From the
Thrombin-catalyzed activation of heterodimeric factor VIII
occurs by limited proteolysis, yielding subunits A1 and A2 derived from
the heavy chain (HC) and A3-C1-C2 derived from the light chain (LC).
The roles of these cleavages in the function of procoagulant activity
are poorly understood. To determine whether LC cleavage contributes to
the potentiation of factor VIII activity, factor VIII heterodimers were
reconstituted from native HC and either thrombin-cleaved LC (A3-C1-C2)
or intact LC and purified by Mono S chromatography. The reconstituted
factor VIII form containing the A3-C1-C2 subunit had a specific
activity (2 units/µg) that was
Factor VIII, the component of blood coagulation deficient or
defective in individuals with hemophilia A, is synthesized as a 300-kDa
precursor protein
(1, 2) with the domain structure
A1-A2-B-A3-C1-C2
(3) . It is processed to a series of
Me
Factor VIII circulates in plasma as a
procofactor associated with a carrier protein, von Willebrand factor
(vWf)
(8) . Binding is noncovalent and involves both
electrostatic and hydrophobic interactions
(9) . A primary
vWf-binding site is contained within the NH
Factor VIII functions in the
intrinsic Xase complex as a cofactor for factor IXa in the
surface-dependent conversion of factor X to factor Xa. This activity is
dependent upon conversion of factor VIII to the active cofactor form,
factor VIIIa.
The functions of the thrombin-catalyzed
cleavages in factor VIII are not well understood. Cleavage of the LC is
believed to be responsible for dissociation of factor VIIIa from vWf
(16) . This cleavage may also add to the cofactor activity of
factor VIIIa
(17, 18, 19) , although this
hypothesis is controversial
(20) . In this study,
reconstitutions of factors VIII and VIIIa from purified subunits were
utilized to determine the contribution of LC cleavage to factor VIIIa
activity. Results indicate that the thrombin cleavage of factor VIII LC
contributes to the potentiation of factor VIIIa activity by directly
affecting the conformation of the factor IXa active site.
Factor
VIIIa was reconstituted in a two-step process. First, the A1 subunit
(300 n
M) was reacted at room temperature with either A3-C1-C2
(400 n
M) or the LC (400 n
M) in buffer A containing 10
m
M MnCl
In the second step,
the A1/A3-C1-C2 or A1/LC dimer was diluted at least 3-fold in buffer A
containing 0.5 mg/ml bovine serum albumin and incubated with the A2
subunit. Dilution of the dimer was performed to decrease the
concentration of Mn
For these experiments, the A1
subunit was reacted with a slight excess (1.3-fold) of either the LC or
A3-C1-C2 in the presence of 10 m
M MnCl
A time course of
factor VIIIa reconstitution was performed by incubating either A1/LC or
A1/A3-C1-C2 with the A2 subunit. Results from this experiment are shown
in Fig. 4. Reconstitutions of A2 with either A1/LC or A1/A3-C1-C2
yielded saturable levels of factor VIIIa activity. However, the
activity associated with the heterotrimer composed of A1/LC/A2 (
Activation of factor VIII is a prerequisite for its role as
the cofactor for factor IXa in the intrinsic Xase complex. Thrombin,
the physiologic activator of factor VIII
(34) , cleaves this
substrate at Arg
This study focuses on the potential contribution to
cofactor activity following LC cleavage. Independent of any effect on
activity, cleavage at this site is required to dissociate factor VIII
from its physiologic carrier protein, vWf, thus allowing for
interaction with a thrombogenic surface. A primary binding site for vWf
is contained within the acidic region defined by LC residues
1649-1689
(10, 16) . Cleavage at this site results
in a marked reduction in the affinity of factor VIII for vWf
(37, 38) . However, recent observations of the failure
of this cleavage fragment to bind vWf
(39) as well as the
capacity for factor VIII C2 domain fragments to inhibit the factor
VIII-vWf interaction
(40, 41) suggest that this
interaction is more complex than originally perceived.
In this
study, we present data that indicate that the LC cleavage of factor
VIII contributes to the potentiation of factor VIIIa activity. We have
done this utilizing purified factor VIII(a) subunits and taking
advantage of the capacity for these isolated subunits to be
reconstituted to produce active material. The result of reconstitution
of native HC and A3-C1-C2 is a factor VIII molecule with
A factor VIIIa molecule derived from A1/LC/A2
possessed 3-fold less activity than factor VIIIa derived from
A1/A3-C1-C2/A2. However, reaction of either the hybrid heterodimer
(HC/A3-C1-C2) or the heterotrimer (A1/LC/A2) with thrombin resulted in
a further potentiation of activity, yielding values equivalent to those
of native factor VIIIa. Therefore, we conclude that cleavage of the LC
by thrombin contributes to the potentiation of factor VIIIa activity,
and cleavage at both chains is required to yield the maximally
activated molecule.
Several earlier reports have attempted to
determine whether LC cleavage contributed to cofactor activity. Shima
et al. (19) isolated DNA from a patient with mild
hemophilia A and found an Arg
Using site-directed mutagenesis, Pittman
and Kaufman
(18) demonstrated a requirement for thrombin
cleavage at both the HC and LC sites for factor VIII activation.
Thrombin-resistant factor VIII molecules were created by making
conservative changes at either Arg
Studies using the snake
venom protease from Bothrops jararacussu (20) , which
produces a thrombin-like cleavage of factor VIII HC, but does not
cleave the LC, show that cleavage of the HC alone leads to a factor
VIIIa activity that is 60% that of factor VIII cleaved by thrombin.
Although it was concluded that LC cleavage was not necessary for
potentiation of activity, this observation is in agreement with our
results, indicating that factor VIII requires LC cleavage for maximal
activation.
Bihoureau et al. (17) attempted to
dissect the relative contributions of each thrombin cleavage to changes
in factor VIII activity. In these studies, factor VIII concentrates
were activated with thrombin, and at various time points, the thrombin
was inactivated by the addition of hirudin. Factor VIII was then
purified by ion-exchange chromatography and analyzed by clotting assay
and by SDS-PAGE. Initial reaction with thrombin yielded partially
(5-fold) activated factor VIII molecules possessing cleaved LC but
intact HC as judged by silver staining of the gels. Further thrombin
activation of these factor VIII molecules resulted in a 15-fold
(overall) activated factor VIIIa and generation of polypeptides
corresponding to the A1, A2, and A3-C1-C2 subunits. These results
suggested that LC cleavage by thrombin potentiated factor VIII
activity. However, the potential for low levels of HC cleavage
contributing to the enhanced activity could not be excluded.
Results
from this study exclude several possible mechanisms by which LC
cleavage might potentiate factor VIIIa activity. First, both the HC/LC
and HC/A3-C1-C2 heterodimers were activated at apparently similar rates
by thrombin and factor Xa, suggesting that initial cleavage of the LC
does not improve subsequent cleavage of the HC. This result is
consistent with the finding that the presence of vWf, which binds to
this portion of the LC, does not affect the catalytic efficiency of
thrombin cleavage of the HC
(43) . Second, the intrinsic
stability of the A1/LC/A2 heterotrimer, as judged by activity
measurements following dilution of the sample, was similar to that
observed for the native factor VIIIa heterotrimer. Thus, the affinity
of the A2 subunit for either dimer form appeared equivalent. This
result is interesting in that a primary binding site for A2 is defined
by the COOH-terminal acidic region of the A1 subunit
(30) .
Thus, the presence of an additional acidic residue-rich region located
in the NH
Other laboratories have shown that
cofactor binding involves changes in the active site of coagulant
enzymes. Ye et al. (44) have shown that the binding of
thrombomodulin derivatives to DEGR-thrombin causes a change in the
emission intensity of the dansyl probe bound in the thrombin active
site. This apparent conformational change in the thrombin active site
is consistent with the change in substrate specificity of the thrombin
such that it is now able to activate the anticoagulant protein C. The
cofactor factor Va has been shown to cause conformational changes in
the factor Xa active site as well as to affect the distance of the
active site above the membrane surface
(45) . Armstrong et
al. (46) have recently shown that factor Va also can cause
a moderate shift in the active site of the activation intermediate
meizothrombin. Therefore, factor Va may function to alter the
conformation and/or orientation of both enzyme and substrate.
Using
similar types of fluorescence studies, Mutucumarana et al. (47) determined that the active site of DEGR-factor IXa is far
above the membrane surface (>70 Å) and that the binding of
factor VIIIa does not alter this distance, although a change in
fluorescence intensity and in the anisotropy of the probe was observed.
Duffy et al. (33) showed that the binding of factor
VIII to an active site-labeled factor IXa produces a small anisotropy
change, whereas a significantly larger change in anisotropy is produced
by activating factor VIII with thrombin. This result appears to
correlate with the role of factor VIIIa in increasing
k
We have utilized the
sensitivity of fluorescence anisotropy of an active site-labeled factor
IXa to factor VIII structure to evaluate the role of LC cleavage. The
anisotropy of Fl-FFR-factor IXa was increased incrementally in the
presence of HC/LC (
We thank Drs. James Brown and George Mitra and the
Cutter Division of Miles Inc. for the therapeutic concentrates used to
prepare factor VIII subunits and Debra Pittman and the Genetics
Institute for the gift of recombinant factor VIII. We also thank Tammy
L. Beattie for excellent technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
3-fold greater than that of the
reconstituted factor VIII form containing native LC (0.6 units/µg).
Factor Xa generation assays using the hybrid heterodimer showed an
initial rate that was unaffected by the presence of von Willebrand
factor and a reduced lag time when compared with the native
heterodimer. The A1/A3-C1-C2 dimer was dissociated by chelation, and
the purified A1 subunit was reacted with either the A3-C1-C2 subunit or
the LC in the presence of Mn
to reconstitute the
dimer. Factor VIIIa heterotrimers were reconstituted from either
A1/A3-C1-C2 or A1/LC plus the A2 subunit. The authentic factor VIIIa
heterotrimer (A1/A3-C1-C2/A2) had 3-fold greater activity than the form
containing the LC. However, upon reaction with thrombin, the activity
of the latter form was increased to that of the factor VIIIa form
containing native subunits. The incremental increase in fluorescence
anisotropy of fluorescein-Phe-Phe-Arg chloromethyl ketone-modified
factor IXa was markedly greater in the presence of HC/A3-C1-C2
(
r = 0.037) compared with HC/LC (
r = 0.011) and approached the value obtained with factor
VIIIa (
r = 0.051). These results suggest that
cleavage of factor VIII LC directly contributes to the potentiation of
coagulant activity by modulating the conformation of the factor IXa
active site.
-linked heterodimers
(4, 5, 6) produced by cleavage at the B-A3 junction, generating a
light chain (LC)
(
)
(A3-C1-C2 domains) and a heavy
chain (HC) (A1-A2-B domains). Additional cleavage sites within the B
domain result in variable sized heavy chains
(3) minimally
represented by the contiguous A1-A2 domains. The two chains can be
separated by chelating reagents
(4, 6) and isolated
following ion-exchange chromatography. The separated chains have no
activity, but factor VIII activity can be reconstituted by combining
the subunits in the presence of divalent metal ion
(6, 7) .
-terminal
portion of factor VIII LC
(10) , and this association prevents
factor VIII from binding to phospholipid surfaces
(11, 12, 13) .
(
)
Thrombin, the principal activator
of factor VIII, cleaves factor VIII HC at Arg
, which
liberates the B domain fragments, and at Arg
, which
splits the HC into the A1 and A2 subunits
(14) . Thrombin also
cleaves factor VIII LC at Arg
(14) , liberating
an acidic rich region and creating a new NH
terminus. Thus,
factor VIIIa is a heterotrimer of subunits designated A1, A2, and
A3-C1-C2. The A1 and A3-C1-C2 subunits retain the Me
linkage and can be isolated as a stable A1/A3-C1-C2 dimer. The A2
subunit is only weakly associated with the dimer. Factor VIIIa activity
can be reconstituted from the isolated A1/A3-C1-C2 dimer and the A2
subunit
(15) .
Reagents
Human factor VIII concentrates
(Koateand Koate-HP
) were generously provided
by the Cutter Division of Miles Inc. Recombinant factor VIII was a
generous gift from Debra Pittman (Genetics Institute). The reagents
-thrombin and factor IXa
(Enzyme Research Labs),
DEGR-CK and PPACK (Calbiochem), fluorescein-Phe-Phe-Arg chloromethyl
ketone-modified factor IXa (Fl-FFR-factor IXa) (Molecular Innovations),
and the chromogenic substrates S-2238
( H-
D-phenylalanyl-
L-piperyl-
L-arginyl- p-nitroanilide)
and S-2765
( N
-benzyloxycarbonyl-
D-arginyl-
L-glycyl-
L-arginyl- p-nitroanilide
dihydrochloride) (Kabi Pharmacia/Chromogenix) were purchased from the
indicated vendors. Phospholipid vesicles were prepared as described
previously
(21) .
Proteins
Factor IXa was inactivated with the
active-site inhibitor DEGR-CK as described previously
(22) .
Factor VIII
(23) , factor VIII subunits
(23) , factor
VIIIa subunits
(15) and vWf
(24) were prepared as
described previously. Recombinant factor VIII was used as the starting
material for some of the subunit preparations, where it behaved
equivalently compared with the plasma-derived material. The A3-C1-C2
subunit was prepared by reacting factor VIII LC (3.78 µ
M)
with thrombin (70 n
M) for 12 h in buffer A (20 m
M Hepes, pH 7.2, 5 m
M CaCl, and 0.01% Tween 20)
containing 100 m
M NaCl. These conditions resulted in complete
conversion of the LC to A3-C1-C2 as determined by SDS-PAGE and silver
staining. Thrombin was subsequently inactivated by adding PPACK in
half-molar equivalents until no amidolytic activity was detected using
the chromogenic substrate S-2238. Following inactivation of thrombin,
the reaction was dialyzed for 4 h against buffer A containing 100
m
M NaCl. A control reaction, which did not contain thrombin,
was performed in parallel with the complete reaction.
Reconstitution of Factor VIII(a) Forms
Factor VIII
activity was reconstituted from A3-C1-C2 (700 n
M) or native LC
(700 n
M) by incubation with native HC (920 n
M) at
room temperature in buffer A containing 30 m
M MnCl. Reconstituted factor VIII was diluted 1:3 in
buffer B (10 m
M histidine, pH 6.0, 5 m
M CaCl
, and 0.01% Tween 20) and applied to a Mono S
column equilibrated with buffer B containing 100 m
M NaCl.
Factor VIII was eluted with a 30-ml linear gradient of 100 m
M to 1
M NaCl in buffer B, and 1-ml fractions were
collected. Factor VIII activity was measured by a one-stage clotting
assay using plasma that had been chemically depleted of factor VIII
activity
(25) . Protein concentrations were determined by the
Coomassie Blue dye binding method of Bradford
(26) .
. Progress of the
Me
-dependent linkage was monitored by reacting an
aliquot of the reaction mixture with the A2 subunit for 10 min at room
temperature and assaying for clotting activity.
since divalent metal ions
interfere with the association of the A2 subunit with the dimer
(15) . Factor VIIIa activity was determined by clotting assay,
and optimal activity was achieved within 10 min.
Factor Xa Generation Assays
The rate of conversion
of factor X to factor Xa was monitored in a purified system. Assays
contained reconstituted factor VIII(a) (2 n
M), factor IXa (1
n
M), factor X (200 n
M), and 100 µg/ml
phospholipid vesicles in buffer A containing 100 m
M NaCl.
Selected reactions also contained vWf. Aliquots were removed at the
indicated times, and EDTA (50 m
M final concentration) was
added to stop the reaction. Initial rates of factor Xa generation were
determined by the addition of the chromogenic substrate S-2765 (460
µ
M). Reactions were read continuously at 405 nm for 2 min
using a Beckman DU650 spectrometer.
Fluorescence Anisotropy Measurements
All
fluorescence anisotropy measurements were made using a Spex Fluorolog
212 spectrophotometer operated in the L format. Excitation and emission
wavelengths were 495 and 520 nm, respectively, and polarizers were
manually rotated. Fluorescence measurements were made using a microcell
(200 µl) initially containing Fl-FFR-factor IXa (20 n
M)
and phospholipid vesicles (10 µg/ml) in buffer A containing 100
m
M NaCl. The anisotropy was determined by acquiring data at
each polarizer position for 30 s. Three measurements were made over an
3-min time period and averaged for each determination. Either
HC/LC or HC/A3-C1-C2 (40-50 n
M) was added, and the
anisotropy was determined as described above. Due to the concentration
of the reconstituted factor VIII forms, the total volume in the
microcell increased
25% after the addition of factor VIII. Control
reactions indicated that this dilution factor did not affect the values
determined for anisotropy. Activation of the factor VIII forms was
achieved following the addition of thrombin (4 n
M). Reactions
were run for 1 min, and the anisotropy was again determined as
described above.
Reconstitution of Factor VIII from Isolated
Subunits
Active factor VIII heterodimers can be reconstituted
from isolated subunits in the presence of divalent metal ion
(27) , and this property was used to determine whether the
thrombin-catalyzed cleavage of the LC affected factor VIII activity.
Factor VIII was reconstituted from either A3-C1-C2 or the LC plus
native HC in the presence of Mn(Fig. 1). Using
these reaction conditions, reconstitution of factor VIII forms was
complete by
20 min as judged by a leveling of regenerated cofactor
activity. The factor VIII form reconstituted with A3-C1-C2
(HC/A3-C1-C2) exhibited
3-fold greater activity than reconstituted
factor VIII derived from native LC (HC/LC).
Figure 1:
Reconstitution of factor VIII activity
from cleaved or native LC and native HC. Reconstitution of factor VIII
from native HC (920 n
M) and A3-C1-C2 (700 n
M; )
or LC (700 n
M;
) was carried out as described under
``Materials and Methods.'' Aliquots were removed at the
indicated time points and assayed for factor VIII
activity.
Purification of Reconstituted Factor VIII
The
enhanced activity observed for the HC/A3-C1-C2 heterodimer compared
with the HC/LC heterodimer could result from either an increased extent
of reconstitution or a higher intrinsic specific activity. To
differentiate between these alternatives, HC/A3-C1-C2 and HC/LC were
run separately on a Mono S column to purify the reconstituted
heterodimers. In each case, two protein peaks were eluted from the
column (Fig. 2). SDS-PAGE analysis of the two peaks showed that
the first one consisted of free HC, while the second contained both the
HC and LC (or A3-C1-C2) (data not shown). Densitometric scans of the
stained gels showed that the HC and LC (or A3-C1-C2) in the latter peak
were present at equivalent stoichiometries (data not shown), suggesting
that these subunits are linked to form factor VIII heterodimers. Column
fractions were assayed for factor VIII activity, which was present only
in the heterodimer fraction. The peak HC/A3-C1-C2 activity was observed
to elute one fraction later (fraction 17) than the peak HC/LC activity
(fraction 16). This result likely reflects the reduction in acidic
character of HC/A3-C1-C2 following loss of residues 1649-1689.
Furthermore, the peak activity of this fraction was 3-fold greater
than the HC/LC activity. Protein determinations across both sets of
fractions from the column revealed similar concentrations. The specific
activity of HC/A3-C1-C2 was determined to be 2 units/µg, while that
of HC/LC was 0.6 units/µg. This result suggests that the difference
in activity is not due to a variance in the extent of factor VIII
reconstitution, but rather reflects a higher intrinsic specific
activity for the heterodimer containing thrombin-cleaved LC.
Figure 2:
Mono S chromatography of reconstituted
factor VIII. Factor VIII was reconstituted from native HC and native LC
( top panel) or A3-C1-C2 ( bottom panel) and purified as described under ``Materials
and Methods.'' Absorbance was measured at 280 nm (). Factor
VIII activity (
) was determined in a one-stage clotting
assay.
The
elevated specific activity of HC/A3-C1-C2 compared with HC/LC suggested
a partially activated factor VIII molecule. To further examine the
activation state of this factor VIII form, the rates and extents of
cofactor activation were assessed. Equivalent concentrations of the two
factor VIII forms were reacted with thrombin. Both HC/A3-C1-C2 and
HC/LC reached peak activation within 2 min (data not shown). But, while
HC/LC was activated 10-fold, HC/A3-C1-C2 was activated
3-fold. The final potentiated activity levels of the two forms
were equivalent. This result was expected since once HC/A3-C1-C2 and
HC/LC are activated by thrombin, both forms consist of an
A1/A3-C1-C2/A2 subunit structure. Furthermore, the similarity in rates
of activation (data not shown) suggested that the increased specific
activity of the HC/A3-C1-C2 heterodimer did not result from it
possessing the capacity for enhanced reactivity with thrombin.
Activity of HC/LC and HC/A3-C1-C2 in a Purified
System
An alternative explanation for the enhanced activity of
the hybrid factor VIII form could reflect a sequestering of native
factor VIII by endogenous vWf present in the plasma used for assay.
Thus, the higher apparent activity of HC/A3-C1-C2 could result from its
inability to bind vWf. To eliminate this variable, a series of
experiments were performed using a factor Xa generation assay composed
of purified reagents. In these assays, factor VIII derived from native
HC and intact or thrombin-cleaved LC was used to assemble the intrinsic
Xase complex. Reactions were run in the absence of exogenous thrombin,
thereby relying on feedback activation of the factor VIII forms by
endogenously formed factor Xa. Both the HC/LC- and
HC/A3-C1-C2-containing reactions showed a lag time preceding the
initial rate of product formation (Fig. 3). This lag reflects the
structure of factor VIII and illustrates the requirement for activation
of the cofactor by limited proteolysis
(28) . However, the
observed lag time for the hybrid form was about one-third that observed
for HC/LC. After 15 min, similar rates of factor Xa generation were
observed, suggesting that both factor VIII forms had been fully cleaved
following feedback activation by generated factor Xa. The enhanced
activity of the hybrid factor VIII form did not result from altered
reactivity during proteolytic activation since both factor VIII forms
were activated at the same rates and to the same extent by factor Xa
(data not shown).
Figure 3:
Factor
Xa generation assay using factor VIII forms in the presence and absence
of vWf. HC/A3-C1-C2 (2 n
M; and
) or HC/LC (2
n
M;
and
) was preincubated with either vWf (100
n
M;
and
) or the equivalent amount of bovine
serum albumin (
and
) for
2 h prior to the addition
of factor IXa (1 n
M), factor X (200 n
M), and 100
µg/ml phospholipid vesicles in buffer A containing 100 m
M NaCl. Reactions were performed as described under ``Materials
and Methods.''
Inclusion of vWf (Fig. 3) did not affect the
factor Xa-generating activity of the HC/A3-C1-C2 form, whereas its
presence markedly reduced the amount of factor Xa generated in the
reaction containing the native factor VIII subunits. This result was
consistent with the requirement for dissociation of factor VIII from
vWf in the expression of cofactor activity and the observation that
factor Xa is a poor activator of vWf-bound factor VIII
(29) .
That low levels of factor Xa generation are observed in this latter
reaction may reflect the presence (and subsequent activation) of
unbound factor VIII, possibly resulting from competition for vWf
binding by the phospholipid surface
(12) .
Reconstitution of Factor VIIIa Activity
To
determine the effect of LC structure on the activity of heterotrimeric
factor VIIIa, the following experiments were performed. Heterotrimeric
molecules were reconstituted from isolated A1 and A2 subunits derived
from factor VIII HC with either native LC or A3-C1-C2. Reconstitution
of the factor VIIIa heterotrimer involved a two-step reaction whereby
the divalent metal ion linkage was formed between A1 and either the LC
or A3-C1-C2, creating a stable dimer, followed by the metal
ion-independent association of the A2 subunit with the dimer to yield
the heterotrimer. The presence of all three subunits is required for
factor VIIIa activity
(15) .
and at
moderate ionic strength (Fig. 4). Reaction conditions were chosen to
help ensure saturation of the A1 subunit since this subunit contains
the primary interactive site for the A2 subunit
(30) as well as
to mimic our previously defined conditions that promote factor VIII
reconstitution from the HC and LC
(27) .
20
units/ml) was 3-fold less than the activity associated with the
authentic factor VIIIa heterotrimer (
60 units/ml). This result was
consistent with the above observation using heterodimeric factor VIII
that suggested that native LC possessed inherently lower specific
activity than the cleaved form.
Figure 4:
Reconstitution of factor VIIIa activity.
A1/A3-C1-C2 (100 n
M; ) or A1/LC (100 n
M;
)
was incubated with A2 (200 n
M) as described under
``Materials and Methods''. At the indicated time points, an
aliquot was removed and assayed for factor VIIIa activity. After 90 min
at room temperature, an aliquot of each reaction was reacted with
thrombin (10 n
M) and, at the indicated time points, assayed
for factor VIIIa activity.
To ensure that this observation did
not reflect a less efficient reconstitution of the A1/LC/A2
heterotrimer and hence a lower concentration of active material, the
two factor VIIIa forms were reacted with thrombin (Fig. 4).
Exposure of the heterotrimer formed from A1/A3-C1-C2 and A2 to thrombin
had no effect on its activity, consistent with its subunit structure
being derived from this protease. However, reaction with thrombin
enhanced the activity of the A1/LC/A2 heterotrimer such that its
potentiated value was equivalent to that of native factor VIIIa. Since
the LC was the only subunit now cleaved in this form of the molecule,
this result directly indicates that its cleavage is required for
maximal expression of factor VIIIa activity.
Factor Xa Generation Assay with Factor VIIIa
Forms
Factor Xa generation assays similar to those described for
the factor VIII forms were performed using factor VIIIa forms derived
from the native HC components (A1 and A2 subunits) and thrombin-cleaved
or intact LC (Fig. 5). Linear regression analysis of the data points
for the reaction containing A1/A3-C1-C2/A2 had a correlation value of
0.995, indicating the absence of a lag time for the generation of
factor Xa in this reaction. A lag time was observed for the reaction
containing A1/LC/A2, suggesting that this form of the cofactor is less
than fully activated. However, this lag time appeared somewhat shorter
than that for the reaction containing HC/LC, consistent with A1/LC/A2
possessing an intermediate level of cofactor activity.
Stability of Reconstituted Factor VIIIa
Factor
VIIIa activity is highly labile at low concentration and physiologic pH
as a result of dissociation of the A2 subunit from the dimer
(15, 31) . To determine whether the
NH-terminal region of the LC influenced factor VIIIa
stability, factor VIIIa was reconstituted from either A1/A3-C1-C2 or
A1/LC plus the A2 subunit at pH 7.2, and the loss of activity upon
dilution was monitored. In addition, a similar set of assays was
performed in the presence of DEGR-factor IXa, which has been shown to
stabilize factor VIIIa under similar reaction conditions
(22, 32) . Inclusion of the latter experiment was to
evaluate whether the NH
-terminal region of the LC affected
the interaction of factor VIIIa with factor IXa. The results of these
experiments are shown in Fig. 6. The loss of factor VIIIa
activity upon dilution was similar with both factor VIIIa forms,
suggesting that the NH
-terminal region of the LC does not
contribute to the association with the A2 subunit. Furthermore, the
equivalent loss of activity of the two factor VIIIa forms in the
presence of DEGR-factor IXa suggested that this region of the LC did
not affect the ability of factor VIIIa to bind to the protease.
Figure 6:
Stability of factor VIIIa in the presence
and absence of DEGR-factor IXa. Factor VIIIa was reconstituted from
either A1/A3-C1-C2 (100 n
M) or A1/LC (100 n
M) and the
A2 subunit (100 n
M) in buffer A containing 500 µg/ml
bovine serum albumin for 10 min. Factor VIIIa derived from A3-C1-C2
( and
) or LC (
and
) was incubated in the
presence (
and
) or absence (
and
) of
DEGR-factor IXa in buffer A and assayed for factor VIIIa activity.
Reactions were then diluted 50% with buffer A containing 180 m
M NaCl and 500 µg/ml bovine serum albumin, incubated for 5 min,
and reassayed. This procedure was repeated until factor VIIIa was
diluted
12.5-fold. The 100% levels of activity were A1/A3-C1-C2/A2
(26.2 units/ml), A1/A3-C1-C2/A2 + DEGR-factor IXa (29.3 units/ml),
A1/LC/A2 (6.9 units/ml), and A1/LC/A2 + DEGR-factor IXa (9.1
units/ml).
Effects of Factor VIII Forms on Fluorescence Anisotropy
of Fl-FFR-factor IXa
Recently, Duffy et al. (33) observed an incremental increase in fluorescence anisotropy
following association of factor VIII with factor IXa modified at its
active site with a fluorescent probe. This anisotropy value was further
increased following activation of factor VIII by thrombin. Thus, this
parameter may be used as an indicator of the activation state of the
cofactor. Results presented in indicate the fluorescence
anisotropy values for Fl-FFR-factor IXa alone, in the presence of
either HC/LC or HC/A3-C1-C2, and following activation of the
heterodimer forms by thrombin. The fluorescence anisotropy value of
Fl-FFR-factor IXa was increased 0.01 upon binding HC/LC. A further
increase was observed following activation by thrombin, resulting in an
overall incremental increase of
0.05. These effects were similar
to the original observations in the prior study
(33) . In
contrast, the fluorescence anisotropy value for Fl-FFR-factor IXa was
increased 0.037 upon HC/A3-C1-C2 binding and was followed by a
relatively small increase upon activation by thrombin such that the
final anisotropy value was equivalent to that obtained following
activation of the native heterodimer. Thus, the observation that
interaction with the hybrid dimer form results in an initial anisotropy
increase that markedly exceeds that for the native dimer and approaches
the magnitude of that for factor VIIIa suggests that this factor VIII
form may be factor VIIIa-like in its effect on the conformation of the
factor IXa active site.
and Arg
in the HC and
Arg
in the LC, converting the inactive procofactor to
the active form. Little is known about the role(s) of these cleavages
and their contribution to the potentiated activity of factor VIIIa. In
the factor Xase complex, the cofactor binds both the (phospholipid)
surface and factor IXa. However, these activities are present in the
unactivated molecule. A high affinity interaction of factor VIII with
phospholipid vesicles ( K
2 n
M (35) ) is mediated through residues located at the
COOH-terminal end of the LC (C2 domain residues 2300-2332
(36) ), and this interaction is likely unaffected by thrombin
cleavage. Recent results of Duffy et al. (33) indicate
a K
of
2 n
M for the factor
VIIIa-factor IXa interaction. A similar high affinity interaction was
observed by these investigators when factor VIII was substituted for
factor VIIIa.
3-fold
higher specific activity than factor VIII reconstituted from native HC
and native LC (2 versus 0.6 units/µg). It must be noted
that the specific activity of purified factor VIII (either
plasma-derived or recombinant) is
2-4 units/µg. The
reason for the lower specific activity of reconstituted factor VIII
(0.6 units/µg) is not known, but may result from partial
denaturation of the subunits during the metal ion chelation and/or
subsequent subunit purification steps. The hybrid material showed a
markedly reduced lag time compared with factor VIII reconstituted from
native subunits, when assayed without prior activation in a purified
factor Xa generation system. The lag observed in this assay is
attributed to the time required for the low levels of factor Xa
generated to feedback activate factor VIII, yielding the active
cofactor
(28) . Both factor VIII forms followed similar rates of
activation by factor Xa (data not shown), suggesting that the more
factor VIIIa-like activity of the hybrid form does not result from
differential rates of activation. In addition, the activity of the
hybrid form was unaffected by the presence of vWf, consistent with it
lacking the primary vWf interactive site. On the other hand, authentic
factor VIII showed a reduced catalytic rate in the presence of vWf,
consistent with factor Xa being a poor activator of vWf-bound factor
VIII
(29) .
Cys mutation,
creating a thrombin-resistant HC. Partially purified factor VIII from
this patient had 3% that of normal plasma factor VIII activity, which
could still be activated 5-fold with thrombin. Activation correlated
with cleavage of the LC, and since there was no vWf present in these
preparations, the increase in factor VIII activity did not result from
cofactor release of vWf. Although only limited regions of the factor
VIII gene from this patient were examined and therefore other mutations
may have gone undetected, the results presented in this study support
the notion that cleavage of factor VIII LC contributed to the increase
in factor VIIIa activity.
(in the LC) or
Arg
(in the HC). These factor VIII molecules were
resistant to activation by thrombin at the altered site, while thrombin
cleavage at the unmodified site was not affected. These investigators
concluded that both cleavages were required for factor VIII activation.
However, a general concern of studies involving site-directed
mutagenesis is that even conservative substitutions may affect protein
characteristics such as folding and/or stability as well as
interprotein interactions
(42) .
-terminal region of intact LC does not appear to
influence the association of the A2 subunit. Finally, results on the
capacity of active site-modified factor IXa to enhance the association
of A2 with either A1/LC or A1/A3-C1-C2 suggest that this region of the
LC is not involved and/or does not interfere with the association of
factor VIIIa with factor IXa.
for the factor IXa-dependent conversion of
factor X by several orders of magnitude.
r = 0.011) and was further
increased following activation to factor VIIIa (
r = 0.051), consistent with the earlier results of Duffy
et al. (33) . However, in the presence of HC/A3-C1-C2,
the initial incremental increase in anisotropy (
r = 0.037) was
4-fold greater than that observed with
HC/LC and approached the value observed with the fully activated
cofactor. Subsequent conversion of the hybrid factor VIII form to
factor VIIIa yielded an anisotropy value equivalent to that obtained
following activation of HC/LC. These results suggest that HC/A3-C1-C2
is more factor VIIIa-like than the native heterodimer with respect to
its effect on the conformation in and around the factor IXa active site
and hence its effect on the catalytic efficiency of factor IXa in the
tenase complex. We propose that this alteration in conformation is
dependent upon LC cleavage and explains mechanistically the observed
higher specific activity and more efficient factor Xa-generating
properties of the hybrid factor VIII heterodimer as well as the
requirement for this cleavage to yield maximally activated cofactor.
Table: Effect of factor VIII forms on the
fluorescence anisotropy of Fl-FFR-factor IXa
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.