From the Departments of Biochemistry and Biophysics
and § Medicine, University of Rochester School of
Medicine and Dentistry, Rochester, New York 14642
Received for publication, September 24, 2002, and in revised form, November 6, 2002
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ABSTRACT |
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Factor VIIIa consists of subunits designated A1,
A2, and A3-C1-C2. The limited cofactor activity observed with the
isolated A2 subunit is markedly enhanced by the A1 subunit. A truncated A1 (A1336) was previously shown to possess similar
affinity for A2 and retain ~60% of its A2 stimulatory activity. We
now identify a second site in A1 at Lys36 that is cleaved
by factor Xa. A1 truncated at both cleavage sites (A137-336) showed little if any affinity for A2
(Kd>2 µM), whereas factor VIIIa
reconstituted with A2 plus A137-336/A3-C1-C2 dimer
demonstrated significant cofactor activity (~30% that of factor
VIIIa reconstituted with native A1) in a factor Xa generation assay.
These affinity values were consistent with values obtained by
fluorescence energy transfer using acrylodan-labeled A2 and
fluorescein-labeled A1. In contrast, factor VIIIa reconstituted with
A137-336 showed little activity in a one-stage clotting
assay. This resulted in part from a 5-fold increase in
Km for factor X when A1 was cleaved at
Arg336. These findings suggest that both A1 termini are
necessary for functional interaction of A1 with A2. Furthermore, the C
terminus of A1 contributes to the Km for factor X
binding to factor Xase, and this parameter is critical for activity
assessed in plasma-based assays.
Factor VIII, a plasma protein that participates in the blood
coagulation cascade, is deficient or defective in individuals with
hemophilia A. Factor VIII functions as a cofactor for the serine
protease, factor IXa, in the anionic phospholipid
surface-dependent conversion of factor X to Xa. Factor VIII
is synthesized as a multi-domain, single chain molecule
(A1-A2-B-A3-C1-C2) (1) with a molecular mass of ~300 kDa (2, 3).
Factor VIII is processed to a series of divalent metal ion-linked
heterodimers by cleavage at the B-A3 junction, generating a heavy chain
consisting of the A1-A2-B domains and a light chain consisting of the
A3-C1-C2 domains. This procofactor is activated by cleavage at
Arg372, Arg740, and Arg1689 by
thrombin and factor Xa, converting the dimer into the factor VIIIa
trimer composed of the A1, A2, and A3-C1-C2 subunits (4, 5). The
resulting factor VIIIa heterotrimer retains the metal ion-dependent linkage between the A1 and A3-C1-C2 subunits,
whereas A2 is associated with a weak affinity by electrostatic
interactions (5, 6). Factor VIIIa is unstable, and loss of activity is due to the dissociation of the A2 subunit from the A1/A3-C1-C2 dimer
(5-7). Under physiological conditions, the Kd for
this interaction is ~260 nM (8, 9); however, at slightly acidic pH and low ionic strength, this interaction is facilitated by an
~10-fold increase in the affinity (Kd = ~30
nM) (8).
The role of factor VIIIa in the intrinsic factor Xase is to bind factor
IXa, which increases the kcat for factor Xa
formation by several orders of magnitude compared with factor IXa alone (10). Interactive sites for factor IXa are localized to A2 and A3
domains (11-13). Recent studies have shown that modulation of factor
IXa by the isolated A2 subunit enhances the kcat
for factor Xa activation by ~100-fold (14) and that the isolated A1
subunit synergizes this effect (>15-fold) to alter the interaction of A2 subunit with the protease (15, 16).
Early evidence suggested that the C-terminal acidic region of A1
subunit (residues 337-372) represented an A2-interactive site and
participated in A2 retention following thrombin activation (17). This
region is also implicated in the binding of factor X (18), although the
significance of this cofactor-substrate interaction is not well
understood. The A1 subunit is cleaved at Arg336 by
activated protein C (19), factor IXa (20, 21), and factor Xa (19).
Proteolysis at this site correlates with inactivation of factor VIIIa.
Thus, this cleavage is thought to represent a mechanism for the
dampening of factor Xase activity. A recent study demonstrated that a
truncated subunit lacking the C terminus of A1 (A1336)
possessed similar affinity for A2 subunit and retained ~60% of the
A2 stimulatory activity compared with native A1 (16). However, that
study also suggested that the C-terminal region of A1 appeared critical
for proper orientation of A2 subunit in factor VIIIa relative to factor
IXa (16).
In this study, we examine the intersubunit interactions of factor VIIIa
employing a purified A1 subunit form possessing a newly identified
cleavage site and designated as A137-336, following
cleavage at both termini (Lys36 and Arg336) by
factor Xa. These results demonstrate a role for the N terminus of the
A1 subunit in preserving a conformation of the subunit necessary for
functional interaction with the A2 subunit. Furthermore, these results
define a role for the A1 C-terminal region in contributing to the
Km for substrate factor X binding to factor Xase. Thus, specific mechanisms for the loss of factor VIIIa function and
down-regulation of factor Xase can now be attributed to individual cleavages of the A1 subunit by factor Xa.
Reagents--
Purified recombinant factor VIII preparations were
generous gifts from Bayer Corp. (Berkeley, CA) and the Genetics
Institute (Cambridge, MA). The monoclonal antibody 58.12 recognizing
the N-terminal end of A1 (22) was a gift from Dr. James Brown, and monoclonal antibody C5 recognizing the C-terminal of A1 (23) was a gift
from Drs. Carol Fulcher and Zaverio Ruggeri. Phospholipid vesicles
containing 20% PS,1 40% PC,
and 40% PE (Sigma) were prepared using
N-octylglucoside (24). TAP was a gift from Dr. S. Krishnaswamy. The reagents human Isolation of Factor VIII Subunits--
Each factor VIII subunit
was isolated from recombinant factor VIII. Factor VIII (1.5 µM) was treated overnight at 4 °C in buffer containing
10 mM MES, pH 6.0, 0.25 M NaCl, 50 mM EDTA, and 0.01% Tween 20, and the light chain and heavy
chain were isolated following chromatography on SP-Sepharose and
Q-Sepharose columns (Amersham Biosciences) as previously described
(25). The purified heavy chain was cleaved by thrombin, and the A2 and
A1 subunits were purified by fast protein liquid chromatography using a
Hi-Trap Heparin column and a Mono-Q column as reported previously (16). The A3-C1-C2 subunit was prepared as previously described (18). The
A1/A3-C1-C2 dimer was isolated from thrombin-treated factor VIIIa using
a Mono-S column chromatography, and residual A2 subunit was removed
using an anti-A2 subunit monoclonal antibody coupled to Affi-Gel 10. The A3-C1-C2 subunit was further purified following dissociation of
dimer by EDTA and chromatography on Mono-Q (8). SDS-PAGE of the
isolated subunits showed >95% purity. The protein concentrations were
determined by the method of Bradford (26).
Cleavage of A1 by Factor Xa to Form
A137-336--
Human factor Xa was added to the purified
A1 (11 µM) in a 1:10 (mol/mol) ratio in a buffer
containing 20 mM HEPES, pH 7.2, 0.1 M NaCl, 5 mM CaCl2, and 0.01% Tween 20 and reacted
overnight at 22 °C. The reaction was quenched using TAP at an
~5-fold molar excess relative to factor Xa. The cleaved A1 subunit
was fractionated by fast protein liquid chromatography using a
Pharmacia Hi-Trap chelating column (1.0 ml), to which 100 µl of 0.1 M CuCl2 was added beforehand (27). The
truncated A1 subunit was eluted at 0.2-0.3 M glycine using
a linear glycine gradient (0-1.0 M) in 20 mM
HEPES, pH 7.2, 0.3 M NaCl, and 0.01% Tween 20. The purity of truncated A1 subunit (A137-336) was >95% as
determined by SDS-PAGE. A chromogenic assay revealed <0.1% residual
factor Xa activity or TAP present in the purified A137-336. A truncated A1336 subunit was
prepared as described earlier (16).
Labeling of A1 and A2 Subunits--
To obtain
fluorescein-labeled A1 (Fl-A1) or A137-336, purified A1
(20 µM) or A137-336 (6.5 µM)
subunits in 20 mM HEPES, pH 7.2, 100 mM NaCl, 1 mM CaCl2, and 0.01% Tween 20 were reacted with
a 50-fold molar excess of fluorescein 5-maleimide in the dark at
22 °C for 4 h. The unbound fluorescein 5-maleimide was removed
by dialyzing the reaction mixture at 4 °C overnight in the above
buffer. Acrylodan-labeled A2 (Ac-A2) was obtained from reacting factor
VIII (1.5 µM) in the above buffer with a 10-fold molar
excess of acrylodan. Labeling conditions and removal of unbound
acrylodan were as described above. Isolated Ac-A2 subunit was purified
from labeled, EDTA-treated factor VIII following thrombin cleavage
using the combination of SP-Sepharose, Q-Sepharose, and Hi-Trap heparin
chromatography steps as described above. Approximately 0.9-1.1 mol of
fluorescein 5-maleimide was incorporated/mol of A1 or
A137-336 subunit. The incorporation of acrylodan into A2
subunit was 1.0-1.2 mol/subunit.
Reconstitution of Factor VIIIa Activity--
The A1/A3-C1-C2
dimer was reconstituted with 500 nM A3-C1-C2 and variable
levels of A1 form overnight at 4 °C in 20 mM HEPES, pH
7.2, 0.3 M NaCl, 25 mM CaCl2, and
0.01% Tween 20. The dimer solution was diluted 10-fold, and factor
VIIIa was formed by the addition of 20 nM A2 subunit in 20 mM MES, pH 6.0, 100 µg/ml bovine serum albumin, and
0.01% Tween 20. The reconstitution reactions were run at 22 °C, and
the resulting factor VIIIa activity was determined after 15 min by a
one-stage clotting assay using factor VIII-deficient plasma (28).
Factor Xa Generation Assays--
The rate of conversion of
factor X to factor Xa was monitored in a purified system (29). For
assays examining the stimulation of isolated A2 subunit by A1 subunit
forms, 400 nM A2 subunit was first reacted with variable
levels of A1 form for 15 min followed by incubation with 5 nM factor IXa and 10 µM PS-PC-PE vesicle in
the presence of 20 mM HEPES, pH 7.2, 50 mM
NaCl, 5 mM CaCl2, 100 µg/ml bovine serum
albumin, and 0.01% Tween 20 for 10 min. For assays examining factor
VIIIa reconstitution from A2 subunit plus A1/A3-C1-C2 dimers comprised
of different forms of A1, the dimers and 20 nM A2 subunit
were reconstituted and incubated with 20 nM factor IXa and
10 µM PS-PC-PE vesicle for 30 s in the above buffer.
The reactions for either assay were initiated with the addition of 500 nM factor X. The aliquots were removed at appropriate times
to assess initial rates of product formation and added to tubes
containing EDTA (final concentration, 50 mM) to stop the reaction. The rates of factor Xa generation were determined by the
addition of the chromogenic substrate, S-2765 (final concentration, 0.46 mM). The reactions were read at 405 nm using a
Vmax microtiter plate reader (Molecular Devices,
Sunnyvale, CA). All of the reactions were run at 22 °C.
Fluorescence Energy Transfer--
Fl-A1/A3-C1-C2 or
Fl-A137-336/A3-C1-C2 dimer was formed by reacting 2 µM A3-C1-C2 and 2 µM Fl-A1 or
Fl-A137-336 as described above. Ac-A2 (100 or 200 nM) was reconstituted with varying amounts of the dimer or
isolated Fl-A1 subunit forms for 30 min at 22 °C in 20 mM MES, pH 6.0, 0.01% Tween 20, and 200 µg/ml bovine
serum albumin. Fluorescence measurements were conducted using an
Amico-Bowman Series 2 Luminescence Spectrometer (Thermo Spectronic,
Rochester, NY). The reconstitution reactions were excited at 395 nm,
and emission spectra were monitored at 420-550 nm. Emission scans were
run at 3 nm/s in triplicate, and the average values of the spectra were
recorded. All of the spectra were corrected for background fluorescence
resulting from the reaction buffer and the fluorescein-labeled
dimer or Fl-A1 form. The percentage of donor quenching was calculated
from integrated fluorescence intensities at Electrophoresis and Western Blotting--
SDS-PAGE was performed
on a 8% gel using the procedure of Laemmli (30). Electrophoresis was
carried out using a Bio-Rad minigel apparatus at 150 V for 1 h.
The bands were visualized following staining with GelCode Blue Stain
Regent (Pierce). Alternatively, the protein was transferred to a
polyvinylidene difluoride membrane using a Bio-Rad mini-transblot
apparatus at 50 V for 2 h in buffer containing 10 mM
CAPS, pH 11, and 10% (v/v) methanol. The protein was probed using the
58.12 and C5 monoclonal antibodies followed by goat anti-mouse alkaline
phosphatase-linked secondary antibody. The signal was detected using
the ECF system (Amersham Biosciences), and the blots were scanned at
570 nm using Storm 860 (Molecular Devices).
NH2-terminal Sequence Analysis--
N-terminal
sequence analysis of the factor Xa-cleaved A1 subunit was performed
using an Applied BioSystems Procise Sequenator by the Protein
Sequencing Facility at Cornell University. The subunit was blotted onto
polyvinylidene difluoride and stained with Ponceau S, and the band was
excised. The sample was subjected to 10 cycles of sequencing.
Isolation and Purification of Factor Xa-cleaved A1
Subunit--
Factor Xa cleaves the A1 subunit (residues 1-372) of
human factor VIIIa at Arg336 yielding a truncated subunit
lacking the C-terminal domain rich in acidic amino acids (19). Because
this cleavage inactivates cofactor activity, an examination of the
A1-associated activities following interaction of the isolated subunit
with factor Xa was undertaken. We observed that an extended interaction
of the subunit with protease (10:1 molar ratio) converted A1 into a
subunit of ~40 kDa, which was significantly smaller than the A1
subunit truncated at Arg336 (~45 kDa). Pretreatment of
factor Xa with a molar excess of TAP resulted in no generation of
either fragment, indicating that both cleavages were catalyzed by
factor Xa (results not shown).
Purification of this fragment was problematic because it did not bind
to either Mono S or Mono Q columns under low salt conditions. However,
it was adsorbed by a Cu2+-bound Hi-Trap chelating column
utilizing the copper binding of His residues (27) and was eluted
quantitatively and with high purity (>95%) with 0.2-0.3
M glycine. Fig. 1A
shows gel electrophoresis of A1, A1336, and the ~40-kDa
subunit. A chromogenic assay revealed <0.1% residual factor Xa
activity or TAP (used to inactivate the factor Xa) present in the
purified fraction. This fragment was further characterized by Western
blot analysis, which employed anti-A1 domain monoclonal antibodies
specific for the N- and C-terminal sequences. Although native A1 and
A1336 subunits were reactive with monoclonal antibody 58.12 (an antibody directed to residues 1-12 of the A1 sequence), the 40-kDa
A1 was not identified (Fig. 1B, left panel). Use
of the C5 antibody (specific for residues contained with 337-372)
showed reactivity with the native A1 subunit only and did not react
with either the 40-kDa A1 form or A1336 (Fig.
1B, right panel). These findings indicated that
the 40-kDa A1 subunit was truncated at both termini.
Identification of the N-terminal Cleavage Site--
To identify
the site of cleavage that results in the N-terminal truncation, the
~40-kDa A1 form was subjected to 10 cycles of automated N-terminal
sequence analysis (Table I). Results from
this analysis indicated that this A1 form was derived from cleavage at
Lys36-Ser37. Thus, cleavage at this site was
consistent with no reactivity using the monoclonal antibody specific
for the A1 N terminus. This 40-kDa A1 subunit was designated as
A137-336. Interestingly, the residue specified at cycle
number 5 (Asn41) was not identified. Because this Asn is
contained within the N-X-(T/S) N-linked
glycosylation site consensus sequence (31), failure to identify this
residue provides direct support for utilization of Asn41
for N-linked glycosylation.
Stimulation of A2 by A1 Forms--
We previously demonstrated that
the isolated A2 subunit possesses limited cofactor activity in
stimulating factor IXa-catalyzed activation of factor X (14), and this
activity is markedly enhanced in the presence of A1 subunit (15, 16).
To gain insights into critical regions of A1 required for this
stimulation of A2, the cofactor activity of A2 in the presence of the
native form and the two truncated A1 forms were compared using a factor
Xa generation assay. Variable levels of A1 forms and 400 nM
A2 subunit were combined, and the reactions were initiated by the
addition of 5 nM factor IXa and 500 nM factor X
as described under "Materials and Methods." The results for the
stimulation of A2 by each A1 form are shown in Fig.
2 and Table
II. Negligible levels of activity were
obtained in the absence of A2 (data not shown) and were corrected for
prior to curve fitting. Similar to the recent observations of Koszelak
Rosenblum et al. (16), the A1336 subunit
stimulated A2 activity to ~60% the level observed for native A1,
whereas similar affinities of the two A1 forms for A2 were observed. In
contrast, the Vmax value obtained for
A137-336 subunit in stimulating A2 cofactor activity
(~0.3 min Reconstitution of Factor VIIIa Forms--
Although the
A137-336 possessed little ability to directly stimulate
the A2 subunit, an additional series of experiments was performed to
assess the capacity of this A1 form to reconstitute factor VIIIa
following association of A3-C1-C2 and A2 subunits. Factor VIIIa was
reconstituted in a two-step procedure. In the first step the
A1/A3-C1-C2 dimer form was prepared by reacting varying amounts of the
A1 forms and 500 nM A3-C1-C2 subunit in the presence of
Ca2+ overnight at 4 °C. The resultant dimer solutions
were diluted 10-fold and reacted with the A2 subunit (20 nM) to generate the factor VIIIa heterotrimers.
Reconstituted factor VIIIa activity was determined in a factor Xa
generation assay initiated by the addition of factor IXa (20 nM) and factor X (500 nM) as described under
"Materials and Methods." These data are illustrated in Fig. 3 and Table II. The
Vmax values obtained at saturating levels of
factor VIIIa reconstituted from native A1 and A1336 with
A3-C1-C2 plus A2 subunits were 26.9 ± 0.9 and 13.0 ± 0.7 min
Interestingly, although isolated A137-336 had little
ability to directly stimulate the A2 subunit, likely in part a
consequence of a weak affinity interaction for A2, reconstitution of
A137-336/A3-C1-C2 dimer plus A2 subunits yielded a form of
factor VIIIa that demonstrated significant cofactor activity. This
activity (8.0 ± 0.3 min Reconstituted Factor VIIIa Activity Measured in a
One-stage Clotting Assay--
Activity of the reconstituted factor
VIIIa composed of either native A1 or A137-336 forms was
measured in a one-stage clotting assay as described under "Materials
and Methods." Compared with factor VIIIa reconstituted with native A1
form, little if any activity was observed for that with the
A137-336 form (Fig. 4). This
result was in contrast to the results obtained in a factor Xa
generation assay with factor VIIIa comprised of the
A137-336 form, which yielded ~30% of the activity of
factor VIIIa containing native A1. Similarly, we previously reported no
activity of factor VIIIa composed of A1336 in a one-stage
clotting assay (16), whereas this form showed ~60% of native
activity in a factor Xa generation assay in the current study. Thus,
the failure to observe factor VIIIa activity with trimer reconstituted
from the A137-336 form using a one-stage clotting assay
was consistent with the earlier result. Overall these results suggest
that the lack of the C-terminal region of A1 leads to disparate results
in the two assay systems.
Activity of Factor VIIIa Forms at Variable Substrate
Concentration--
Because the above results suggest altered
activities of the cofactor forms possessing C-terminal truncations in
A1 subunit and because this region of factor VIII contains a factor
X-interactive site (18), a study was undertaken to evaluate the role of
substrate factor X concentration relative to factor VIII function. We
hypothesized that an increase in Km for factor X as
a result of a C-terminal truncation in factor VIIIa leading to a
disrupted interaction with factor X would have a more pronounced effect
on the one-stage assay in which the factor X concentration is ~25%
the plasma level or ~40 nM compared with the factor Xa
generation assay, which uses a concentration of substrate yielding
Vmax conditions (29) (500 nM as
described under "Materials and Methods").
For this analysis, 500 nM native A1 or
A137-336 subunit and A3-C1-C2 subunit were reconstituted
as described above. The preformed dimers were diluted 10-fold and
reacted with 20 nM A2 subunit, followed by the addition of
20 nM factor IXa. The reactions were initiated using
varying amounts of factor X as described under "Materials and
Methods." The results of this experiment are shown in Fig.
5. The Vmax values
obtained from the fitted curve for factor VIIIa forms composed of
native A1 and A137-336 subunits were 21.8 ± 4.1 and
7.2 ± 0.3 min Energy Transfer between Ac-A2 and Fl-A1 Subunit Forms--
In an
earlier report, we demonstrated that reconstitution of factor VIIIa
using fluorophore-labeled subunits can be assessed and intersubunit
affinity can be quantitated by following fluorescence resonance energy
transfer (32). A similar approach was undertaken to examine
interactions of isolated A2 with the native and truncated A1 subunit
forms. The A2 subunit and the A1 subunit forms were labeled with the
sulfhydryl-specific probes, acrylodan and fluorescein-5-maleimide, respectively, utilizing the presence of a free cysteine residue in the
A1 (Cys310) and A2 (Cys692) domains (33).
Because modification of the isolated A2 subunit with acrylodan results
in an inactive subunit that failed to yield functional factor VIIIa in
a reconstitution assay (32), the Ac-A2 subunit was isolated from
acrylodan-labeled intact factor VIII as described under "Materials
and Methods." The incorporation of acrylodan into A2 subunit was
1.0-1.2 mol/subunit. Direct labeling of isolated A1 subunit with
fluorescein 5-maleimide yielded an active subunit with similar
incorporation of fluorescein 5-maleimide into A1 or
A137-336 subunit (0.9-1.1 mol/subunit). Reconstitution of
the Ac-A2 subunit and Fl-A1 form/A3-C1-C2 dimer retained >80%
specific activity compared with factor VIIIa prepared from unlabeled
subunits (data not shown).
Fluorescence experiments were conducted at pH 6.0, which enhances the
intersubunit affinity (8), using 200 nM Ac-A2 and varying
amount levels of the Fl-A1 form as described under "Materials and
Methods." The fluorescence emission spectrum of the Ac-A2 subunit
(fluorescence donor) overlaps with the excitation spectrum of the Fl-A1
form (fluorescence acceptor). Upon binding of Ac-A2 to the Fl-A1 form,
the fluorescence intensity of the acrylodan is quenched, and the extent
of this quenching is an indicator of the spatial separation between
donor and acceptor fluorophores (34). The relative fluorescence
intensities were recorded and integrated at Influence of A3-C1-C2 in the Energy Transfer between Ac-A2 and
Fl-A1 Subunits--
To further assess the role of the A3-C1-C2 subunit
in the inter-A1-A2 interaction, we performed energy transfer analysis
between Ac-A2 and the A1/A3-C1-C2 dimer reconstituted from the Fl-A1
subunit forms and A3- C1-C2. Fl-A1/A3-C1-C2 and
Fl-A137-336/A3-C1-C2 dimers were formed by reacting 2 µM A3-C1-C2 with 2 µM Fl-A1 or
Fl-A137-336, respectively. Ac-A2 (100 nM) was
reconstituted with varying amounts of the dimer forms as described
under "Materials and Methods," and the results are shown in Fig.
7 and Table III. Donor
quenching was observed for either dimer form, indicative of association of Ac-A2 subunit. The levels of the percentage of donor quenching at
saturating Fl-labeled dimer consisting of A1 and
A137-336 were 55.6 ± 2.5 and 29.9 ± 1.6%,
respectively. These results suggest an altered spatial separation
between the fluorophores with an apparent closer separation existing
for the native subunits. This result is consistent with an altered
conformation in the trimer formed with the truncated A1 form that may
reflect reduced cofactor activity.
The Kd values calculated for interaction of Ac-A2
with Fl-A1/A3-C1-C2 and Fl-A137-336/A3-C1-C2 dimer were
similar (72.2 ± 7.8 and 85.2 ± 10.8 nM,
respectively) and ~5-fold less when A3-C1-C2 was included. Comparison
of binding energy values calculated for the interaction of A2 with
native A1 (Kd = 432 nM, 8.7 kcal
mol Factor Xa, a potent activator of factor VIII (35), has been shown
to cleave human factor VIII at sites identical to those attacked by
thrombin during cofactor activation. Factor Xa also attacks
Arg1721 in the A3 domain of the A3-C1-C2 subunit and has
been shown to cleave the C-terminal region of A1 at a site tentatively
identified as Arg336 (19). Earlier studies employing a
factor VIII light chain cleaved by factor IXa at Arg1721
showed that cleavage at this site was benign to cofactor function following reconstitution of factor VIII with native heavy chain (7).
However, cleavage at Arg336, a site also attacked by
activated protein C (19, 36) and factor IXa (20, 21), correlates with
inactivation of the cofactor and down-regulation of factor Xase
activity. The mechanisms for activity loss following cleavage at
Arg336 are not fully understood but include reduced
interaction of factor VIIIa with substrate factor X (37) and altered
interaction with A2 subunit, as evidenced by reduced stimulation of
activity associated with the A2 subunit (16). Thus, subsequent
proteolytic attack of the cofactor by generated factor Xa would yield a
reaction product limiting further factor Xa generation by a
self-dampening mechanism.
In this work we examined the intersubunit interactions of factor VIIIa
employing an A1 subunit form following terminal digest with factor Xa.
Electrophoretic analysis of a terminal A1 cleavage fragment revealed
truncation at its C terminus consistent with the loss of residues
337-372, as well as cleavage at a second prominent factor Xa-catalyzed
site at Lys36-Ser37 as determined by N-terminal
sequence analysis. The resultant A137-336, truncated by 36 residues at both its termini, showed markedly reduced affinity for the
A2 subunit and no apparent capacity to stimulate the limited cofactor
activity associated with A2 subunit. This result is in contrast to
observations in this report as well as an earlier study (16) using an
A1 subunit truncated only at its C terminus, A1336, which
demonstrated similar affinity of this A1 form compared with native A1
for A2 subunit and showed a high level (~60%) of A2 stimulatory
activity. Thus, these results identify the N-terminal region of A1 as
participating either directly or indirectly in the interaction with A2 subunit.
The results obtained from the energy transfer studies comparing Ac-A2
association with Fl-A1 in the absence or presence of A3-C1-C2 suggest
that ~90% of the binding energy for A2 association within the factor
VIIIa heterotrimer is derived from direct interaction with the A1
subunit. This result is consistent with earlier studies demonstrating a
lack of competition of isolated factor VIII light chain (A3-C1-C2) with
the A1/A3-C1-C2 dimer for A2 subunit in a functional, factor VIIIa
reconstitution assay (8, 17). This value of 90% may represent a
minimum value, because A3-C1-C2 appears to alter the conformation of A1
so as to enhance its interaction with A2. This observation was
suggested by the weak (undetermined) affinity of isolated
A137-336 for A2 subunit as assessed by the energy transfer
assay, whereas association of this truncated subunit with A3-C1-C2
restored a high affinity interaction of A2 with the dimer.
Although the molecular basis for the loss of functional interaction
between the isolated A1 and A2 subunits with cleavage at the N-terminal
site is not well understood, examination of the homology model (38)
indicates that A1 residues Pro25-Phe30
juxtapose the A2 domain and that this interaction may be significant. Specifically, Val26 and Asp27 are separated
from A2 residues 537-541 by ~2.5-5 Å. These potential contacts in
A2 include Arg541 and Val537, and point
mutations at these two sites have been shown to result in hemophilia A
(HAMSTeRs data base; europium.csc.mrc.ac.uk). However, no point
mutations within this sequence in A1 have been identified to yield a
hemophilia phenotype. Thus, we speculate that the first 36 residues of
A1 may either directly contact A2 and/or promote a conformation of A1
important to its physical interaction with A2. Interestingly, this
effect is somewhat ameliorated in the presence of the A3-C1-C2 subunit.
Reconstitution of factor VIIIa using the A137-336 form
yielded ~30% of the activity of the cofactor formed with native A1
subunit and ~60% of the activity of the cofactor formed with
A1336, truncated only at its C terminus. These results
suggested that any putative conformation defect attributed to cleavage
at Lys36 is somewhat compensated for following the
reassociation of A1 with the A3-C1-C2 dimer. This restoration of
cofactor function in the presence of A3-C1-C2 was expected based upon
earlier observations employing phospholipid-independent, factor Xa
generation assay indicating that A1-A3 contacts subsequently positively
influence the interaction of A1 with A2 subunits (39). However, the
disparate extents of fluorescence quenching we observed for the
acrylodan-labeled A2 subunit by the Fl-A1 and Fl-A137-336
forms in the presence of A3-C1-C2 suggested an altered (increased) interspatial separation of fluorophores in the factor VIIIa form possessing the cleaved A1. We suggest that these changes in
conformation are responsible for reduced cofactor activity and
reflective of altered interaction between the A1 and A2 subunits.
Although factor Xa generation assays showed the truncated A1 forms
retained significant activity following reconstitution reactions to
yield factor VIIIa, the one-stage clotting assay failed to report
significant activity using these forms of the cofactor. We observed a
similar result with the one-stage clotting assay using factor VIIIa
reconstituted from the A1336 form in an earlier report
(16). We now identify a factor contributing to this assay discrepancy.
Kinetic analyses using factor Xa generation show a
Km for factor X of ~40 nM using factor
Xase composed of native factor VIIIa, whereas this value was increased
5-fold for factor Xase comprised of the C-terminal truncated A1.
Because the typical factor Xa generation assay uses concentrations of substrate that yield (near) Vmax reaction rates
(29), the rates are independent of the concentration of factor X. However, because the plasma concentration of factor X is ~120
nM and the plasma is diluted 4-fold in the one-stage assay,
the limiting amount of substrate factor X (~20% the
Km value for factor Xase comprised of the cleaved A1
forms) would markedly depress the rate of factor Xase activity and
subsequent clot formation for reactions run using these A1 forms. These
results contribute to understanding the basis for cofactor inactivation
following cleavage at Arg336 by enzymes such as activated
protein C.
The kinetic studies ascribe a role for the A1 C terminus 337-372 in
the interaction of factor Xase with substrate factor X. This functional
effect is consistent with earlier studies suggesting that this sequence
represented a factor X interactive site (18). In a complementary study,
zero length cross-linking employing either factor VIIIa or the
A1/A3-C1-C2 showed a critical salt bridge between the C-terminal region
of A1 and the protease domain of factor X (40). The requirement for the
A1 C-terminal region was based upon absence of a cross-linked product
using the A1336/A3-C1-C2 dimer form. Interestingly, a
region in the protease domain distinct from the activation peptide
sequence was identified based upon persistence of the linkage following
zymogen activation by the X activator in Russel's viper venom. Thus,
we speculate that factor Xa may also interact with the C-terminal
region of the A1 subunit. Indeed, attack at Lys36 appears
dependent upon an intact C terminus of the A1 subunit, indicating an
ordered reaction pathway in factor Xa-catalyzed inactivation of factor
VIIIa.2
The study by Parker et al. (41) provides a detailed analysis
of the proteolytic activation of porcine factor VIII by factor Xa. In
addition to cleavage at the thrombin-susceptible sites Arg372, Arg740, and Arg1689, factor
Xa also cleaves at Arg219 within A1 and Arg490
within A2 to yield a pentameric factor VIIIa. Although inactivation of
cofactor was not studied in that report, no cleavage at
Arg336 was noted. Interestingly, Arg219 in A1
is unique to the pig protein and is a Gln residue at the homologous
site in the human, dog, and mouse proteins (42-44). Conversely, the
Lys36 cleavage site we report in this study is replaced by
Gly (pig and dog) and Thr (mouse) in the animal proteins, indicating
that this factor Xa site is restricted to the human factor VIII.
Parker et al. (41) also noted that the catalytic efficiency
(kcat/Km) for factor Xase
comprised of factor Xa-activated cofactor is severalfold lower than
that for factor Xase comprised of thrombin-activated factor VIIIa, a
result consistent with an earlier study from that laboratory (35). The
reason for this is the combination of an ~3-fold increase in the
Km for factor X plus a similar fold decrease in
Vmax. These parameters approach the deficiencies
in kcat and Km as well as the overall reduced catalytic efficiency (~15-fold) we observed for factor Xase wherein the reconstituted cofactor was altered solely within its A1 subunit. Thus, it is tempting to speculate that cleavage
of porcine factor VIIIa at Arg219 in the A1 subunit is
primarily responsible for the reduced catalytic efficiency relative to
thrombin-activated factor VIIIa as observed by these investigators.
In summary, we show that cleavage of the A1 subunit by factor Xa occurs
at two distinct sites, Lys36 and Arg336, which
are localized on opposite faces of the A1 domain according the homology
model of Pemberton et al. (38). This spatial orientation suggests a complex interaction of protease with this subunit in attack
of the scissile bonds. Both cleavages reduce, by degrees, the intrinsic
capacity of A1 subunit to stimulate the cofactor activity associated
with the A2 subunit. Although the activity loss resulting from cleavage
of A1 at Arg336 persists following reconstitution with the
A3-C1-C2 subunit, the activity loss attributed to cleavage at
Lys36 is somewhat restored upon reassociation in the dimer.
This result suggests that a component of the contribution of residues
1-36 is in maintaining an active conformation of the A1 subunit.
Importantly, a prominent mechanism for the loss of cofactor activity
following terminal digest of the A1 subunit reflects a markedly
increased Km for factor X, a property that likely
severely limits factor Xase activity in plasma.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-thrombin, factor IXa, factor X,
and factor Xa (Enzyme Research Laboratories, South Bend, IN) and the
chromogenic Xa substrate S-2765
(N-
-benzyloxycarbonyl-D-arginyl-glycyl-L-arginyl-p-nitroanilide-dihydrochloride; DiaPharm Group, Westchester, OH) were purchased from the indicated vendors. Acrylodan and fluorescein-5-maleimide were obtained from Molecular Probes (Eugene, OR).
= 460-480 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (25K):
[in a new window]
Fig. 1.
SDS-PAGE and Western blots of native and
cleaved A1 forms. A, the purified subunits were run on an
8% gel followed by staining with GelCode Blue. Lane 1,
molecular mass markers; lane 2, native A1 subunit;
lane 3, A1336; lane 4,
A137-336. B, Western blot of the A1 (lane
1), A1336 (lane 2), and
A137-336 (lane 3) using anti-A1 monoclonal
antibody specific for the N-terminal region (58.12,
left panel) and for the C-terminal region (C5,
right panel).
Amino-terminal sequence analysis of the ~40-kDa A1 subunit
1) was only slightly greater than the value
obtained with A2 subunit alone (~0.1 min
1; data not
shown). Although a functional Kd value was difficult
to quantitate given the low levels of activity, these data suggest a
weak intersubunit interaction for this A1 form with A2
(Kd > 2 µM), consistent with a marked
reduction in the capacity of A137-336 to bind A2. Taken
together, these results indicate that the N-terminal region of A1 is
required for high affinity interaction with A2 subunit and subsequent
stimulation of A2 activity.
View larger version (15K):
[in a new window]
Fig. 2.
Stimulation of A2 activity by A1 forms.
Cofactor activity of the isolated A2 subunit was measured in a factor
Xa generation assay. A2 subunit (400 nM) was reacted with
indicated levels of A1 (open circles), A1336
(closed circles), or A137-336
(squares) subunit. After incubation with 5 nM
factor IXa and 10 µM PS-PC-PE, the reaction was initiated
with the addition of 500 nM factor X as described under
"Materials and Methods." The initial rates of factor Xa generation
were plotted as functions of A1 concentration and fitted to the
quadratic equation by nonlinear least squares regression (45).
Kinetic and binding parameters for interactions of A1 and A2 subunits
1, respectively. These relative activity values were
similar to results obtained above for the direct stimulation of the A2
subunit. The Kd value for the intersubunit
interaction with A2 using native A1/A3-C1-C2 dimer was 202 ± 21 nM, a value similar to the earlier reported values obtained
under physiological conditions (~260 nM) (8, 9).
Furthermore, the Kd value (299 ± 51 nM) for A2 subunit interaction with the
A1336/A3-C1-C2 dimer was similar to that of native A1.
View larger version (16K):
[in a new window]
Fig. 3.
Factor VIIIa reconstituted with A1
forms. The A1 form/A3-C1-C2 dimer was reconstituted with 500 nM A3-C1-C2 and the indicated levels of A1 (open
circles), A1336 (closed circles), or
A137-336 (squares) overnight at 4 °C. The
reaction was diluted 10-fold, and factor VIIIa was formed by addition
of 20 nM A2 subunit. After incubation with 20 nM factor IXa and 10 µM PS-PC-PE, the
reaction was initiated with the addition of 500 nM factor X
as described under "Materials and Methods." The initial rates of
factor Xa generation were plotted as a function of A1 concentration and
fitted to the quadratic equation by nonlinear least squares
regression.
1) was ~30% the value
compared with factor VIIIa formed with native A1 and ~60% of the
activity formed with A1336. Furthermore, the
A137-336/A3-C1-C2 dimer showed equivalent affinity for the
A2 subunit (Kd = 245 ± 35 nM)
compared with the values obtained for the native dimer and
A1336/A3-C1-C2 dimer. This association of the
A137-336 subunit with A3-C1-C2 resulted in a >10-fold
affinity enhancement compared with that observed for isolated
A137-336 subunit. These results indicate that both a
physical and functional interaction of the A137-336
subunit with A2 requires association of this A1 form with the A3-C1-C2
and suggests that this interaction is mediated by an A3-C1-C2-dependent change in the conformation of the
A137-336 subunit.
View larger version (11K):
[in a new window]
Fig. 4.
Activity of reconstituted factor VIIIa forms
in a one-stage clotting assay. The A1/A3-C1-C2 (open
circles) or A137-336/A3-C1-C2 (closed
circles) dimer forms were reconstituted as described in the legend
to Fig. 3. The reactions were diluted 10-fold, and factor VIIIa was
reconstituted with 20 nM A2 subunit. Cofactor activity was
determined in a one-stage clotting assay using factor VIII-deficient
plasma as described under "Materials and Methods."
1, respectively, consistent with the
data described above. The Km value for factor X
obtained with factor Xase comprised of the A1-containing factor VIIIa
(41.3 ± 3.3 nM) was 5-fold lower compared with that
for A137-336-containing factor VIIIa (206 ± 19 nM). Overall, these parameters result in a marked reduction
in catalytic efficiency
(Vmax/Km) for factor Xase
composed of the truncated A1 (0.035 min
1
nM
1) compared with native factor Xase (0.53 min
1 nM
1). These results also
indicate that for reactions containing the latter factor VIIIa form,
the factor X concentration is ~20% the Km value
in the one-stage assay. Taken together, these results support a role
for the C-terminal region of A1 in factor Xase substrate binding and
are consistent with the hypothesis that the disparity observed in the
two assays when using C-terminal truncated factor VIIIa forms reflects,
in part, a rate-limiting substrate concentration in the plasma-based
assay.
View larger version (13K):
[in a new window]
Fig. 5.
Kinetics of factor VIIIa forms for factor
X. A3-C1-C2 (500 nM) was reconstituted with 500 nM A1 (open circles) or A137-336
(closed circles) overnight at 4 °C. Factor VIIIa was
formed by incubation of 50 nM dimer form and 20 nM A2. After incubation with 20 nM factor IXa
and 10 µM PS-PC-PE, the reactions were initiated with
variable levels of factor X as described under "Materials and
Methods." The initial rates of factor Xa generation were plotted as a
function of factor X concentration, and the data were fitted by
nonlinear least squares analysis.
= 460-480 nm, and
percentage of donor quenching was calculated based on the intensity of
Ac-A2 in the absence and presence of the Fl-A1 form. These data are
shown in Fig. 6 and Table
III. Saturable donor quenching
(46.1 ± 3.0%) was observed following titration with Fl-A1,
indicative of association of Ac-A2 subunit, and these data were used to
calculate a Kd value (432 ± 66 nM). This value obtained using the physical assay was equivalent to intersubunit affinity determined using a functional assay
(Kd = 428 nM; see Table II). However,
little donor quenching was observed for the
Ac-A2-Fl-A137-336 subunit pairing (~7%), and this
effect was not saturable over the range of acceptor concentrations
employed, suggestive of little association of Ac-A2 with the truncated
A1. Thus, the affinity values obtained with the physical assay support
the functional affinities determined by the factor Xa generation assay
and are consistent with a marked increase in Kd
(>4-fold) following cleavage of Lys36.
View larger version (13K):
[in a new window]
Fig. 6.
Energy transfer between Fl-A1 form and
Ac-A2. Ac-A2 (200 nM) and varying levels of Fl-A1
(open circles) or Fl-A137-336 (closed
circles) was reacted for 30 min as described under "Materials
and Methods." The emission intensity of Ac-A2 was measured at
= 460-480 nm and recorded in the absence and presence of
indicated levels of the Fl-A1 form. % Donor Quenching
refers to the fluorescence intensity of Ac-A2 in the presence of the
Fl-A1 form relative to that of Ac-A2 alone. Unlabeled A1 had no effect
on the fluorescence emission of Ac-A2. The percentage of quenching was
plotted as a function of Fl-A1 concentration, and the data were fitted
by nonlinear least squares analysis.
Binding affinity and donor quenching parameters derived from energy
transfer between F1-A1 and Ac-A2 subunits
View larger version (14K):
[in a new window]
Fig. 7.
Energy transfer between Fl-A1 form/A3-C1-C2
dimer and Ac-A2. Fl-A1/A3-C1-C2 or
Fl-A137-336/A3-C1-C2 dimer was reconstituted by reacting 2 µM A3-C1-C2 and 2 µM Fl-A1 (open
circles) or Fl-A137-336 (closed circles).
Ac-A2 (100 nM) was reacted with varying amounts of each
dimer for 30 min as described under "Materials and Methods."
% Donor Quenching refers to the fluorescence intensity of
Ac-A2 reconstituted with the dimer relative to that of Ac-A2 alone. The
data were fitted as described above.
1) and with A1/A3-C1-C2 (Kd = 72 nM, 9.7 kcal mol
1) indicate that ~90% of
the binding energy for A2 association within the factor VIIIa
heterotrimer is derived from interaction with the A1 subunit.
Furthermore, the N- and C-terminal truncations in A1 contribute
little to the direct binding of A2 in the factor VIIIa heterotrimer.
Therefore, the lack of interaction of A2 with the isolated
A137-336 compared with high affinity binding of A2 to the
dimer containing these A1 truncations suggest that interactions within
the dimer promote a change in conformation in A1, facilitating its
interaction with A2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Debra Pittman of Genetics Institute and Lisa Regan of Bayer Corporation for the gifts of recombinant factor VIII.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL 38199 and HL 30616.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, P. O. Box 610, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-6576; Fax: 585-473-4314; E-mail: Philip_Fay@urmc.rochester.edu.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M209811200
2 K. Nogami and P. J. Fay, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; TAP, tick anticoagulant peptide; MES, 4-morpholineethanesulfonic acid; Ac-A2, A2 subunit labeled with acrylodan; Fl-A1, A1 labeled with fluorescein; Fl-A137-336, A137-336 labeled with fluorescein; CAPS, 3-(cyclo-hexylamino)-1-propanesulfonic acid.
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