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INTRODUCTION |
Factor VIII, a plasma protein deficient or defective in
individuals with hemophilia A, functions as a cofactor for the serine protease, factor IXa, in the anionic phospholipid
surface-dependent conversion of factor X to Xa (1). Factor
VIII is synthesized as an ~300-kDa precursor with domain structure
A1-A2-B-A3-C1-C2 and is processed to a series of divalent metal
ion-dependent 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 (2-4). Factor
VIII is activated by thrombin or factor Xa by limited proteolysis at
the A1-A2 domain junction (Arg372), A2-B junction
(Arg740), and near the N terminus of the light chain
(Arg1689) (5). The resultant factor VIIIa, a heterotrimer
composed of A1, A2, and A3-C1-C2 subunits (6, 7), retains the metal ion-dependent linkage between the A1 and A3-C1-C2 subunits,
whereas A2 is associated with weak affinity (Kd
~260 nM (8)) by primarily electrostatic interactions (7,
9). Thus at physiological concentrations (~1 nM), factor
VIIIa is unstable, and loss of activity is attributed to the
dissociation of the A2 subunit from the A1/A3-C1-C2 dimer (7, 9,
10).
The association of factor VIIIa with factor IXa increases the
kcat for factor Xa formation by several orders
of magnitude compared with factor IXa alone (11). Recent studies have
shown that modulation of factor IXa by A2 subunit enhances the
kcat for factor Xa activation by ~100-fold
(12) and that A1 subunit synergizes this effect (>15-fold) to alter
the interaction of A2 subunit with factor IXa (13, 14). The C-terminal
acidic region of the A1 subunit (residues 337-372) appears to
represent an A2-interactive site and participates in the orientation of A2 yielding maximal stimulation of factor IXa (14). This region is also
implicated in the binding of factor X (15), and recent evidence
suggests that this contributes to the Km value for
factor X interaction with factor Xase (16). Several proteases including
activated protein C (5, 17), factor IXa (18, 19), and factor Xa (5)
attack this site cleaving at Arg336 in the A1 subunit, and
this event correlates with inactivation of the cofactor. Although the
mechanism for inactivation following cleavage at this site is not fully
understood, component effects include altered interaction of the A2
subunit with the truncated A1 and an increase in the
Km value for substrate factor X. Recently, a second
cleavage site specific for factor Xa, Lys36, has been
identified in the A1 subunit (16). Attack at this site is also
inactivating, and this effect results in part from altered conformation
of A1 limiting productive interaction with the A2 subunit (16).
Although factor Xa functions as an activator of factor VIII, subsequent
factor Xa-catalyzed cleavage(s) within the A1 subunit of factor VIIIa
may contribute to the dampening of factor Xase activity. This potential
role may be significant given that the protease is the product of the
reaction catalyzed by factor Xase. However, the interactions of factor
Xa with substrate factor VIIIa (A1 subunit) are poorly understood. In
this study, we report on the catalytic mechanism during proteolytic
inactivation due to cleavage within the A1 subunit by factor Xa. By
using isolated subunits of the cofactor and by employing functional
reconstitution assays and SDS-PAGE analyses, we show the initial
reaction of the A1 subunit with the protease yields two distinct
intermediates indicative of separate pathways leading to the terminal
A137-336 product. Furthermore, specific cleavages at
Lys36 and Arg336 are differentially modulated
following interaction of the protease with the A3-C1-C2 subunit, as
well as the acidic C-terminal region of the A1 subunit.
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MATERIALS AND METHODS |
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 (20) was a gift from Dr. James Brown, and monoclonal antibody C5 recognizing the C-terminal end of A1 (21) was a
gift from Drs. Carol Fulcher and Zaverio Ruggeri. Phospholipid vesicles
containing 20% phosphatidylserine, 40% phosphatidylcholine, and 40% phosphatidylethanolamine (Sigma) were prepared using
N-octyl glucoside (22).
TAP1 was a gift from Dr.
Sriram Krishnaswamy. The reagents human
-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. The synthetic peptide corresponding to factor VIII residues Met337-Arg372 (designated as
peptide337-372) was prepared by the Biotechnology
Analytical and Synthesis Facility at Cornell University and has been
described previously (23). Peptide337-359 and
peptide360-372 were derived from a tryptic digest of
peptide337-372 and purified as described previously (23).
The peptide351-365 was a gift from Dr. Paul Foster.
Isolation of Factor VIIIa Subunits--
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 and heavy
chains were isolated following chromatography on SP-Sepharose and
Q-Sepharose columns (Amersham Biosciences) as described previously
(24). 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 (14). The A3-C1-C2 subunit was prepared as described previously (15). The
A1/A3-C1-C2 dimer was isolated from thrombin-treated factor VIIIa using
a Mono-S column chromatography, and the residual A2 subunit was removed
using an anti-A2 subunit monoclonal antibody coupled to Affi-Gel 10 (20). The A3-C1-C2 subunit was further purified following dissociation
of the dimer by EDTA and chromatography on Mono-S (15). A truncated
A11-336 subunit was prepared as described earlier (14).
SDS-PAGE of the isolated subunits showed >95% purity. Protein
concentrations were determined by the method of Bradford (25).
Cleavage of A1 or A1/A3-C1-C2 by Factor Xa--
The
A1/A3-C1-C2 dimer was reconstituted from isolated A1 and A3-C1-C2
subunits (2.4 µM each) overnight at 4 °C in 20 mM HEPES, pH 7.2, 0.3 M NaCl, 25 mM
CaCl2, and 0.01% Tween 20. Human factor Xa was added to
the isolated A1 subunit or A1/A3-C1-C2 dimer in a 1:10 ratio (mol/mol)
in a buffer containing 20 mM HEPES, pH 7.2, 0.1 M NaCl, 5 mM CaCl2, and 0.01%
Tween 20, and the reactions were run at 22 °C. Samples were taken at
the indicated times, and the reactions were immediately terminated and
prepared for SDS-PAGE by adding SDS and boiling for 3 min.
Factor Xa Generation Assays--
The rate of conversion of
factor X to factor Xa was monitored in a purified system (26). To
prepare A1/A3-C1-C2 dimers containing the cleaved A1 subunit, the
cleavage reaction was quenched using TAP at an ~5-fold molar excess
relative to factor Xa. This mixture was adjusted to 500 nM
and was reconstituted with 500 nM A3-C1-C2 subunit. Factor
VIIIa was reconstituted from the A1/A3-C1-C2 dimer form(s) (5 nM) plus A2 subunit (20 nM), and reacted with
20 nM factor IXa and 10 µM
phosphatidylserine-phosphatidylcholine-phosphatidylethanolamine vesicle
for 30 s in 20 mM HEPES, pH 7.2, 0.05 M
NaCl, 5 mM CaCl2, 100 µg/ml bovine serum
albumin, and 0.01% Tween 20. Reactions were initiated with the
addition of 500 nM factor X. 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. Rates of factor Xa generation were determined by
the addition of the chromogenic substrate, S-2765 (final concentration;
0.46 mM). Reactions were read at 405 nm using a
Vmax microtiter plate reader (Molecular Devices,
Sunnyvale, CA). All reactions were run at 22 °C.
Electrophoresis and Western Blotting--
SDS-PAGE was performed
with 8% gels using the procedure of Laemmli (27). Electrophoresis was
carried out using a Bio-Rad minigel apparatus at 150 V for 1 h.
Bands were visualized following staining with GelCode Blue Stain
Reagent (Pierce). For Western blotting, the proteins were 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. Proteins were 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 a Storm 860 instrument (Molecular Devices).
Densitometric scans were quantitated using ImageQuant software
(Molecular Devices).
Data Analysis--
The concentrations of remaining intact A1 and
factor Xa-cleaved A1 products were calculated from band densities using
the formula A1 = (density of A1 at the indicated time/density of
initial A1) × initial A1 concentration, or A1 fragment = (density of A1 fragment at the indicated time/density of initial
A1) × (mol mass A1/mol mass fragment) × initial A1
concentration, respectively. In order to evaluate the catalytic
efficiency of factor Xa, we calculated the cleavage rate constants
based on the densitometric values. Assuming the cleavage event and
release of products are rapid, the concentration of free factor Xa
should be constant. Therefore, the rate constant correlates with the
concentration of substrate. The apparent rate constant (k)
for A1 subunit cleavage by factor Xa was determined by employing
Equation 1,
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(Eq. 1)
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where [A]t is the intact A1 subunit
concentration at time point t.
The apparent rate constant values (k11,
k12, k21, and
k22) in the following Scheme 1 are based
on parallel and series reactions for intact A1 subunit cleavage
by factor Xa and were estimated by nonlinear least squares regression
using multiple equations as described in Equations 2-5.
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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(Eq. 5)
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where [A]t, [B1]t,
[B2]t, and [C]t are the
concentrations at time point (t) of intact A1 and A1
fragments consisting of residues 37-372 (A137-372),
residues 1-336 (A11-336), and residues 37-336
(A137-336). We observed a reduction in total staining
intensity for these fragments as the time course progressed, in
parallel with the appearance of diffuse, low molecular mass staining
material. For this reason it was necessary to incorporate expressions
for rate values for these small degradation products (designated
D1-D4). Because the initial loss of mass was greatest at the initial
time points, we assume k3 > k4. Furthermore, rate values for
k4 could not be differentiated for species
designated B1, B2, and C.
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RESULTS |
Proteolysis of A1 Subunit by Factor Xa--
We recently
demonstrated that factor Xa cleaves the A1 subunit (residues 1-372) of
factor VIIIa at Lys36 (16) in addition to
Arg336 (5). Identification of reaction products was
determined following reaction of the isolated A1 subunit (2.4 µM) with factor Xa (0.24 µM). Fig.
1A shows the stained gel
following electrophoresis of intact A1 and the products of A1 subunit
cleavage by factor Xa after reaction for 4 h at 22 °C. Three
fragments were observed in addition to the intact subunit. These
fragments were further characterized by Western blot analysis employing
anti-A1 domain monoclonal antibodies specific for the N- or
C-terminal sequences. Although the intact A1 (fragment 1) and fragment
3 were reactive with monoclonal antibody 58.12 (an antibody directed to
residues 1-12 of the A1 sequence), fragments 2 and 4 were not
identified (Fig. 1B, a). Use of the C5 antibody
(specific for residues contained within the C-terminal acidic region of
A1) showed reactivity with the intact A1 (fragment 1) and fragment 2 but did not react with either fragments 3 or 4 (Fig. 1B,
b). These results, taken together with identified factor Xa
cleavage sites for intact A1 (A11-372) at
Lys36 and Arg336, identify fragment 2 as A1
residues 37-372 (A137-372), fragment 3 as A1 residues
1-336 (A11-336), and fragment 4 as A1 residues 37-336
(A137-336). Fig. 1C schematically illustrates
the products of A1 digestion by factor Xa. Pretreatment of factor Xa
with a molar excess of TAP failed to yield any proteolysis of A1
subunit, indicating that both cleavages were catalyzed by factor Xa
(data not shown).

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Fig. 1.
SDS-PAGE and Western blot of native and
factor Xa-cleaved A1 forms. A, purified A1 subunit
(2.4 µM) was reacted with factor Xa at 22 °C for
4 h as described under "Materials and Methods." Samples were
run on an 8% gel and stained with GelCode Blue. Lane
a, intact A1 subunit; lane b, A1
following reaction with factor Xa. B, Western blot of
the factor Xa-treated A1 using anti-A1 monoclonal antibodies 58.12 specific for the N-terminal region (lane a) and C5 specific
for the C-terminal region (lane b). C,
schematic of the cleaved A1 fragments generated following reaction with
factor Xa.
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Factor Xa Cleavage of A1 Subunit Occurs by Two Pathways--
Time
course assays were used to correlate rates of cleavages in the A1
subunit and residual cofactor activity following the reconstitution of
the factor Xa-treated A1 with other factor VIIIa subunits. Fig.
2, A and
B, shows the results of SDS-PAGE of the cleavage
reactions. Densitometry scanning of the stained gel was employed to
quantitate band density of the substrate A1 and each cleavage product.
The A11-372 subunit gradually diminished following
addition of factor Xa, and degradation was essentially complete by
8 h. Appearance of the A137-372 and
A11-336 fragments correlated with loss of
A11-372. However, rates of generation and degradation of
these two forms were not identical. Generation of A137-372
appeared somewhat faster compared with the A11-336 based
upon the initial slope of the curve, and the former was obtained in
greater yield based upon total band density. Subsequently, complete
degradation of A137-372 occurred within 10 h of peak
level, while the A11-336 persisted at ~30% of peak
level for 18 h, suggesting a markedly reduced rate of cleavage of
the latter compared with the former. The A137-336,
truncated at both termini, appeared after a lag of ~2 h, and its
generation increased over the remainder of the time course (24 h). This
band showed no loss in density over an extended (24 h) reaction time
suggesting the fragment represented a terminal digestion product.

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Fig. 2.
Time course of proteolysis and cofactor
activity of A1 subunit following reaction with factor Xa.
A, SDS-PAGE of A1 subunit (2.4 µM) was
reacted with factor Xa (0.24 µM) as described under
"Materials and Methods." At the indicated times, the reaction was
terminated, and samples were run on an 8% gel and stained with GelCode
Blue. The cleaved A1 sample (adjusted at 500 nM) was
reconstituted with the A3-C1-C2 (500 nM), followed by
reaction with A2 subunit (20 nM). Reconstituted factor
VIIIa activity was measured using a factor Xa generation assay as
described under "Materials and Methods." B,
quantitative evaluation by densitometry. The concentration of each A1
form was determined from scanning densitometry of the stained gel shown
in A. The symbols used are as follows: open
circles, A11-372; closed circles,
A137-372; open squares,
A11-336; closed squares, A137-336; and open triangles, total
concentration of A1 and A1-derived fragments based upon summing the
staining density. Data were fitted to the model described under
"Materials and Methods." The inset shows factor VIIIa
activity (% of control) (open circles). C,
schematic of proposed pathways for A1 cleavage by factor Xa. The
arrows show the site of factor Xa cleavage.
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The simultaneous generation of A11-336 and
A137-372 indicated the presence of two pathways for A1
cleavage of factor Xa to yield the terminal product, and these
alternatives are schematically illustrated in Fig. 2C.
Estimation of apparent rate constants for each cleavage reaction was
derived from fitting the data shown in Fig. 2B to equations
by a parallel series reaction as described under "Materials and
Methods." Based upon staining density, the total concentration of
intact A1 plus the fragments A11-336,
A137-372, and A137-336 (Fig. 2B,
triangles) decreased during the reaction time course. This
result suggested additional, minor cleavage pathways and was supported
by the appearance of diffuse staining, low molecular mass fragments in
the gels (data not shown). To account for these auxiliary fragments,
the model incorporated conversion of the initial substrate,
intermediates, and terminal fragment to degradation products designated
D1-D4 as described under "Materials and Methods." Because the
greatest rate of loss occurred within the first 2 h, this result
suggested the rate constant for D1 derived from degradation of intact
A1 subunit was greater than those constants reflecting generation of
D2-D4. We assume these latter rates (k4) are
equivalent to fairly compare the rate constants
(k11, k12, k21, and k22) for the
primary cleavage pathways. The data are summarized in Table
I. By using the isolated A1 subunit,
there appeared to be a modest (2-fold) preference for factor Xa to
cleave Lys36 compared with Arg336. However, the
apparent cleavage rate at Lys36 in the intact A1 subunit
(k11) was ~8-fold greater than that observed
for the A11-336 substrate (k22).
This result suggested that the C-terminal region (residues 337-372)
represented a critical region for factor Xa-dependent cleavage at this site. Conversely, the N-terminal region of A1 subunit
(residues 1-36) showed little influence on cleavage at the
Arg336 site (k12 ~2-fold greater
than k21).
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Table I
Relative rate constants estimated for A1 subunit cleavage by factor Xa
Parameter values were calculated from the fitted data shown in Figs. 2
and 3 using the formula presented under "Materials and Methods."
All data are represented as rate constant values (k) × 1000.
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The analysis of A1 cleavage was complemented with assessment of the
change of cofactor activity for factor VIIIa reconstituted from the
cleaved A1. For this analysis, cleavage of A1 was quenched at the
indicated time points by addition of TAP, and the A1 forms were
reconstituted with an equimolar concentration of A3-C1-C2 as described
under "Materials and Methods." The resultant A1/A3-C1-C2 dimer
forms were diluted and reacted with A2 (20 nM) to generate factor VIIIa heterotrimers, and cofactor activity was measured in a
factor Xa generation assay. The amount of exogenous factor Xa resulting
from the cleavage of A1 subunit was measured in a control assay, and
the data were corrected prior to plotting. Loss of cofactor activity as
a result of A1 cleavage was correlated with proteolysis of the
A11-372 and the appearance of the A137-336
via the intermediates A137-372 and A11-336
(Fig. 2, A and B). However, this inactivation was
incomplete, representing ~40% of the activity for cofactor
reconstituted with intact A1. This level of residual activity was
consistent with the value obtained for factor VIIIa reconstituted with
purified A137-336 (16).
Effect of A3-C1-C2 Subunit on A1 Cleavages by Factor Xa--
The
above results showed slow rates of cleavage of the isolated A1 subunit
at either terminus. Earlier studies have demonstrated optimal reaction
rates for factor Xa activity directed against factor VIII substrates
require a phospholipid surface (5). Furthermore, a factor Xa
interactive site was recently mapped to the C2 domain of the cofactor
(28), which also contains residues required for the interaction of
cofactor with anionic phospholipid surfaces (29). Thus, these factors
likely contribute to catalytic efficiency of factor Xa-catalyzed
cleavage of A1. In order to determine the roles for C2 and
phospholipid, isolated A1 subunit was reconstituted with isolated
A3-C1-C2, and the resultant A1/A3-C1-C2 dimer was employed as substrate
for factor Xa in reactions performed in the presence of phospholipid.
Comparison of factor Xa cleavage of isolated A1 with that for A1
reconstituted in the dimer is shown in Fig.
3. Time course experiments
were run using 2.4 µM of either substrate and 0.24 µM factor Xa at 22 °C as described under "Materials
and Methods." Data obtained from the stained gel (Fig. 3A)
were subjected to densitometry scans (Fig. 3B) and are
summarized in Table I. The cleavage of A1 in the dimer was rapid
compared with the isolated subunit. Generation of significant levels of
the A11-336 fragment from this substrate appeared within 2 min and reflected an ~6-fold increase in constant
k21(+A3C1C2) compared with constant
k21(
A3C1C2). Subsequent conversion of this
fragment to the terminal digestion product showed little if any
influence of the A3-C1-C2 based upon near equivalence of the estimated
rate constants
(k22(+A3C1C2)/k22(
A3C1C2)
~1). In contrast to the rapid appearance of the A11-336
fragment following cleavage of the dimer, generation of the
A137-372 was not significantly enhanced by the presence of
the A3-C1-C2 subunit
(k11(+A3C1C2)/k11(
A3C1C2)
~1.8). However, subsequent conversion to the A137-336
terminal fragment was markedly enhanced by this association
(k12(+A3C1C2)/k12(
A3C1C2) ~6). These results indicate that the cleavage at Arg336
rather than at Lys36 is markedly affected by inclusion of
the A3-C1-C2 subunit and the surface, suggesting a selective
role for interaction of protease via binding to the C2 domain in
catalyzing cleavage at the C-terminal site. Moreover, the cleavage at
Arg1721 at the A3-C1-C2 subunit by factor Xa appeared
within 2 min and was almost complete by 120 min as described
previously (5).

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Fig. 3.
Effect of A3-C1-C2 subunit on cleavage of A1
by factor Xa. A, SDS-PAGE of A1 subunit (2.4 µM) either reconstituted with equimolar A3-C1-C2 subunit
or alone reacted with factor Xa (0.24 µM) in the absence
or presence of phospholipid vesicles (10 µM). Reactions
were performed as described under "Materials and Methods." At the
indicated times, the reactions were terminated, and samples were run
on an 8% gel followed by staining with GelCode Blue.
B, quantitative evaluation by densitometry. The
concentration of each A1 form was determined from scanning densitometry
of the stained gel shown in A. The symbols used are as
follows: open circles, intact A11-372;
closed circles, A137-372; open
squares, A11-336; closed squares,
A137-336. Data were fitted to the model described under
"Materials and Methods."
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Kinetics of A1 Proteolysis by Factor Xa--
A series of
experiments was performed to quantitate the kinetics of A1 cleavage
(initial event) and the contributions made by other factor VIIIa
subunits and the phospholipid surface. Fig. 4 shows the rate of loss of intact A1
subunit under the different experimental conditions. Results were
derived from scanning densitometry of the stained gels, and data points
were fitted using Equation 1 as described under "Materials and
Methods." The rate of cleavage of the isolated A1 subunit (2.4 µM) by factor Xa (0.24 µM) was independent
of phospholipid (8.12 ± 0.39 (×10
3)
min-1 and 7.59 ± 0.25 (×10
3)
min
1, in the presence and absence of phospholipid,
respectively). Association of A1 subunit with the A3-C1-C2 yielded an
~3-fold increased rate of A1 cleavage (19.2 ± 1.09 (×10
3) min
1), and this rate was further
increased ~2-fold (40.7 ± 3.04 (×10
3)
min-1) in the presence of phospholipid. Reconstitution of
the factor VIIIa heterotrimer by subsequent addition of the A2 subunit
resulted in no further increase in the rate of A1 subunit cleavage
(42.6 ± 4.23 (×10
3) min
1). Overall,
the incorporation of A1 subunit in the A1/A3-C1-C2 dimer bound to the
phospholipid resulted in an ~6-fold increase in cleavage rate
constant compared with isolated A1 subunit alone.

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Fig. 4.
Effect of other factor VIIIa subunits on the
cleavage of A1 subunit by factor Xa. A1 subunit (2.4 µM) in the presence (circles) or absence
(squares) of A3-C1-C2 subunit (2.4 µM) was
reacted with factor Xa (0.24 µM) in the presence
(open symbols) or absence (closed symbols) of
phospholipid vesicles (10 µM) for 2 h as described
under "Materials and Methods." In addition, A1/A3-C1-C2 dimer
reconstituted with the A2 subunit (2.4 µM) was reacted
with factor Xa (triangles). Samples were run on an 8% gel
followed by staining with GelCode Blue, and the concentration of
remaining intact A1 was calculated using the formula described under
"Materials and Methods." Data were plotted and fitted using
Equation 1 described under "Materials and Methods."
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Cleavage at Lys36 in A11-336 by Factor
Xa--
Results presented above indicate a slow rate of proteolysis of
the A11-336 to the A137-336 terminal
fragment. In order to further study the mechanism for this cleavage, a
purified A11-336 fragment was used as substrate for factor
Xa. A11-336 was isolated from intact A1 following cleavage
by activated protein C using Mono-S-Sepharose as described previously
(14). Cleavage of this intermediate (2.4 µM) by factor Xa
(0.24 µM) was evaluated by SDS-PAGE and densitometry
(Fig. 5). Surprisingly, the purified A11-336 subunit persisted throughout an extended time
course with ~80% of material uncleaved following a 15-h reaction
with factor Xa. This observation is consistent with results presented
in Fig. 2 showing the slow rate of loss of the A11-336
intermediate when derived from initial cleavage from A1. Furthermore, the apparent rate of this cleavage was not affected by the presence of
the A3-C1-C2 subunit. These results support the hypotheses that the
acidic C-terminal region of the A1 subunit (residues 337-372) is
necessary for the effective cleavage at Lys36, and this
cleavage is independent of the A3-C1-C2 subunit. Furthermore, these
results suggest that this C-terminal region may represent an
interactive site for factor Xa.

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Fig. 5.
Cleavage of purified A11-336
subunit by factor Xa. The isolated A11-336 (2.4 µM) or A11-336/A3-C1-C2 dimer (2.4 µM) was reacted with factor Xa (0.24 µM).
Samples were removed at the indicated times and run on an 8% gel
followed by staining with GelCode Blue.
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Synthetic Peptide 337-372 Inhibits the Cleavage of the A1 Subunit
by Factor Xa--
To examine further the possibility that this
C-terminal region of A1 contained an interactive site for factor Xa, a
synthetic peptide to this region (residues 337-372) was evaluated as a
potential inhibitor of A1 proteolysis by factor Xa. In this experiment, the A1 subunit (2 µM) was reacted for an extended period
with factor Xa (0.2 µM) in the absence and presence of
increasing concentrations of the peptide. Reaction products were
visualized by SDS-PAGE and quantitated by scanning densitometry (Fig.
6). In the absence of peptide, A1 subunit
was essentially fully converted to the A137-336 terminal
product. Increasing concentrations of peptide resulted in a diminution
of the terminal product and concomitant increase in the concentration
of the A11-336 intermediate, and this effect was
dose-dependent (Fig. 6A). Fig. 6B
illustrates the relative concentrations of the terminal product and
intermediate. At 1 mM peptide, >80% of the intermediate
persisted indicating significant inhibition of factor Xa-catalyzed
cleavage at Lys36. From these data, an IC50
value for this inhibition was calculated to be 230 µM.
Low levels of intact A1 subunit and A137-372 also
persisted at the highest levels of peptide employed (~10% of the
total protein), suggesting that the peptide had a marginal effect in
inhibiting the cleavage at Arg336. Interestingly, neither
the three overlapping peptides derived from the 337-372 sequence and
representing residues 337-359, 351-365, and 360-372 nor the C5
antibody which binds residues 351-365 (21) inhibited the cleavage of
the A1 subunit by factor Xa using the above conditions (results not
shown). Taken together, these results suggest that the C-terminal
sequence of A1 comprises an extended interactive surface for binding
factor Xa, and this association is required for the selective
proteolysis of A1 subunit at Lys36.

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Fig. 6.
Inhibition of factor Xa-catalyzed cleavage of
A1 subunit by factor VIII peptide337-372. A mixture
of A1 subunit (2 µM) and serial dilutions of
peptide337-372 was reacted with factor Xa (0.2 µM) at 22 °C overnight. Samples were run on an 8% gel
followed by staining with GelCode Blue. A, SDS-PAGE;
lane 1 shows intact A1. Lanes 2-7 show A1
subunit plus factor Xa in the presence of peptide (0, 62.5, 125, 250, 500, and 1000 µM, respectively). B,
densitometry scan of the stained gel shown in A. Density of
the A137-336 band in lane 2 of the stained gel
was used to represent the 100% level for the terminal product. The
symbols used are as follows: open circles,
A137-336; and closed circles,
A11-336.
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Heparin Blocks A1 Cleavage by Factor Xa--
A hallmark of the
C-terminal region of A1 subunit is the high content of acidic amino
acids (13 Glu + Asp/36). Thus, this region could define an interactive
region for an anion-binding site on the protease. Indeed, a
heparin-binding exosite on factor Xa was described recently (30). In
order to test whether this exosite contributed to the cleavage of the
A1 subunit at Lys36 and/or Arg336, an
experiment was performed to evaluate proteolysis of A1 in the presence
of heparin (100 and 500 units/ml). Results shown in Fig.
7 indicated a dose-dependent
inhibition of factor Xa-catalyzed cleavage of A1 by heparin. Although
both cleavages appeared to be inhibited by heparin, it appeared that
cleavage of Lys36 was somewhat more reduced than cleavage
at Arg336, based upon relative concentrations of the
A11-336 intermediate and A137-336 terminal
product. These observations support a role for the heparin-binding exosite of factor Xa in the proteolytic inactivation of factor VIIIa as
mediated through cleavages at Arg336 and
Lys36.

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Fig. 7.
Effect of heparin on A1 subunit proteolysis
by factor Xa. The A1 subunit (2.5 µM) was
preincubated with heparin for 15 min followed by reaction with factor
Xa (0.25 µM) and CaCl2 (5 mM) at
22 °C overnight. Samples were run on an 8% gel followed by staining
with GelCode Blue. Lane 1 shows intact A1. Lanes
2-4 show the cleavage of A1 by factor Xa in the presence of
heparin (0, 100, 500 units/ml, respectively).
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DISCUSSION |
Activation of human factor VIII by thrombin or factor Xa results
from limited proteolysis at identical sites. However, the latter
protease attacks additional sites in the factor VIII/VIIIa substrate.
Cleavage within the A3 domain at Arg1721 (5) appears benign
based upon factor VIIIa reconstitution studies using factor VIII light
chain cleaved at this site in place of the thrombin-cleaved chain (31).
Three additional sites have been mapped within the A1 domain/subunit of
factor VIII/VIIIa. Porcine factor VIII is cleaved at Arg219
as a part of the activation process, yielding a factor VIIIa form
possessing two subunits derived from the original A1 domain (32). This
residue is Gln in other factor VIII molecules and thus is not attacked.
Interestingly, porcine factor Xase composed of factor Xa-activated
factor VIIIa shows a severalfold lower catalytic efficiency than factor
Xase composed of the thrombin-activated cofactor. Factor Xa also
cleaves at Arg336 (5), preceding the anionic C-terminal
region and Lys36 (16). Proteolysis at these two sites
correlates with inactivation of cofactor function. Furthermore, these
effects appear additive with factor VIIIa composed of A1336
and A137-336 possessing ~60 and ~30% of native factor
VIIIa activity, respectively (16). Cleavage at Arg336
alters the interaction of A1 relative to A2 subunit reflecting reduced
kcat values (14), as well as resulting in a
severalfold increase in Km of factor Xase for
substrate factor X (16). Loss of activity resulting from cleavage of A1
at Lys36 is less well understood but appears to alter its
conformation markedly affecting the affinity of this subunit for A2
subunit (16).
In this study we examine the mechanisms of interaction of factor Xa
resulting in the limited proteolysis of the A1 subunit, thus reflecting
proteolytic pathways leading to inactivation of the cofactor. The
identification of two intermediates, A137-372 and
A11-336, indicated alternative pathways, designated
I and II (Fig. 2C), respectively, leading to the formation
of the terminal product, A137-336. Facile determination of
the intermediates was achieved following Western blotting with
monoclonal antibodies specific for the N and C termini. By using
isolated A1 subunit as substrate, pathway I resulting in initial
cleavage at Lys36 (k11) demonstrated
an ~2-fold faster relative rate than initial cleavage at
Arg336 (k21). However, whereas
cleavage of A137-372 (k12) occurred
at an appreciable rate, cleavage of the A11-336
intermediate (k22) was markedly reduced. This
result suggested a role for the C-terminal acidic region (residues
337-372) in this catalytic step. This conclusion was supported by
several observations including a slow rate of cleavage of
Lys36 using a purified A11-336 subunit and
selective inhibition of cleavage at this site by the 337-372 peptide.
Reconstitution of A1 subunit with the A3-C1-C2 subunit to form the
A1/A3-C1-C2 dimer and inclusion of a phospholipid surface resulted in
disparate effects on the cleavages catalyzed by factor Xa. Although the
rate for initial cleavage at Lys36 was marginally increased
in the dimer
(k11(+A3-C1-C2)/k11(-A3-C1-C2) ~1.8), the rate for initial cleavage at Arg336 was
increased ~8-fold. This result suggested a differential regulation in
the two cleavage reactions and suggested that cleavage of
Arg336 was facilitated by interaction of the protease with
the surface-bound substrate. Recently, a factor Xa interactive site was
identified in the C2 domain of factor VIII (28). Results
presented in that study showed the C2 domain-specific antibody
NMC-VIII/5 did not block factor Xa-catalyzed activation of factor VIII
(cleavage at Arg372). Interestingly, examination of the gel
presented in that report shows no subsequent cleavage of the A1 subunit
in the presence of the antibody. This result suggested that (i)
interaction of factor Xa with C2 may facilitate cleavage of A1 subunit,
and (ii) factor Xa may interact differentially with this domain in
catalyzing cleavages that result in activation compared with those
resulting in inactivation.
Although cleavage at Arg336 appears to be selectively
enhanced by contribution of protease binding C2, cleavage at
Lys36 appears to be modulated by the acidic C-terminal
region of the A1 subunit. Earlier studies have indicated that this
region constitutes a factor X interactive site. Solid phase binding
analyses indicated an affinity of factor X for A1 subunit of ~1-3
µM (15) and showed that cleavage of A1 at
Arg336 by activated protein C abrogated this interaction
(33). Zero-length cross-linking studies identified a salt bridge(s)
between residues in this region of A1 and a portion of the protease
domain of the zymogen exclusive of the activation peptide (34). Based
upon these observations, and the results of the present study showing markedly reduced rates of cleavage at Lys36 using the
A11-336 substrate compared with intact A1 and specific
inhibition of attack at this site by the 337-372 peptide, we suggest
that this C-terminal region of A1 constitutes an interactive site for
factor Xa as well as factor X.
Previous studies (30) have identified a series of basic residues in the
heparin-binding exosite of factor Xa. In addition to binding heparin,
some of these residues contribute to factor Xa interaction with
cofactor and/or substrate recognition by prothrombinase. Results from
the current study show selective inhibition by heparin in factor
Xa-catalyzed cleavage at the Lys36 and Arg336
sites. Although high concentrations of heparin inhibited proteolysis at
both sites, a lower level of heparin selectively blocked cleavage at
the Lys36 site. These results suggest involvement of the
heparin-binding exosite of the protease in the catalysis of A1
cleavage. Furthermore, given the highly anionic nature of the
C-terminal region of A1, we suggest that this sequence interacts with
the protease via residues that form its heparin-binding exosite.
Factor VIII is homologous to factor V (35), the activated form of which
serves as cofactor for factor Xa in the prothrombinase complex.
Although factor V shows no acid-rich region separating the A1 and A2
domains, a recent report indicates an acidic residue-rich sequence
localized to the N-terminal region of the A2 domain of factor Va
(residues 323-331, net charge =
3) represents a binding site
for factor Xa (36). This conclusion was based upon
peptide-dependent inhibition of prothrombinase activity as
well as direct inhibition of the factor Va-factor Xa interaction
determined by fluorescence techniques. Although this enzyme-cofactor
binding interaction in prothrombinase clearly differs functionally from
the association of enzyme and substrate described in this study, the
similarities in these interactions relative to location of sites and
sequence characteristics of the cofactor proteins are noteworthy and
suggest homologous sites of interaction.
The physiological significance of factor Xa-catalyzed cleavage of A1
subunit resulting in inactivation of cofactor function remains to be
determined. Although the catalytic rates for these reactions are slow,
the concentrating effect of the thrombogenic surface in restricting
reactants to a two-dimensional space and the generation of high local
concentrations of protease by the factor Xase complex
(kcat ~200 min
1 (12)) support
the hypothesis that factor Xa, the product of factor Xase, may
contribute to the dampening of this enzyme complex.