From the Department of Biochemistry and Biophysics
and the § Department of Medicine, University of Rochester
School of Medicine, Rochester, New York 14642
Received for publication, October 18, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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Factor VIII circulates as a noncovalent
heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B
domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B
junctions generates the A1 and A2 subunits of factor VIIIa. Although
the isolated A2 subunit stimulates factor IXa-catalyzed generation of
factor Xa by ~100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa
and X resulted in an increase in factor IXa activity because of
conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by
thrombin or factor Xa yielded similar products, although factor Xa
cleaved at a rate of ~1% observed for thrombin. HC showed little
inhibition of the A2 subunit-dependent stimulation of
factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the
fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the
presence of factor X, whereas thrombin-cleaved HC yielded a marked
increase in this parameter. These results indicate that HC cleavage by
either thrombin or factor Xa is essential to expose the factor
IXa-interactive site(s) in the A2 subunit required to modulate protease activity.
Factor VIII, the plasma protein deficient or defective in
individuals with hemophilia A, is synthesized as a 300-kDa precursor (1, 2) with the domain structure A1-A2-B-A3-C1-C2 (3). It is processed
to a series of divalent metal ion-dependent heterodimers after cleavage at the B-A3 junction, generating a
HC1 (A1-A2-B domains) and a
LC (A3-C1-C2 domains). Additional cleavage sites within the B domain
result in variably sized HCs minimally represented by contiguous A1-A2
domains. The two chains can be separated by chelating reagents (4, 5)
and isolated after ion exchange and/or immunoaffinity chromatography.
Factor VIII activity can be reconstituted from the separated chains by
combining them in the presence of a divalent metal ion (6).
Factor VIII functions in the intrinsic factor Xase complex as a
cofactor for the serine protease, factor IXa in the
surface-dependent conversion of factor X to Xa. This
activity is dependent upon conversion of factor VIII to the active
cofactor form, factor VIIIa, by thrombin or factor Xa. These enzymes
cleave factor VIII HC at Arg-740, removing the B domain (or fragments)
and at Arg-372, bisecting the HC into the A1 and the A2 subunits (7).
The proteases also cleave factor VIII LC at Arg-1689 (7), liberating an
acid-rich region and creating a new NH2 terminus. Thus,
factor VIIIa is a heterotrimer of subunits designated as A1, A2, and
A3-C1-C2 (8, 9). The A1 and A3-C1-C2 subunits retain the divalent metal
ion-dependent linkage, whereas the A2 subunit is weakly associated with the A1-A3-C1-C2 dimer by primarily electrostatic interactions (9, 10).
Two regions of factor VIII have been identified as interactive sites
for factor IXa. A high affinity site (Kd~15 nM (11)) was localized to the A3 domain of the LC in and
around residues 1811-1818 (12). A second, lower affinity site
(Kd~300 nM (13)) was localized within
the (isolated) A2 domain and comprises residues 558-565 (14).
Recently, isolated A2 subunit was shown to stimulate the
kcat for factor IXa-catalyzed conversion of
factor X by ~100-fold (13). This property appeared unique to A2 and was not observed for either the isolated A1 or A3-C1-C2 subunit. However, the A1 subunit synergistically increased the cofactor activity
of the isolated A2 subunit by ~10-fold (15).
Proteolysis of the LC during activation is responsible for the
dissociation of factor VIIIa from its carrier protein, von Willebrand
factor (16). This cleavage also appears to increase the cofactor
activity of factor VIIIa (17, 18). The function of HC cleavage (at
Arg-372) is not well understood. However, this step is essential for
generating cofactor activity based upon mutations at this which result
in severe hemophilia (19).
The isolated A2 subunit of factor VIIIa shows a several hundredfold
weaker affinity for factor IXa and ~1% of the cofactor activity
compared with intact factor VIIIa (13). It is unknown whether these low
level activities are intrinsic to the HC or whether cleavage of the HC
is necessary to manifest them. In this report we examine the capacity
of isolated HC and derived subunits to modulate the catalytic activity
of factor IXa in a purified system. Studies were performed in the
absence of LC to preclude any interactions of this chain with factor
IXa or factor X. Results show that intact HC possesses no detectable
cofactor-like activity and fails to compete with isolated A2 subunit
for interaction with factor IXa. However, its resultant cleavage by
thrombin or (less efficiently) by factor Xa generates active subunits
that show cofactor activity and modulation of factor IXa activity
similar to that observed previously using the purified subunits. These results indicate that a primary role for HC cleavage during cofactor activation is the exposure of a functional factor IXa-interactive site.
Reagents--
The reagents Factor VIII Subunits--
Recombinant factor VIII preparations
were gifts from the Bayer Corporation and the Genetics Institute.
Factor VIII HC was prepared as described previously (23) and is
illustrated in Fig. 1. Potential trace
levels of factor VIII LC present in the HC preparation were removed
following chromatography using antibody 10104 coupled to Affi-Gel 10 as
described previously (6). The A2 subunit was prepared following
fractionation of factor VIIIa using Mono S as described previously
(9).
Factor Xa Generation Assays--
The rate of conversion of
factor X to factor Xa was monitored in a purified system (24). HC forms
were reacted with factor IXa in 20 mM Hepes pH 7.2, 50 mM NaCl, 5 mM CaCl2, and 0.01%
Tween 20 (buffer A) in the presence of 100 µg/ml bovine serum albumin and 10 µM phospholipid vesicles. Reactions were initiated
with the addition of factor X (for reactant concentrations, see figure legends). Aliquots were removed at appropriate times to assess the
initial rates of product formation and added to tubes containing EDTA
(80 mM final concentration) to stop the reaction. Rates of factor Xa generation were determined by the addition of the chromogenic substrate, S-2765 (0.46 mM final concentration). Reactions
were read at 405 nm using a Vmax microtiter
plate reader (Molecular Devices).
Electrophoresis and Western Blotting--
SDS-polyacrylamide gel
electrophoresis was performed using the method of Laemmli (25)
with a Bio-Rad minigel system. Electrophoresis was performed at 200V
for 1 h. Proteins were stained using Gel-Code Blue (Pierce).
Alternatively, the proteins were transferred to a polyvinylidene
difluoride membrane using a Bio-Rad mini-transblot apparatus at 0.5 amps for 30 min in a buffer containing 10 mM CAPS, pH 11, and 10% (v/v) methanol. Western blotting used the R8B12 monoclonal
followed by goat anti-mouse horseradish peroxidase-conjugated secondary
antibody. The secondary antibody signal was detected using the ECL
system (Amersham Pharmacia Biotech) with luminol as substrate, and the
blots were exposed to film for various times. Films were scanned, and
band densities (obtained from a linear exposure range) were quantitated
by using ImageQuant software (Molecular Devices).
Rates of HC cleavage were calculated from the linear portion (initial
time points) of the plotted data. Initial rates were estimated using
best fit lines through points where Fluorescence Spectroscopy--
Fluorescence anisotropy
measurements were conducted using an Amico-Bowman series 2 spectrometer
equipped with automatic polarizers arranged in an L-format.
Reactions (0.2 ml) were run at room temperature in buffer A containing
90 nM Fl-FFR-IXa, 460 nM HC form, and 50 µM PSPCPE vesicles in the absence or presence of 500 nM factor X. Samples were excited at 495 nm, and the
emission intensity was monitored at 520 nm (band pass = 4 nm) for
2 s at each polarizer position. Anisotropy values were calculated
automatically after subtraction of blank readings. Data were averaged
for at least five anisotropy measurements.
Cleavage of Isolated HC Is Required for Cofactor
Activity--
Earlier we showed that the isolated A2 subunit of factor
VIII stimulated the kcat for factor
IXa-catalyzed conversion of factor X by ~1% the level observed for
factor VIIIa (13). The effect was enhanced further by 1 order of
magnitude in the presence of saturating A1 subunit (15). Isolated
factor VIII HC was assessed similarly for cofactor activity using a
factor Xa generation assay and employing purified components. HC was
obtained from EDTA-treated factor VIII as described under "Materials
and Methods" and was essentially free from LC. Titration of intact HC
in the factor Xa generation assay yielded virtually no activity
increase under the reaction conditions described in Fig.
2. However, prior cleavage of HC by
thrombin to yield the HCIIa (free A1 and A2 subunits) resulted in a marked increase in the rate of factor Xa formed which was
dose-dependent and saturable with respect to the cleaved HC. The concentration of substrate factor X used in these reactions (500 nM) represented near Vmax
levels (data not shown). The extent of factor IXa stimulation observed
at near saturating levels of HCIIa (1 µM;
kcat ~ 9 nM factor Xa
generated/min/nM factor IXa) was similar
to that observed for factor IXa in the presence of an equivalent
concentration of purified A2 plus A1 subunits
(kcat ~14 factor Xa
generated/min/nM factor IXa) (15). These results indicate
that intact HC possesses no detectable factor IXa-stimulating activity
and that this property is expressed only after cleavage of the HC to
component subunits.
To help exclude any contribution of trace factor VIII to these results,
a series of experiments was performed in which HC was reacted first
with one of three anti-factor VIII monoclonal antibodies prior to
treatment with thrombin (Table I).
Antibody 10104, a potent inhibitor that binds factor VIII LC (5),
showed no effect in inhibiting HCIIa activity, consistent
with the absence of functional factor VIII in the assay. R8B12, an
anti-A2 domainal monoclonal with little inhibitory activity, also
showed no effect. However, antibody 413, which binds an epitope defined
by A2 domain residues 484-508 (22) and blocks the interaction between
isolated A2 subunit and factor IXa (26), completely eliminated the
cofactor activity of HCIIa. These results support the
conclusion that the activity observed was attributed to
HCIIa and was not the result of trace contamination of the
HC preparation with functional factor VIII.
Interestingly, although no activity of HC was detected using the early
time points after its addition to the factor Xa generation assay,
prolonged incubation of these reaction components yielded the
appearance of factor Xa-generating activity (Fig.
3). In this experiment, HC was reacted in
the presence of factor IXa, factor X, and PSPCPE vesicles. At the
indicated times, an aliquot of this mixture was removed and assessed
for the rate of factor Xa generation by a chromogenic assay and for HC
cleavage by SDS-polyacrylamide gel electrophoresis and Western blotting
using the anti-A2 domain monoclonal antibody R8B12. Progress curves for
factor Xa formation and HC cleavage began with an apparent lag phase
followed by progressive increases in reaction rate and product
appearance. The extent of factor Xa-generating activity paralleled
cleavage of HC to form the A2 subunit. These results were reminiscent
of earlier data illustrating factor Xa generation and cleavage of
intact cofactor in a purified system lacking any factor X activator
(27). Because no thrombin was present in the reaction, we attributed HC
cleavage to factor Xa formed by factor IXa, the activity of which was
accelerated after generation of the A1 and A2 subunits. Control
experiments indicated the absence of HC cleavage when factor X was
omitted from the reaction mixture (results not shown), thus precluding
factor Xa formation as well as indicating that factor IXa did not
utilize HC as a substrate. Furthermore, a marked reduction of HC
cleavage was observed when the reaction was supplemented with the
factor Xa-specific inhibitor, tick anticoagulant peptide (results not
shown). Taken together, these results indicate that cleavage of HC
resulted from factor Xa generated in the reaction mixture.
Cleavage of HC by Thrombin and Factor Xa--
The above results
demonstrated that the isolated HC was a substrate for factor Xa which,
similar to thrombin, can convert the inactive precursor to an active A2
subunit. To compare isolated HC as a substrate for thrombin and factor
Xa, cleavage rates were determined from time course reactions. 300 nM HC in the presence of buffer A containing 10 µM phospholipid was reacted with either 5 nM thrombin or 50 nM factor Xa. This substrate
concentration was chosen to parallel HC levels used in the factor Xa
generation assays. Desitometry analyses of the Western blots reacted
with R8B12 were used to calculate the concentration of epitope present as either HC or the A2 subunit. As observed in the inset in
Fig. 4, intact HC is represented by a
series of bands (~210-130 kDa) that are first cleaved to an
intermediate of ~90 kDa (contiguous A1-A2 domains). This intermediate
is then cleaved to yield the ~40-kDa A2 subunit plus A1. We
originally observed this processing step for the HC of factor VIII by
thrombin (5). Interestingly, factor Xa follows a similar reaction
pathway. Because the factor VIII heterodimer comprising a 90-kDa HC is
activable by thrombin to the same extent as heterodimers with higher
Mr HC, the density attributed to the 90-kDa band
was combined with these HC forms. Thus, the rates of cleavage reflect
conversion to the final product, A2 subunit.
The rate of A2 formation catalyzed by thrombin was significantly
greater than that observed for factor Xa. Examination of initial time
points ( Effects of HC and HCIIa on the Fluorescence Anisotropy of
Fl-FFR-Factor IXa--
The above results using a functional assay
demonstrate essentially no effect of isolated HC on the catalytic
activity of factor IXa. To determine whether this chain influences the
active site of the protease, fluorescence anisotropy using a
Fl-FFR-labeled factor IXa was performed. Earlier results demonstrated
that this parameter was affected little if any by isolated A2 subunit,
whereas a significant increase in anisotropy was observed in the
presence of substrate factor X (13). Inclusion of isolated HC showed no
effect on the anisotropy of Fl-FFR-factor IXa, either in the absence or
presence of factor X (Table II). However,
prior cleavage of HC with thrombin resulted in a marked increase in
anisotropy observed in the presence of factor X, consistent with the
generated A2 subunit altering the factor IXa active site. These results indicate the uncleaved HC does not affect the active site of factor IXa, consistent with its lack of affect in enhancing catalytic activity.
Competition Analysis between HC and A2 Subunit for Binding Factor
IXa--
The isolated A2 subunit binds factor IXa with a
Kd ~ 300 nM, whereas the A1 subunit
does not appear to bind the protease directly (13) nor affect the
affinity of A2 subunit for factor IXa (15). Thus any potential factor
IXa-interactive site in the HC is likely contained within the A2
domain. To assess the relative affinity of HC and the derived A2
subunit for factor IXa, a competition assay was performed using
stimulation of factor IXa activity by A2 subunit as an indicator of the
interaction. The effect of increasing amounts of HC on this stimulation
was subsequently determined. Results in Fig.
5 show stimulation of factor
IXa-catalyzed generation of factor Xa in the presence of 100 nM A2 subunit. Addition of HC resulted in little change in the A2-dependent stimulation of activity. At 1 µM HC, a 10-fold excess relative to A2 subunit, the
A2-dependent activity was reduced by ~20%. This result
suggests that the affinity of factor IXa for HC is at least 1 order of
magnitude weaker than that for the A2 subunit.
To gain insights into the contribution made by HC cleavage to
cofactor activation, a series of experiments was performed using purified HC in the absence of LC to preclude any interaction of the
latter with components of the assay. In this report we show that
cleavage of isolated factor VIII HC is essential for subsequent interaction with factor IXa and stimulation of its catalytic activity. The isolated A2 subunit of factor VIIIa is capable of binding factor
IXa and stimulating the activation of factor X (13); however, the
isolated HC (i) showed no stimulation of factor IXa in the generation
of factor Xa, (ii) did not affect the conformation in and around the
active site of the enzyme in the absence or presence of substrate, and
(iii) failed to compete effectively with the A2 subunit for binding
factor IXa as judged by a functional assay. Thus, these results
indicate that (isolated) HC is devoid of the basal cofactor-like
activities associated with its component subunits. These results are
consistent with the observed lack of cofactor activity in the
unactivated factor VIII heterodimer (27).
It is of interest to note that both thrombin and factor Xa cleave the
isolated HC, although with markedly disparate efficiency. The slow
cleavage of HC by low levels of factor Xa in the Xa generation assay
resulted in the time-dependent stimulation of the reaction rate. This can be attributed to production of A2 subunit, which could
then serve as cofactor to factor IXa. In a more controlled experiment,
we show that HC cleavage by factor Xa occurred at ~1-2% the rate
observed for thrombin. These reactions contained a phospholipid
surface, which is requisite for optimal factor Xa activity. However,
the absence of factor VIII LC precluded association of HC with surface
and this condition severely retarded cleavage by the protease.
Consistent with this observation was the recent identification of a
factor Xa-interactive site within the C2 domain of factor VIII (28). In
the absence of LC, we speculate that the factor X site localized to HC
residues 337-372 (29) may serve as an interactive site for (solution
phase) factor Xa binding and cleavage of the subunit. Support for this
speculation comes from studies employing a zero-length cross-linker
that indicated that this site was bound by a region of the
protease-forming domain of factor X distinct from the activation
peptide sequence (30).
Using intact porcine factor VIII as substrate, Lollar et al.
(27) showed that the catalytic efficiency
(kcat/Km) for cofactor
cleavage was ~5-fold greater for thrombin compared with factor Xa.
Results of that study also indicated that factor VIIIa activity
generated by thrombin was ~3-fold greater than that generated by
factor Xa. Our data do not suggest that the isolated (human) HC cleaved
by factor Xa possesses significantly less activity than
thrombin-cleaved HC. Results obtained in Fig. 2 showed that the
complete conversion of 300 nM HC by factor Xa yielded
activity similar to that observed earlier with an equivalent concentration of isolated A2 plus A1 subunits prepared following cleavage of factor VIII by thrombin (15). Thus, the
activator-dependent disparity in cofactor activity may
reflect a differential effect and/or contribution of the factor VIII LC
cleavage, or it may indicate a species difference.
Recently, Lollar and co-workers (31) characterized more fully factor
Xa-activated (porcine) factor VIIIa and noted cleavage sites in the HC
at Arg-219 (A1 domain) and Arg-490 (A2 domain) in addition to those HC
sites cleaved by thrombin (Arg-372 and Arg-740). Although the Arg-490
site is present in the human protein, residue 219 is Gln and thus would
not be attacked efficiently by the protease. However, Arg-490 in the
isolated human HC did not appear to be cleaved at any appreciable level
because the fragment generated (residues 491-740) would retain
reactivity with the R8B12 antibody, and this fragment was not detected
in our blots. We did note, however, that the spacing between the A2
doublet bands was somewhat greater for the Xa-cleaved material, reflecting a slightly smaller, lower Mr band in
the doublet. The reason for this is unclear at the present time.
The results presented in this study are compatible with, at best, a
weak affinity interaction between HC and factor IXa. In an earlier
report, Lenting et al. (11) used a solid phase binding assay
in the absence of phospholipid to show that isolated factor VIII LC
bound factor IXa with high affinity (Kd ~15
nM). However, no detectable factor IXa binding was observed
using HC up to ~300 nM. A factor IXa site has been
localized to residues 558-565 in the A2 subunit (14). Recently, we
showed that isolated A2 subunit bound factor IXa in a
phospholipid-containing system with a functional Kd
~300 nM (13). In the absence of a surface, saturation of
factor IXa with A2 subunit is not readily achieved reflecting a
Kd >5
µM.2 Thus
the phospholipid surface likely orients factor IXa such that collisions
with A2 subunit are more productive. In this report, we show that a
10-fold molar excess of HC relative to A2 subunit marginally inhibited
the A2-dependent stimulation of factor IXa activity,
suggesting that the affinity of isolated HC for the enzyme is at least
10-fold weaker (>3 µM) than that of the derived A2 subunit.
One model consistent with these observations is that cleavage of HC is
required to expose the factor IXa-interactive site(s). Several lines of
evidence demonstrate a conformational change in factor VIII HC
following the conversion of factor VIII to factor VIIIa. For example,
reaction of factor VIIIa with the zero-length cross-linker, EDC,
resulted in formation of a covalent linkage between A1 and A2 subunits
(32), indicating the presence of a salt bridge at the site of
cross-linking. However, treatment of factor VIII with EDC prior to
cleavage by thrombin showed no linkage between the subunits, suggesting
that the interdomain salt bridge was not present in the unactivated
form. In addition, examination of binding of the apolar probe
bisanilinonapthalsulfonic acid to isolated factor VIII and factor VIIIa
subunits revealed two exposed hydrophobic sites on the isolated HC (33)
possessing affinities (Kd values) of 0.21 and
1.4 µM. However, these sites contrast the single sites
localized to the isolated A1 subunit (0.77 µM) and A2
subunit (0.11 µM), suggesting a change in conformation in
and around these regions following thrombin cleavage. Finally, CD
studies suggest an increase in Little evidence exists for a specific conformational change in and
around the factor IXa-interactive site in A2 after cofactor activation.
Suggestive evidence comes from observations of the inactivation of
factor VIII and factor VIIIa by activated protein C. In collaboration
with Walker, we showed that the bovine protease binds the cofactor near
the COOH-terminal region of the A3 domain in the light chain (35) and
preferentially attacks Arg-562 (21) within the factor IXa-interactive
site. Interestingly, the rate of cleavage at this site in factor VIIIa
is ~5-fold faster than its cleavage in factor VIII (21). In factor
VIIIa, this site is protected from cleavage by factor IXa (36).
However, no factor IXa-dependent protection was observed
using factor VIII (37). These results are compatible with differential
exposure of the scissile bond at Arg-562 in the two substrates, with
this region partially masked in the unactivated form. Taken together
with the results of this study, these observations suggest that the factor IXa site localized to residues 558-565 in the A2 subunit is not
fully formed in the contiguous A1-A2 domains of uncleaved HC. This lack
of a functional factor IXa site in HC likely represents a primary
requirement for cofactor activation at this domain junction and
provides an explanation for the molecular basis of severe hemophilia
attributed to cleavage-resistant mutations at Arg-372.
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, factor Xa (Enzyme Research Laboratories), and Fl-FFR-factor IXa
(Molecular Innovations) were purchased from the indicated vendors.
Phospholipid vesicles composed of 20% PS, 40% PC, and 40% PE (Sigma)
were prepared using octyl glucoside as described previously (20). Tick
anticoagulant peptide was a gift from Dr. S. Krishnaswamy. The
anti-factor VIII monoclonal antibody, R8B12, which recognizes the
COOH-terminal portion of the A2 domain (21), was prepared as described
(9). Antibody 10104, an inhibitory monoclonal that binds the
NH2-terminal region of factor VIII light chain (5), was
obtained from QED BioScience. Monoclonal antibody 413 binds an epitope
defined by residues 484-508 (22) and was a generous gift from Dr. Leon Hoyer.
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Fig. 1.
SDS-polyacrylamide gel electrophoresis of
factor VIII and purified HC. Factor VIII (lane 1) and
HC (lane 2) (1 µg each) were visualized following
electrophoresis and staining as described under "Materials and
Methods."
50% of the substrate had been
converted to product. The concentration of HC remaining was calculated
from band densities using the formula HC = (density of HC/(density
of HC + density of A2 subunit)) × initial HC concentration. The
concentration of A2 subunit formed following HC cleavage was calculated
using the formula A2 subunit = (1
(density of HC/(density of HC + density of A2 subunit))) × initial HC concentration.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Factor Xa-generating activity of intact and
thrombin-cleaved HC. Factor Xa generation assays contained 5 nM factor IXa, 10 µM PSPCPE vesicles, 500 nM factor X, and the indicated concentrations of HC
(squares) or HC that had been treated for 5 min with 20 nM thrombin followed by quenching of the thrombin with
hirudin (circles). Reactions were run for less than 2 min at
room temperature at which time the amount of factor Xa formed was
quantitated as described under "Materials and Methods." Data points
represent the mean of at least three separate determinations.
Effects of anti-factor VIII antibodies on the stimulation of factor IXa
activity by thrombin-cleaved HC
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Fig. 3.
Correlation of rate of factor Xa generation
with cleavage of HC. The reaction mixture contained 300 nM HC, 1 nM factor IXa, 500 nM
factor X, and 10 µM PSPCPE vesicles. At the indicated
times, aliquots were removed and assessed for factor Xa generated
(open circles) and HC cleavage using SDS-polyacrylamide gel
electrophoresis (closed circles) as described under
"Materials and Methods." The inset shows the gel,
blotted with R8B12, used to obtain densitometry data on A2 subunit
formation. Lanes 1-7 correspond to time points starting at
the 0 min point.
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Fig. 4.
Time course of HC cleavage by thrombin and
factor Xa. Isolated HC (300 nM) was reacted in buffer
A plus 10 µM PSPCPE vesicles and either 5 nM
thrombin (open circles) or 50 nM factor Xa
(closed circles). At the indicated times, aliquots were
removed, the reactions were terminated by boiling in gel sample buffer,
and samples were subjected to electrophoresis and blotting as described
under "Materials and Methods." The molar amount of heavy chain
remaining was calculated from densitometry scans of the blot.
Insets, bands were visualized following reaction with the
R8B12 antibody. Lanes 1-9 correspond to time points
beginning at the 0 min point after the addition of thrombin
(left) or factor Xa (right).
50% substrate utilized) using a linear curve fit indicated
rates of ~33.3 min
1 and 0.4 min
1 for thrombin and factor Xa,
respectively. Although these values obtained at a single HC
concentration allow for a limited comparison of rate for substrate
cleavage by the two enzymes under these reaction conditions, cleavage
by factor Xa appeared to occur at ~1-2% the rate observed for
thrombin. One likely reason for this markedly slower rate by factor Xa
is the absence of LC, which is required for association of factor VIII
with the phospholipid surface, a requisite step for efficient cleavage
of factor VIII by this protease (27).
Effects of HC cleavage and substrate on the fluorescence anisotropy of
Fl-FFR-factor IXa
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Fig. 5.
Effect of HC on the A2
subunit-dependent stimulation of factor IXa. Factor Xa
generation assays containing 5 nM factor IXa and 10 µM PSPCPE vesicles in buffer A were run in the presence
(circles) and absence (squares) of 100 nM A2 subunit plus the indicated concentrations of HC.
Factor Xa generated was determined from points taken at <2-min
reaction times. Data points represent the mean of at least three
separate determinations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheet structure in factor VIIIa
formed from factor VIII (34).
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ACKNOWLEDGEMENTS |
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We thank Debra Pittman of the Genetics Institute and James Brown and Lisa Regan of the Bayer Corporation for the gifts of recombinant factor VIII, Sriram Krishnaswamy for the tick anticoagulant peptide, and Leon Hoyer for the monoclonal antibody 413.
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FOOTNOTES |
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* This work was supported by Grants HL 38199 and HL 30616 from the National Institutes of Health.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.: 716-275-6576; Fax 716-473-4314; E-mail: Philip_Fay@urmc.rochester.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M009539200
2 P. J. Fay, M. Mastri, and M. E. Koszelak, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: HC, factor VIII heavy chain; LC, factor VIII light chain; Fl-FFR-FIXa, factor IXa modified in its active site with fluorescein Phe-Phe-Arg chloromethyl ketone; PS, phosphatidylserine, PC, phosphatidylcholine; PE, phosphatidylethanolamine; CAPS, 3-(cyclohexylamino)propanesulfonic acid; HCIIa, thrombin-cleaved HC; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.
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