point mutations at Arg
or Arg
are both associated with the bleeding
disorder hemophilia B(23) . Whereas cleavage of the
Arg
-Val
bond corresponds with the
zymogen-activating site that FIX is sharing with many other serine
protease precursors(24) , cleavage of the
Arg
-Ala
bond is unique for FIX.
-Ala
bond in the activation
of the FIX zymogen. For this purpose FIX
was compared with other
FIX activation products with regard to a number of parameters that are
associated with FIXa enzyme function. These included reactivity toward
synthetic and natural substrates and inhibitors and interaction with
the cofactor FVIII. This approach allowed us to establish that cleavage
at Arg
plays a major role in the assembly of the
FIX-FVIII complex.
Materials
Protein A-Sepharose CL4B and
CNBr-Sepharose CL4B were from Pharmacia LKB Biotechnology AB (Uppsala,
Sweden). Microtiter plates (Immulon) were from Dynatech (Plockingen,
Germany) unless stated otherwise. Glu-Gly-Arg-chloromethyl ketone
(EGR-CK) and dansyl-Glu-Gly-Arg-chloromethyl ketone (DEGR-CK) were from
Calbiochem.
CHSO
-D-Leu-Gly-Arg-p-nitroanilide
(CH
SO
-LGR-pNA), product name CBS 31.39, was
from Diagnostica Stago (Asnières, France). Heparin (grade 1-A)
was obtained from Sigma. p-Nitrophenol p-guanidinobenzoate (NPGB) was from BDH Chemicals Ltd. (Poole,
United Kingdom).
Antibodies
The anti-FVIII antibodies CLB-CAg 12
and CLB-CAg 69 have been described previously(25, 26) .
The murine anti-FIX antibodies CLB-FIX 10 and CLB-FIX 11 were obtained
as outlined previously(22) , employing a screening strategy
based on binding to immobilized FIX in the presence or absence of
calcium ions. Binding of CLB-FIX 10 to FIX was calcium-independent,
whereas binding of CLB-FIX 11 was markedly enhanced in the presence of
calcium ions. Both antibodies CLB-FIX 10 and CLB-FIX 11 strongly
inhibit FIX activity (results not shown). The murine anti-FIX antibody
CLB-FIX D4 has been described elsewhere(27) . This antibody is
directed against the FIX sequence
Asn-Asp
, which comprises the
Arg
-Ala
activation site. By virtue of
the location of its epitope, antibody CLB-FIX D4 distinguishes between
intact FIX and cleaved FIX (i.e. FIX
and
FIXa
)(27) . All FIX antibodies used were from the
IgG
isotype. Monoclonal antibodies were purified from
culture medium employing protein A-Sepharose as recommended by the
manufacturer. Polyclonal antibodies against human FIX were obtained as
described previously (22). Antibodies were conjugated with horseradish
peroxidase as described(28) .
Various Proteins
The human FVIII light chain was
purified as described(22) . FX was prepared as
described(29) . The factor X-activating protein from
Russell's viper venom was purified as described (30) and
coupled to CNBr-Sepharose (2 mg/ml) according to the
manufacturer's instructions. Purified factor XIa (FXIa) was
obtained from Enzyme Research Laboratories. Purified Antithrombin III
(ATIII), C-inhibitor, and human serum albumin (HSA) were
obtained from the Division of Products of our institute. Bovine serum
albumin was from Miles Inc. Purified
-antitrypsin and
-antiplasmin were gifts from Dr. W. Wuillemin,
Department of Autoimmune Diseases of our institute. All proteins used,
including FIX and its activation products (see below) were homogeneous
as assessed by SDS-polyacrylamide gel electrophoresis (PAGE) (see Figs.
3 and 5).
FIX and FIX Cleavage Products
Human FIX was
purified from a concentrate of prothrombin, FIX, and FX (31) obtained from the Division of Products of our institute.
NaCl, benzamidine, and sodium citrate (pH 7.4) were added to the
concentrate to final concentrations of 0.15 M, 0.01 M, and 0.02 M, respectively, and the mixture was
subjected to immunoaffinity chromatography employing the anti-FIX
antibody CLB-FIX D4 (5 mg/ml CNBr-Sepharose). After extensive washing
with 0.15 M NaCl, 0.01 M benzamidine, 0.02 M sodium citrate (pH 7.4), FIX was eluted in a linear gradient
(0-2 M KSCN). FIX-containing fractions were pooled and
stored at -20 °C in 0.1 M NaCl, 0.05 M Tris
(pH 7.4). The specific activity of the FIX preparations ranged between
300 and 350 units/mg.
was prepared by incubating purified
FIX (4 µM) with human FXIa (0.23 µM) for 2 h
at 37 °C in 0.1 M NaCl, 2 mM CaCl
,
0.05 M Tris (pH 7.4). After the reaction was terminated by the
addition of EDTA (0.01 M final concentration), residual FIX
and FIX
were removed from the incubation mixture by
rechromatography on the CLB-FIX D4 affinity column. In this
immunoaffinity step, FIXa
and FXIa did not bind to the column,
whereas FIX and FIX
remained bound (27). Finally, FIXa
and
FXIa were separated employing anion exchange chromatography as
described previously(32) . FIXa
was stored at -20
°C in 50% glycerol, 0.1 M NaCl, 0.05 M Tris (pH
7.4). The FIXa
preparations were more than 90% active as
determined by active site titrations employing NPGB(33) .
was prepared by incubating purified FIX (4 µM)
with human FXIa (16 nM) in the presence of 6.8 mM MnCl
in 0.1 M NaCl, 0.05 M Tris (pH
7.4). After incubation for 2 h at 37 °C, the reaction was
terminated by the addition of EDTA and benzamidine (0.01 M
final concentrations). Under these conditions, approximately 90% of FIX
was converted into FIX
as judged by SDS-PAGE. FIX
was then
separated from FIX and FXIa employing CLB-FIX D4 affinity
chromatography. In this step FXIa and possible traces of FIXa
passed through the column, while FIX
and residual FIX were bound.
After extensive washing, FIX and FIX
were eluted separately in a
linear gradient (0-3 M KSCN)(27) . The
FIX
-containing fractions were pooled and stored at -20
°C in 0.1 M NaCl, 0.05 M Tris (pH 7.4). The
position of cleavage in FIX
was assessed by
NH
-terminal amino acid sequence analysis employing
automated equipment (Applied Biosystems, Warrington, UK; Eurosequence,
Groningen, the Netherlands). The resulting sequence,
Ala-Glu-Thr-Val-Phe, corresponds with the five NH
-terminal
amino acids of the FIX
activation peptide region, demonstrating
that indeed cleavage had occurred at
Arg
-Ala
(11). The FIX
preparations were more than 95% active as determined by NPGB
titration(33) . FIXa
was prepared from purified FIX
essentially as described (17) but modified in that the purified
FX-activating enzyme from Russell's viper venom instead of the
crude snake venom was immobilized on CNBr-Sepharose.
Protein Concentrations
Protein was measured by the
method of Bradford using HSA as a standard(34) . FIX activity
was measured employing a commercially available chromogenic method
(Baxter-DADE, Düdingen, Switzerland). Antigen concentrations of
FIX or its derivatives were quantified by an immunological assay as
described previously (22) but modified in that immunopurified
polyclonal anti-FIX antibodies were immobilized (0.2 µg/well)
instead of monoclonal antibody CLB-FIX 2. Dose-response curves were
transformed by plotting logit absorbance versus log
concentration and were linear between 0.07 and 7 nM. Within
this range the coefficient of variation was approximately 5%. Antigen
values were converted into molar concentrations using the purified FIX
derivatives as standards. Molar concentrations were calculated from
protein concentrations employing M = 57,000
for FIX, FIX
, and FIXa
and M
=
45,000 for FIXa
(2). In experiments in which FIXa
or FIX was
used as competitor for the binding of FIX
to the FVIII light
chain, nonbound FIX
was assayed by a FIX
-specific assay
employing the antibody CLB-FIX D4. Samples containing FIX
and FIX
or FIXa
were incubated with the immobilized antibody (0.5
µg/well) in 3 M NaCl, 0.1% (v/v) Tween-20, 1% (w/v) HSA,
0.05 M Tris (pH 7.2). After washing with 0.15 M NaCl,
0.1% (v/v) Tween-20, 0.05 M Tris (pH 7.2), bound FIX
was
detected employing peroxidase-conjugated polyclonal anti-FIX IgG in the
washing buffer. Under these conditions FIX
but not FIX or
FIXa
binds to the immobilized antibody CLB-FIX D4. Dose-response
curves were transformed by plotting logit absorbance versus log concentration and were linear between 0.1 and 10 nM.
FX Activation
The ability of FIX or its cleaved
derivatives to activate FX was assayed essentially as described
previously (35) employing acetylated FX to prevent cleavage of
the Arg-Ala
bond by the product
FXa(16) . FX was acetylated according to Neuenschwander and
Jesty(36) . The modified FX zymogen had lost more than 95% of
its biological activity, whereas amidolytic activity was fully
maintained.
Hydrolysis of
CH
Cleavage
of CHSO
-LGR-pNA
SO
-LGR-pNA by FIX or its derivatives was
assayed in 0.2% (w/v) HSA, 0.1 M NaCl, 0.01 M CaCl
, 0.05 M Tris (pH 8.4). Substrate
hydrolysis was initiated by the addition of 50 µl of a 2.5 mM solution of CH
SO
-LGR-pNA to a 50-µl
sample in a microtiter plate (Costar, type flat bottom). Initial rates
of substrate hydrolysis were measured at 37 °C by monitoring
absorbance at 405 nm in time. Kinetic parameters of substrate
hydrolysis by FIX cleavage products were determined employing substrate
concentrations between 0 and 15 mM at two different enzyme
concentrations. Absorbance values were converted into molar
concentrations using a molar extinction coefficient of 9.65
10
M
cm
for p-nitroanilide and a pathlength of 0.35 cm for a 100-µl
volume. The experimental data were fitted in the Michaelis-Menten
equation using EnzFitter software (Elsevier, Amsterdam, the
Netherlands) to obtain K
and k
values.
Protein Binding Assays
The binding of FIX or its
cleaved derivatives to the immobilized FVIII light chain and
calculation of binding parameters were performed as
described(22) .
Enzymatic Activity of FIX Activation
Products
The role of the individual cleavages at Arg and Arg
in human FIX was investigated with respect
to the development of enzymatic activity. FIX and its cleaved
derivatives were compared for their ability to activate FX in the
presence of calcium ions, phospholipids, and FVIIIa. As expected, the
enzyme FIXa
efficiently activated FX under these conditions (Fig. 1A). FXa was also generated by the intermediate
FIXa
, although at a lower rate than by FIXa
. In contrast,
both the intermediate FIX
and the FIX zymogen were incapable of
activating FX. These data demonstrate that cleavage at Arg
converts FIX into an active protease but that the additional
cleavage at Arg
develops full proteolytic activity. To
investigate whether limited proteolysis of FIX had a similar effect on
amidolytic activity, the reactivity of the zymogen FIX and its cleaved
derivatives toward the synthetic substrate
CH
SO
-LGR-pNA was tested. No substrate cleavage
occurred in the presence of the FIX zymogen (Fig. 1B).
In contrast, all cleaved forms of FIX, including the intermediate
FIX
, were capable of hydrolyzing this synthetic substrate. The
kinetic parameters for the hydrolysis of
CH
SO
-LGR-pNA by FIXa
, FIXa
, and
FIX
were determined. As listed in , FIXa
and
FIXa
display similar catalytic efficiency. The catalytic
efficiency of FIX
, however, appears to be 4-fold lower, which is
mainly due to a decreased k
. The possibility was
considered that the lower catalytic efficiency could be due to FIX
being only partially active. However, active site titrations employing
the active site titrant NPGB indicated that the extent of the p-nitrophenol burst corresponded to 90-95% of the
protein concentrations of both the FIX
and FIXa
preparations
employed (see ``Experimental Procedures''). It was noted that
titration of FIXa
requires about 4 min to reach
completion(33) , whereas the p-nitrophenol burst lasts
7-8 min for FIX
(results not shown). This slight difference
in reactivity toward NPGB was not further elaborated. Collectively,
these results confirm that the zymogen FIX is an inactive species,
whereas the enzyme FIXa
displays activity toward FX,
CH
SO
-LGR-pNA, and NPGB. With regard to the
activation intermediates, FIXa
equals the enzyme FIXa
in
synthetic substrate hydrolysis but is less efficient in FX activation.
FIX
, however, is extremely inefficient in generating FXa but at
the same time hydrolyzes CH
SO
-LGR-pNA and
reacts with the active site titrant NPGB. This demonstrates that
cleavage at Arg
converts the FIX zymogen into an
enzymatic form that lacks proteolytic activity.
Figure 1:
Activity of FIX and its cleaved
derivatives toward FX and CHSO
-LGR-pNA. A, FX activation was assessed by the incubation of various
concentrations of FIXa
(
), FIXa
(
), FIX
(
), or FIX (
) with acetylated FX (0.2 µM),
phospholipids (0.1 mM), and FVIIIa (0.4 nM) at 37
°C in 3.0 mM CaCl
, 0.1 M NaCl, 0.2
mg/ml bovine serum albumin, 0.05 M Tris (pH 7.4). FXa
generation was detected as described (35). B, amidolytic
activity was assayed by the addition of
CH
SO
-LGR-pNA (1.25 mM final
concentration) to solutions containing various concentrations of
FIXa
(
), FIXa
(
), FIX
(
), or FIX
(
) in 0.2% (w/v) HSA, 0.1 M NaCl, 0.01 M
CaCl
, 0.05 M Tris (pH 8.4). Hydrolysis of
CH
SO
-LGR-pNA was monitored continuously at 405
nm as described under ``Experimental Procedures.'' The data
shown represent the mean values ± S.D. of three to five
experiments. pNA, p-nitroanilide.
Interaction of FIX
Although the concentration of active sites
was in good agreement with the protein concentrations of purified
FIXa and FIXa
with Serine
Protease Inhibitors
and FIX
, these experiments do not fully exclude the
possibility that traces of other serine proteases could contribute to
the observed CH
SO
-LGR-pNA hydrolysis by
FIX
or FIXa
. Therefore, the effect of a number of serine
protease inhibitors was tested. Amidolytic activity of FIX
or
FIXa
appeared to be unaffected by the presence of serine protease
inhibitors including hirudin, soybean trypsin inhibitor,
-antitrypsin,
-antiplasmin, and
C
-inhibitor (data not shown). This demonstrates that the
amidolytic activity is not likely to be associated with the presence of
a variety of potential contaminants. In contrast,
CH
SO
-LGR-pNA hydrolysis was effectively
inhibited in the presence of the monoclonal anti-FIX antibody CLB-FIX
10 (see Fig. 5). Control experiments demonstrated that this
antibody does not inhibit CH
SO
-LGR-pNA
hydrolysis by thrombin, FXa, or FXIa. This strongly suggests that the
observed CH
SO
-LGR-pNA hydrolysis originates
from the enzyme FIXa
or the activation intermediate FIX
.
Figure 5:
Effect of monoclonal anti-FIX antibodies
on FIX amidolytic activity and FVIII light chain binding.
Amidolytic activity (closed symbols) by FIX
(300
nM) was determined as described under ``Experimental
Procedures'' in the presence of various concentrations of antibody
CLB-FIX 10 (
) or CLB-FIX 11 (
). Binding of
EGR-CK-inactivated FIX
(40 nM) to the immobilized FVIII
light chain (open symbols) was investigated in the presence of
varying concentrations of antibody CLB-FIX 10 (
) or CLB-FIX 11
(
) as described (22). Plotted is the percentage of residual
activity or binding versus the molar excess of antibody over
the FIX
concentration. An inhibition constant of 0.7 ± 0.3
nM was calculated for antibody CLB-FIX 11 by analyzing the
data for the FIX
-FVIII light chain interaction employing a model
of competitive inhibition (22). Inset, FIX
was reduced
and subjected to 12.5% (w/v) SDS-PAGE. Total protein was visualized by
silver staining (lane 1). Protein blots were incubated with
antibody CLB-FIX 10 (lane 2), antibody CLB-FIX 11 (lane
3), or the FVIII light chain (lane 4). The bound
antibodies were detected employing peroxidase-labeled goat anti-mouse
antibodies, and the bound FVIII light chain was detected employing
peroxidase-labeled anti-FVIII antibody CLB-CAg
69.
Additional experiments were performed to determine the affinity of
FIX and FIXa
for the inhibitor benzamidine. This reversible
inhibitor is known to inhibit the various coagulation enzymes with K
values ranging from 0.04 to 11 mM(37, 38, 39) and as such could contribute
to the identification of the active species in FIX
. Under the same
experimental conditions as in Fig. 1B, FIX
inhibition proved to be similar to that of FIXa
, with K
values of 2.9 ± 0.3 and 2.0
± 0.2 mM, respectively. These data demonstrate that
FIX
and FIXa
share similar inhibition characteristics by
benzamidine.
and FIX
were
examined in more detail using the synthetic serine protease inhibitor
EGR-CK. Binding of EGR-CK to FIX
or FIXa
was determined by
continuously monitoring CH
SO
-LGR-pNA hydrolysis
by FIX
or FIXa
in the presence of various concentrations of
EGR-CK. As expected, substrate hydrolysis by FIXa
displayed
progress curves that are typical for irreversible enzyme inhibition; in
the steady state situation, all FIXa
had been inactivated by
EGR-CK as p-nitroanilide formation was completely inhibited (Fig. 2A)(40, 41) . In contrast, progress
curves of CH
SO
-LGR-pNA hydrolysis by FIX
displayed residual substrate hydrolysis in the steady state (Fig. 2B), suggesting that FIX
inhibition, unlike
that of FIXa
, is not irreversible. Apparently, FIX
and
FIXa
are both capable of binding EGR-CK but are dissimilar with
respect to the mechanism of inhibition. This notion was further
explored by incubating FIX
and FIXa
with the dansyl-labeled
derivative DEGR-CK. Irreversible binding of DEGR-CK to FIXa
was
clearly visualized by incorporation of the DEGR-moiety into the
FIXa
heavy chain, whereas only a faint band was observed for
FIX
(Fig. 2, inset). This is compatible with the
view that FIX
and EGR-CK associate in a nonirreversible manner.
Figure 2:
Effect of EGR-CK on amidolytic activity of
FIX and FIXa
. 25 µl of FIXa
(A) or FIX
(B) was added to a mixture containing
CH
SO
-LGR-pNA and EGR-CK in 0.2% (w/v) HSA, 0.1 M NaCl, 0.01 M CaCl
, 0.05 M Tris
(pH 8.4) to a final volume of 100 µl. Final concentrations were 250
nM FIXa
, 500 nM FIX
, 4 mM CH
SO
-LGR-pNA and 0 (
), 25
µM (
), 50 µM (▾), or 100
µM (
) EGR-CK. After the addition of FIXa
or
FIX
, p-nitroanilide formation was monitored as described
under ``Experimental Procedures.'' Data represent the mean of
three independent experiments. Inset, the interaction between
EGR-CK and FIX
or FIXa
was examined by incubating FIX
or
FIXa
(both 1 µM) with dansyl-modified EGR-CK (50
µM) in 0.1 M NaCl, 0.01 M CaCl
, 0.05 M Tris (pH 8.4) for 16 h at room
temperature. Subsequently, 1.5 µg of FIX
and 1.5 µg of
FIXa
were reduced and subjected to 12.5% (w/v) SDS-PAGE.
Incorporation of the
dansyl-L-glutamyl-L-glycyl-L-arginine moiety
in the respective active sites was detected by UV light illumination.
FIXa
and FIX
treated with DEGR-CK are presented in lanes
1 and 3, respectively. FIX
treated with the
unmodified EGR-CK is presented in lane 2. pNA, p-nitroanilide.
Finally, the interaction between FIX or FIXa
and the
macromolecular inhibitor ATIII was addressed. In preliminary
experiments it was observed that binding was most efficient in the
presence of heparin, resulting in complete inhibition within 2 min. In
the ATIII inhibition experiments, 20-min incubation periods were
maintained to ensure that residual activities represent true end
points. As shown in Fig. 3, ATIII readily inhibited cleavage of
CH
SO
-LGR-pNA by both FIX
and FIXa
.
FIXa
and FIX
were similar in their interaction with ATIII in
that the stoichiometry was approximately 1:1 in both cases (Fig. 3). The same reaction mixtures were analyzed by SDS-PAGE.
ATIII formed a SDS-resistant complex with FIXa
, whereas no complex
could be visualized for FIX
or the FIX zymogen (Fig. 3, inset). Apparently, the association of the activation
intermediate FIX
with ATIII is different from that of mature
serine proteases such as FIXa
.
Figure 3:
Interaction of ATIII with FIX and
FIXa
. FIX
(
) and FIXa
(
), both 600
nM, were incubated with various concentrations of ATIII for 30
min at 37 °C in the presence of heparin (2 mg/ml). Residual
amidolytic activity was then measured by the addition of 50 µl of
CH
SO
-LGR-pNA to 50-µl samples as described
under ``Experimental Procedures.'' Data represent mean values
± S.D. of three experiments. Final concentrations (mean ±
S.D.) of FIXa
and FIX
were 320 ± 14 nM and
303 ± 12 nM, respectively, as determined employing an
immunological assay, and final concentrations of ATIII are those
indicated. Inset, complex formation between ATIII and FIX,
FIX
, or FIXa
was examined by the incubation of FIX, FIX
,
or FIXa
(all 1 µM) with ATIII (1.8 µM)
in the presence of heparin (2 mg/ml). Subsequently, 2 µg of
unreduced FIX, FIX
, or FIXa
was subjected to 7.5% (w/v)
SDS-PAGE. Protein was visualized by Coomassie staining. The lanes represent the following proteins. Lane 1, ATIII; lane
2, FIX
; lane 3, ATIII + FIX
(no complex); lane 4, FIXa
; lane 5, ATIII + FIXa
(complex); lane 6, FIX; lane 7, ATIII + FIX (no
complex).
Effect of FIX Cleavage on Binding to the FVIII light
chain
Optimal FX activation by FIXa requires complex
formation with the protein cofactor FVIIIa. One of the sites that might
be exposed during FIX activation is the binding site for the FVIII
light chain. Therefore, the contribution of the individual cleavages at
Arg
and Arg
to the affinity for the FVIII
light chain was tested employing a previously established
method(22) . As shown in Fig. 4, binding of FIXa
to
the FVIII light chain was less effective than observed for FIXa
.
In contrast, the interaction of FIX
with the FVIII light chain was
similar to that of FIXa
. The interaction between the FVIII light
chain and FIX
displayed a K
of 11.9
± 1.0 nM and a stoichiometry of 0.8 ± 0.2
mol/mol of FVIII light chain. These parameters are similar to those of
FIXa
(Fig. 4)(22) . For FIXa
the K
and stoichiometry were 49.9 ±
7.4 nM and 0.9 ± 0.1 mol of FIXa
/mol of FVIII
light chain, respectively. This indicates that cleavage at Arg
alone is insufficient for full exposure of the FVIII light chain
binding site.
Figure 4:
Interaction of FVIII light chain with FIX
and its cleaved derivatives. Various amounts of FIXa (
),
FIXa
(
), or FIX
(
) were inactivated with EGR-CK
and subsequently incubated with the immobilized FVIII light chain (1.2
pmol/well) in 1% (w/v) HSA, 0.1% (v/v) Tween-20, 0.1 M NaCl, 5
mM CaCl
, 25 mM histidine (pH 6.2).
Binding was quantified as described elsewhere (22). Binding parameters
for the interaction with the FVIII light chain were derived employing a
model describing the interaction with one single class of binding sites
(22). The calculated dissociation constants were 14.8 ± 3.2,
11.9 ± 1.0, and 49.9 ± 7.4 nM for FIXa
,
FIX
, and FIXa
, respectively. Inset,
EGR-CK-inactivated FIX
(40 nM) was incubated with the
immobilized FVIII light chain (1.2 pmol/well) in the presence of
various concentrations of EGR-CK-inactivated FIXa
(
) or FIX
(
). FIX
binding was measured as described under
``Experimental Procedures'' and expressed as
, which
represents the ratio of FIX
bound in the presence and the absence
of competitor. The drawn lines were obtained from a model
describing competitive inhibition (22). The calculated inhibition
constants for FIXa
and FIX were 10.1 ± 3.2 and 282 ±
19 nM, respectively. Data represent mean values ± S.D.
of three experiments.
The interaction of FIX with the FVIII light chain
was examined more closely in competition experiments. As shown in the inset of Fig. 4, FIXa
inhibited binding of FIX
to the FVIII light chain in a dose-dependent manner. By analyzing these
experimental data in a model of competitive inhibition(22) , the K
was calculated to be 10.1 ± 3.2
nM (mean ± S.D.), which is similar to the K
for the interaction of the FVIII light
chain with FIXa
obtained in direct binding studies (Fig. 4)(22) . The same experimental approach was used to
determine the K
for the interaction of
the FVIII light chain with the zymogen FIX. As presented in the inset of Fig. 4, only minor inhibition became apparent
at the higher concentrations of FIX tested, which was calculated to
reflect a K
of 282 ± 19
nM. This demonstrates that the affinity of FIX for the FVIII
light chain is considerably lower compared with that of FIX
and
FIXa
. In conclusion, these findings imply that full exposure of
the FVIII binding site requires cleavage of the
Arg
-Ala
bond.
Monoclonal Antibodies Distinguish between Amidolytic
Activity and FVIII Light Chain Binding
Cleavage of the
Arg-Ala
bond develops both amidolytic
activity (Fig. 1B) and exposure of the binding site for
the FVIII light chain (Fig. 4). To identify regions involved in
these processes, the monoclonal antibodies CLB-FIX 10 and CLB-FIX 11,
which strongly inhibit FIX activity (see ``Experimental
Procedures''), were investigated for their effect on the FVIII
light chain binding and amidolytic activity. With regard to amidolytic
activity, it appeared that CH
SO
-LGR-pNA
hydrolysis by FIX
was unaffected by the presence of antibody
CLB-FIX 11 but strongly inhibited by antibody CLB-FIX 10 (Fig. 5). With respect to FVIII light chain binding, the opposite
was observed; CLB-FIX 11 strongly interfered in the interaction of
FIX
with the FVIII light chain, whereas CLB-FIX 10 did not (Fig. 5). FIXa
was identical to FIX
in that its
amidolytic activity and FVIII light chain binding also were inhibited
by the same antibodies (not shown). In conclusion, our data demonstrate
that FVIII light chain binding and amidolytic activity are inhibited by
two different monoclonal antibodies, indicating that distinct regions
contribute to these processes. In immunoblotting experiments (Fig. 5, inset), both antibody CLB-FIX 10 and CLB-FIX 11
were found to be directed against the light chain of FIX
. The
notion that the latter antibody also interferes with binding of
FIX
to the FVIII light chain suggests that the light chain of
FIX
is involved in the interaction with the light chain of factor
VIII. This possibility was confirmed in ligand blotting experiments
employing purified FVIII light chain. By this method, the FVIII light
chain was observed to bind to the light chain but not the heavy chain
of FIX
(Fig. 5, inset, lane 4).
Collectively, these findings suggest that distinct FIX
light chain
regions are involved in amidolytic activity and binding to the FVIII
light chain.
-Ala
bond is the first of two
consecutive cleavages catalyzed by the physiological activators FXIa or
factor VIIa(2, 12, 13, 14, 15) .
Cleavage at Arg
alone gives rise to the activation
intermediate FIX
(12, 15) . This intermediate is
known to lack clotting activity(15) , which is in agreement with
our observation that FIX
is incapable of activating FX (Fig. 1A). It was surprising, however, that FIX
did
display enzymatic activity toward the active site titrant NPGB and the
synthetic substrate CH
SO
-LGR-pNA (Fig. 1B and ). Although we considered the
possibility that traces of contaminating serine proteases contributed
to the observed amidolytic activity, several lines of evidence led us
to conclude that substrate hydrolysis indeed originates from FIX
.
First, a series of serine protease inhibitors did not affect FIX
or FIXa
amidolytic activity, although these inhibitors should
interfere with the activity of a variety of potential contaminants.
Second, benzamidine inhibits FIX
and FIXa
activity with a
similar K
of 2-3 mM,
whereas the K
values for other enzymes
are significantly lower (thrombin, FXa, FXIa, factor XIIa, kallikrein,
trypsin, plasmin) or higher (factor
VIIa)(37, 38, 39) . Third, a 1:1 stoichiometry
was observed in active site titrations of the intermediate FIX
employing the active site titrant NPGB or the serine protease inhibitor
ATIII (Fig. 3). Finally, a monoclonal anti-FIX antibody
specifically inhibited amidolytic activity of FIX
and FIXa
(Fig. 5). Together with our finding that FIX
differed from
FIXa
by 4 orders of magnitude in activating FX (Fig. 1A), our results provide strong evidence that the
observed amidolytic activity is an intrinsic property of the
intermediate FIX
.
promotes development of amidolytic activity, cleavage of the
Arg
-Ala
bond is apparently associated
with changes within the FIX protease domain. In this respect, FIX
seems similar to the homologous intermediate prethrombin-2, which
exposes binding sites for the inhibitors
dansylarginine-N-(3-ethyl-1, 5-pentanediyl)-amide and hirugen,
whereas these sites are not exposed in the prothrombin zymogen (42,
43). With regard to the interaction of FIX
with the inhibitors
ATIII and EGR-CK, however, we obtained some intriguing data. In
contrast to FIXa
, FIX
displayed minor incorporation of the
dansyl-labeled EGR-CK into its active site (Fig. 2, inset). In concordance with this observation, FIX
and
FIXa
were also found to be dissimilar in EGR-CK inhibition
studies. FIXa
was inhibited in the expected irreversible manner,
whereas FIX
showed nonirreversible inhibition (Fig. 2).
Although the latter seems unusual, this type of inhibition is
compatible with the general mechanism by which chloromethyl ketone
inhibitors inactivate serine proteases(44, 45) .
Inhibition is initiated by the formation of a noncovalent complex,
after which the methyl carbon of the inactivator is attached to the
active site serine hydroxyl group to form a hemiketal intermediate.
After subsequent conversion of this intermediate into an epoxyether
intermediate, two distinct pathways have been distinguished. First, the
serine-bound epoxyether intermediate may alkylate the active site
histidine residue. This is the generally known pathway that results in
the irreversible, covalent complex as observed for FIXa
.
Alternatively, the epoxyether intermediate may be hydrolyzed by the
water solvent, leading to the formation of a free hydroxymethyl ketone
and to regeneration of the active enzyme(44, 45) .
Generally, serine protease inhibition by chloromethyl ketones follows
both pathways simultaneously(44) . Our finding that FIX
displays residual activity in the steady state indicates that for
FIX
the alternative pathway is predominant over the irreversible
pathway. As spatial alignment of the active site histidine and serine
residues is critical for irreversible inhibition(45) , we
speculate that the intermediate FIX
lacks the optimal alignment
within its catalytic site.
was
strikingly different from FIXa
, because no stable complexes were
formed in the presence of SDS (Fig. 3, inset). Usually,
serpin-serine protease complexes are driven into a stable complex upon
incubation with denaturing agents such as SDS(46, 47) .
However, FIX
is not the sole exception to this general mechanism
in lacking such complex formation. Anomalous behavior has been found to
be associated with serpin variants with mutations in the reactive site
loop(48, 49, 50) , or with modifications in the
active site of the target protease(51, 52) . The
observation that FIX
and ATIII are unable to form a stable complex
in the presence of SDS thus indicates that the active site of FIX
differs from that of FIXa
. In this respect, the data for ATIII and
EGR-CK are fully compatible, because they both suggest that the
FIX
active site is not fully developed. This is in support of the
notion that FIX
is an intermediate in the transition of the FIX
zymogen into the mature enzyme FIXa
.
and FIXa
both
display increased affinity for the FVIII light chain compared with
uncleaved FIX (Fig. 4). The FIX zymogen thus is similar to the
serine protease precursors FX and prothrombin in that proteolytic
activation is associated with the exposure of the respective cofactor
binding sites of these zymogens as well(53, 54) .
Whereas FX and prothrombin require cleavage at the protease domain
amino terminus for full exposure of the cofactor binding
site(53, 54) , this seems to be untrue for FIX. Cleavage
of the Arg
-Val
bond alone results in
a 5-fold lower affinity for the FVIII light chain compared with the
enzyme FIXa
(Fig. 4). This may explain the observation that
FIXa
is less efficient than FIXa
in generating FXa in the
presence of FVIIIa (Fig. 1A)(12, 17) ,
whereas FIXa
and FIXa
are equally potent in generating FXa in
the absence of FVIIIa(17) . Cleavage of the
Arg
-Ala
bond alone is sufficient for
full exposure of the binding site for the FVIII light chain, because
FIX
and FIXa
were similar in binding the FVIII light chain (Fig. 4). This indicates that cleavage at Arg
plays
a major role in the formation of the FIX-FVIII complex. With respect to
the FIX zymogen it is important to note that its plasma concentration
of 35-70 nM(3) is well below the K
of approximately 300 nM for
the interaction with the FVIII light chain and thereby not in favor of
complex assembly. This limitation is overcome after cleavage of FIX at
Arg
, which decreases the K
to approximately 12 nM. Because cleavage of the
Arg
-Ala
bond may be catalyzed by FXa
(16), this may provide a previously unrecognized mechanism that
promotes assembly of the FIX-FVIII complex.
and probably also with binding of FIXa
to FVIIIa.
However, that antibody is directed against the FIX heavy
chain(10) , whereas our antibody is directed against the FIX
light chain (Fig. 5, inset). The presence of an
independent binding site on the FIX
light chain would be in
agreement with our observation that the FVIII light chain interacts
with the FIX
light chain when tested by ligand blotting (Fig. 5, inset). However, we cannot exclude the
possibility that the FVIII light chain binds also to a FIX heavy chain
site that is not resistant to the immunoblotting technique.
Alternatively, it seems conceivable that binding of the FVIII light
chain to the FIX heavy chain is dependent on the conformation of the
FIX light chain. In this respect it should be noted that the antibody
CLB-FIX 10 is directed against the FIX light chain but inhibits
amidolytic activity (Fig. 5), which obviously is associated with
the FIX protease domain. Further studies will be needed to resolve the
mutual interrelation between the various FIX domains with regard to the
interaction with the complete FVIII heterodimer.
Table: Kinetic
parameters for the hydrolysis of CHSO
-LGR-pNA
by FIX
, FIXa
, or FIXa
SO
-LGR-pNA was monitored in the presence of
varying concentrations of CH
SO
-LGR-pNA
(0-15 mM) as described under ``Experimental
Procedures.'' Kinetic parameters were determined at enzyme
concentrations of 200 and 300 nM (FIXa
and FIXa
) or
at 300 and 400 nM (FIX
). Mean values ± S.D. of
3-4 experiments are presented. Catalytic efficiency k
/K
is calculated
from K
and k
.
, factor IX
; FIXa
, factor IXa
; FIXa
,
factor IXa
; FX, factor X; FXa, factor Xa; FXIa, factor XIa;
CH
SO
-LGR-pNA,
CH
SO
-D-leucyl-L-glycyl-L-arginine-p-nitroanilide;
NPGB, p-nitrophenol p-guanidinobenzoate; PAGE,
polyacrylamide gel electrophoresis; EGF, epidermal growth factor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.