(Received for publication, June 21, 1994; and in revised form, November 16, 1994)
From the
The role of Factor VIII light chain cleavage in Factor VIII
activation and subunit interaction was investigated. Purified Factor
VIII was dissociated into its separate subunits, and the isolated light
chain was cleaved by thrombin at position Arg or by
Factor Xa at position Arg
. These Factor VIII light chain
derivatives then were used for reconstitution with purified Factor VIII
heavy chain to obtain heterodimers that were exclusively cleaved within
the light chain. Intact and cleaved light chain could effectively be
reassociated with heavy chain, with concomitant regain of Factor VIII
cofactor function. The association rate constant of Factor Xa-cleaved
light chain was found to be 3-fold lower than that of thrombin-cleaved
or intact light chain, suggesting a role of the region
Ser
-Arg
in subunit assembly.
Dissociation rate constants, however, were independent of Factor VIII
light chain cleavage. Low ionic strength was observed to promote
association but to destabilize the Factor VIII heterodimer. At high
ionic strength, Factor VIII dissociation was extremely slow (k
10
s
) for all Factor VIII light chain
derivatives, indicating that Factor VIII light chain cleavage is not
related to Factor VIII dissociation. Furthermore, Factor VIII light
chain cleavage does not affect enzyme-cofactor assembly, since the
various light chain derivatives proved equally efficient in binding to
Factor IXa (K
15 nM).
Studies in a purified Factor X-activating system demonstrated that
thrombin and Factor Xa activate Factor VIII to the same extent.
However, Factor Xa differed from thrombin in that it cleaved at
Arg
rather than at Arg
. Reassociated
heterodimers of Factor VIII heavy chain and intact light chain did not
promote Factor X activation. In contrast, heterodimers that contained
cleaved light chain exhibited substantial Factor VIIIa activity. These
data demonstrate that a single cleavage at either Arg
or
Arg
converts the inactive Factor VIII heterodimer into
an active cofactor of Factor IXa.
Human blood coagulation Factor VIII (FVIII) ()participates as cofactor for Factor IXa (FIXa) in
activation of Factor X (FX) in the intrinsic pathway of blood
coagulation(1) . FVIII is synthesized as a single-chain
polypeptide displaying a domain structure with the sequence
A1-A2-B-A3-C1-C2(2, 3) . The protein circulates in
plasma as a heterodimer consisting of FVIII heavy chain (FVIII-HC) and
FVIII light chain (FVIII-LC) comprising the domains A1-A2-B and
A3-C1-C2, respectively. Because of limited proteolysis in the B-domain,
FVIII-HC is heterogeneous (M
,
90,000-210,000)(4, 5) . The FVIII heterodimer is
metal ion-dependent, since EDTA destroys FVIII activity concomitant
with dissociation of FVIII-HC and
FVIII-LC(4, 6, 7) . Moreover, the addition of
divalent metal ions to inactivated FVIII or to a mixture of isolated
subunits results in FVIII reassembly(8, 9) and regain
of FVIII activity(8, 9, 10) . Thus, FVIII
activity is restricted to the heterodimeric complex of heavy and light
chain.
FVIII activation is required for cofactor function in the
intrinsic pathway. Activation is accomplished by limited proteolysis of
both the heavy and light chain of
FVIII(11, 12, 13) . Major cleavage sites are
located at or close to the boundaries of the A-domains, in the FVIII-HC
at amino acid positions Arg and Arg
, and in
the FVIII-LC at position Arg
(5) . After cleavage
of FVIII-HC between the domains A1 and A2 at position
Arg
, the A2 domain is noncovalently associated with the
A1 domain and the A3-C1-C2 comprising light
chain(5, 14, 15) . Activated FVIII (FVIIIa)
has transient activity, since the A2 domain readily dissociates from
the A1/A3-C1-C2 dimer(16) . Both thrombin and Factor Xa (FXa)
activate FVIII by limited proteolysis at the three major cleavage
sites(5) . However, FXa cleaves some additional sites, one of
which is located in the heavy chain at position
Arg
(5) . The same site is cleaved by activated
Protein C and FIXa, which inactivate
FVIII(5, 17, 18, 19) . Cleavage at
this site, and the release of the region
Ser
-Arg
, promotes FVIIIa dissociation
and inactivation(20) . These findings have established the
concept that limited proteolysis of FVIII-HC activates FVIII but
simultaneously triggers subunit dissociation and concomitant FVIII
inactivation.
With respect to FVIII-LC cleavage, its role in FVIII
activation and inactivation remains controversial. One problem is that
FVIII-LC is susceptible to cleavage at a number of sites, including the
positions Arg, Arg
, and
Arg
. It seems evident that FVIII activation by thrombin
involves proteolysis at Arg
(5, 21) .
The same site may be cleaved upon activation by FXa, although this
enzyme cleaves at position Arg
as well, in a process
that coincides with inactivation of FVIII(5, 22) .
Inactivation may also be accomplished by FIXa, which cleaves FVIII-LC
at position Arg
(18, 19) . However, a
recent observation suggests that cleavage at this site may be unrelated
to FVIII inactivation(22) . Interpretation of these findings is
further complicated by the notion that the same enzymes that cleave
FVIII-LC also cleave at various positions within FVIII-HC (5, 18, 19, 20, 21, 22) .
Thus, the precise role of FVIII-LC cleavage in FVIII function remains
unclear. In the present study, we addressed this problem employing
FVIII heterodimers that were cleaved exclusively at the FVIII-LC
positions Arg
or Arg
. These were obtained
by assembling isolated FVIII-HC with well defined FVIII-LC derivatives.
Subsequently, these FVIII species were compared with respect to subunit
interaction, FVIII-FIXa complex assembly, and function as cofactor of
FIXa in FX activation.
The extent of FVIII assembly at a given time t thus is the resultant of the simultaneously occurring association and dissociation. Since FVIII association is a slow process (see ``Results''), this is most conveniently analyzed in terms of initial rates of association. Under certain experimental conditions, FVIII dissociation is remarkably slow (see ``Results''). In the initial phase, the concentration of dimer formed remains low, whereas the concentrations of free subunits do not differ substantially from their initial values. The reversible association reaction then reflects the simple, bimolecular association process,
When the initial concentration of FVIII-HC (a) is slightly higher than that of FVIII-LC (b
), the integrated expression, with the limits t = 0 and t = t, has
previously been given as(32) .
Here k represents the association rate
constant, and p is the concentration of FVIII dimer formed at
time t. Association kinetics were derived from the initial 120
min of FVIII assembly experiments. k
was
calculated by fitting of data into employing Enzfitter
software (Elsevier, Amsterdam, The Netherlands).
Here [FVIII] is the FVIII concentration at t = 0, and k
is the first order
rate constant of inactivation of FVIII heterodimers.
The apparent K values were calculated from association and
dissociation rate constants according to the following
equation.
Figure 1:
Purified
FVIII subunit derivatives. FVIII-HC and FVIII-LC derivatives were
reduced and subjected to SDS-polyacrylamide gel electrophoresis on a
7.5% (w/v) polyacrylamide gel. Protein was visualized by silver
staining. Lanes 1 and 2 contain 1 µg of purified
FVIII-HC and 0.25 µg of FVIII-LC, respectively. FVIII-LC was
subjected to limited proteolysis by thrombin and FXa, and cleavage
products were purified (see ``Experimental Procedures'').
Lanes 3 and 4 show 0.25 µg of FVIII-LC and
FVIII-LC
, respectively. NH
-terminal amino
acid sequence analysis of FVIII-LC
and FVIII-LC
was performed to verify the positions of cleavage by thrombin and
FXa. The derived sequences of amino acids are shown together with the
NH
-terminal sequence of intact
FVIII-LC(5) .
In preliminary experiments, the effect of pH and divalent cations
was examined with respect to regeneration of FVIII activity from
isolated subunits. The results were essentially similar to those
described previously by Fay(8) , with the exception that
Ca proved more effective than Mn
with respect to regain of FVIII activity (results not shown).
Conditions were identified that promoted FVIII regeneration from intact
FVIII-LC and an excess of FVIII-HC, yielding a specific activity of 1.9
± 0.2 units/µg (mean ± S.D.). In terms of effectively
assembled protein, thus excluding the excess of FVIII-HC, the specific
activity was 4 units/µg. This value is similar to that of the FVIII
starting material used for the preparation of the isolated subunits.
These conditions, thus, are appropriate for studies on the role of
FVIII-LC cleavage in regeneration of FVIII activity. FVIII-LC
was found to be similar to intact FVIII-LC with respect to the
specific activity of regenerated FVIII (1.8 ± 0.2 units/µg).
In contrast, FVIII-LC
restored FVIII activity to a lower
specific activity (0.9 ± 0.1 units/µg). This suggests that
either FVIII-LC
containing heterodimers have reduced
activity, or that the process of heterodimer formation is less
efficient when the sequence Ser
-Arg
is lacking from FVIII-LC.
Figure 2:
Reconstitution of FVIII-LC derivatives
with FVIII-HC. Varying concentrations of FVIII-LC (),
FVIII-LC
(
), and FVIII-LC
(
)
were tested for complex assembly with FVIII-HC (415 nM) into
active FVIII heterodimers. Subunits were incubated in 400 mM NaCl, 40 mM CaCl
, 1% (w/v) HSA, 20 mM Hepes (pH 7.2). After 30 min of incubation at 22 °C, FVIII
activity (panel A) and FVIII heterodimer formation (panel
B) were assessed as described under ``Experimental
Procedures''.
Figure 3:
Ionic strength dependence of FVIII subunit
interaction. Association (panel A) of FVIII-HC (415
nM) with intact FVIII-LC (125 nM) was performed in
the presence of varying concentrations of NaCl in 1% (w/v) HSA, 20
mM Hepes (pH 7.2), and either 10 mM () or 40
mM (
) CaCl
. After 1 h of incubation at 22
°C, FVIII activity was determined as described under
``Experimental Procedures.'' The same incubation conditions
were used for dissociation of reassociated FVIII (panel B).
FVIII subunits were associated for 20 h in 150 mM NaCl, 1%
(w/v) HSA, 20 mM Hepes (pH 7.2), and either 10 mM (
) or 40 mM (
) CaCl
prior to
dilution to 0.4 nM FVIII activity. Residual FVIII activity was
measured after 3 h of dissociation. Data represent the average ±
S.D. of three experiments.
Association (Fig. 4A) of FVIII-HC with various FVIII-LC derivatives
as well as dissociation (Fig. 4B) of various
reassociated FVIII species was investigated time-dependently.
Association and dissociation data were fitted into models of
noncatalytic interaction and single exponential decay, respectively
(see ``Experimental Procedures''), in order to derive
association (k) and dissociation (k
) rate constants and apparent K
values (Table 1). Comparison between the various FVIII-LC
derivatives demonstrates that FVIII-LC
differs from
FVIII-LC
and intact FVIII-LC in that it displays a
2-3-fold lower association rate constant (Table 1). For
FVIII-LC
and FVIII-LC
, the dissociation
rate constants were found to be equal, and slightly higher than the
dissociation rate constant of heterodimers containing intact FVIII-LC.
Similarly, no differences between FVIII-LC species were apparent under
much more stringent dissociating conditions (Fig. 4B).
As the association process, and thus k
, is
particularly ionic strength-dependent, the same holds for the apparent K
values derived from these data. K
values may be 2-3-fold lower at lower NaCl concentrations (cf. Fig. 3).
Figure 4:
Interaction between FVIII subunit
derivatives. FVIII-HC (415 nM) was reconstituted with 125
nM of intact FVIII-LC (), FVIII-LC
(
), and FVIII-LC
(
) in a buffer
consisting of 400 mM NaCl, 40 mM CaCl
, 1%
(w/v) HSA, 20 mM Hepes (pH 7.2) (panel A). At various
time points FVIII activity was determined, and association rate
constants were calculated from data between 0 and 120 min of
association employing under ``Experimental
Procedures.'' For inactivation studies (panel B), FVIII
first was assembled in 400 mM NaCl, 40 mM CaCl
, 1% (w/v) HSA, 20 mM Hepes (pH 7.2) and
subsequently diluted to 0.4 nM FVIII activity in 1% (w/v) HSA,
20 mM Hepes (pH 7.2) containing 400 mM NaCl and 40
mM CaCl
(solid line) or 7.5 mM NaCl and 10 mM CaCl
(dashed line),
and decay of FVIII activity was determined. Data were fitted into under ``Experimental Procedures,'' and the
resulting curves are shown. Data are given as the average
± S.D. of three experiments.
Figure 5:
FIXa binding to immobilized FVIII-LC
variants. Intact FVIII-LC (), FVIII-LC
(
),
and FVIII-LC
(
) were immobilized to the
anti-FVIII-LC monoclonal antibody CLB-CAg 12 (1 µg/well). The
amount of FVIII-LC derivatives bound to the antibody was quantified
employing an immunological assay (see ``Experimental
Procedures'') and was found to be equivalent (1 pmol/well) for the
FVIII-LC derivatives. Subsequently, FIXa was added, and after 4 h of
incubation FIXa binding was assessed as described
elsewhere(23) . Data represent the average ± S.D. of
three experiments.
Figure 6:
Initial rates of FXa formation. FX (0.56
µM) was activated in a mixture containing FVIII (0.4
nM), FIXa (0.3 nM), phospholipids (100
µM), and Ca (10 mM). FX
activation was started by the addition of various concentrations of
thrombin (
) or FXa (
). Progress curves of FX activation
served to derive the initial rates of FXa formation. Each data point
represents the average ± S.D. of at least three experiments. In
order to detect cleavage fragments, FVIII (32 nM) was
activated for 3 min with thrombin (FIIa) or FXa (both 5 nM)
under the same conditions, except that FIXa and FX were omitted.
Subsequently, activated FVIII was subjected to SDS-polyacrylamide gel
electrophoresis and immunoblotting. FVIII-LC fragments were visualized
employing a polyclonal antibody against FVIII-LC. The positions of the
FVIII-LC cleavage products (73 and 67 kDa) upon incubation with
thrombin or FXa, respectively, are indicated in the inset.
Figure 7:
FXa formation in the presence of
reconstituted FVIII. Intact FVIII-LC (), FVIII-LC
(
), or FVIII-LC
(
) (125 nM)
were reconstituted with FVIII-HC (415 nM) in 400 mM NaCl, 40 mM CaCl
, 1% (w/v) HSA, 20 mM Hepes (pH 7.2) for 24 h at 22 °C. Reconstituted FVIII (0.08
nM) was added to FIXa (0.3 nM), phospholipids (100
µM), CaCl
(10 mM), and acetylated FX
(0.2 µM), and FXa formation was determined (panel
A). In panel B, the same experiment was performed,
employing native FX instead of chemically modified FX. Data are given
as the average ± S.D. of three experiments. The inset shows an immunoblot analysis of FVIII-HC reassociated for 24 h
with intact FVIII-LC (lane 1), FVIII-LC
(lane 2), and FVIII-LC
(lane 3).
FVIII-HC was visualized employing a chicken antibody (see
``Experimental Procedures'') directed against the A1 domain
of FVIII-HC.
Previous studies have established that, in the absence
of exogeneous FVIII activating enzymes such as FXa or thrombin, FVIII
activation is fully dependent on FXa formed during the initial phase of
FX activation (11, 12) . To assess the contribution of
feedback activation caused by FVIII-HC cleavage by FXa, the experiments
of Fig. 7A were also performed using native, unmodified
FX as the substrate. As shown in Fig. 7B, FVIII
containing FVIII-LC or FVIII-LC
displayed
a 4-fold higher rate of FXa formation than observed in the presence of
chemically modified FX (Fig. 7A). This implies that
under the conditions of Fig. 7, feedback cleavage of FVIII-HC by
FXa contributes to the activity of fully activated FVIII. Moreover, the
same rate of FXa formation was observed in the presence of heterodimers
containing FVIII-LC
or FVIII-LC
,
demonstrating that these two FVIII-LC derivatives are functionally
equivalent. Fig. 7B further shows that the initial rate
of FXa formation (<3 min) was more than 10-fold reduced when the
reconstituted FVIII heterodimer contained intact instead of cleaved
FVIII-LC. Apparently, FVIII-LC cleavage, either by thrombin or by FXa,
is essential for developing the full cofactor potential of FVIII-HC.
The objective of the present study was to assess the role of
FVIII-LC cleavage by thrombin and FXa, with special reference to FVIII
subunit interaction and FVIII activation and inactivation. Our
experimental approach employed FVIII-LC derivatives obtained by limited
proteolysis using thrombin and FXa (Fig. 1). Subsequently, these
cleavage products were reassembled with FVIII-HC in order to obtain
FVIII heterodimers that were cleaved in one subunit only. Isolated
FVIII subunits could be effectively reassociated into active
heterodimers (Fig. 2). For intact FVIII-LC, our results are
similar to those of previous studies of FVIII
assembly(8, 16) . To describe subunit interaction in
more detail, we have assessed association and dissociation kinetics.
Low ionic strength was found to promote FVIII subunit association but
simultaneously to destabilize the heterodimer (Fig. 3). The same
ionic strength dependence has recently been reported for the subunit
association of activated FV (FVa)(37) , which is similar to
FVIII in that it also comprises a heterodimer of the domains A1-A2 and
A3-C1-C2(37, 38) . For FVa subunit interaction, K values have been reported (38) that are
at least one order of magnitude lower than for the interaction between
FVIII-HC and intact FVIII-LC (Table 1). Since K
values for FVIII subunit interaction represent apparent
equilibrium constants that are highly dependent on ionic strength (Fig. 3), nonequilibrium rate constants may be considered as
more appropriately reflecting subunit interaction. Comparison between
the association rate constants demonstrates that assembly of FVIII
heterodimer is about 50-fold slower than of FVa (Table 1, (39) ). However, the dissociation rate constants of associated
FVIII and FVa subunits indicate that, except at relatively low ionic
strength, dissociation is negligibly slow for both FVIII and FVa (Fig. 4B, (39) ). It may seem contradictory
that subunit association rates are lower at the higher ionic strength (Fig. 3), whereas optimal regain of FVIII activity from isolated
subunits requires the same condition (Table 1; cf. (8) ). Apparently, the extent of heterodimer formation is fully
controlled by the dissociation process.
The dissociation rate
constant of uncleaved FVIII-HC and FVIII-LC (Table 1) is
1000-fold lower than that of the interaction between the A2 domain and
the A1/A3-C1-C2 dimer(16) . This supports the concept that
limited proteolysis of FVIII-HC, besides activating FVIII, also
triggers subunit dissociation and concomitant FVIII inactivation. We
have considered the possibility that, in analogy with FVIII-HC,
FVIII-LC cleavage also affects subunit interaction. Although
FVIII-LC proved to be less efficient than intact
FVIII-LC and FVIII-LC
in heterodimer assembly and regain
of FVIII activity from isolated subunits (Fig. 2), heterodimer
dissociation rates were strikingly similar (Table 1, Fig. 4B). Therefore, we conclude that the FVIII-LC
fragment Ser
-Arg
, although promoting
subunit association, has no effect on dissociation of FVIII into its
constituent units.
In the current model of FVIII regulation, FXa is
believed to cleave FVIII-LC at position Arg prior to
Arg
(5) . The latter cleavage thus may be a
secondary event that is associated with FVIII inactivation and as such
could serve the same function as FVIII-HC cleavage at
Arg
(21, 22) . Our data, however, do not
support this view. First, heterodimers containing FVIII-LC
do have FVIII activity (Fig. 2). Second, FVIII containing
FVIII-LC
displays the same subunit dissociation rate
constant as heterodimers containing FVIII-LC
or
uncleaved FVIII-LC (Table 1, Fig. 4B). Moreover,
FVIII-LC
and FVIII-LC
have the same
affinity for FIXa (Fig. 5) and contribute to FVIIIa activity to
the same extent (Fig. 7). Collectively, these data suggest that
thrombin and FXa follow distinct pathways in cleaving FVIII, which
yield different but functionally indistinguishable FVIIIa species.
Whereas this view could be confirmed in a system of purified components (Fig. 6), the physiological mechanism of FVIII activation seems
to be more complex. The notion that a point mutation at the thrombin
cleavage site Arg
leads to FVIII dysfunction and
hemophilia A (21, 40, 41, 42) implies that
cleavage at Arg
by FXa does not compensate for the lack
of cleavage at Arg
by thrombin. This raises the question
of whether Arg
is susceptible to proteolysis by FXa when
FVIII is in complex with its physiological carrier protein von
Willebrand Factor (vWF). Several studies have reported that vWF indeed
interferes with events involving the amino-terminal portion of
FVIII-LC, such as the activation of FVIII by FXa (43) and the
interaction of FIXa with the FVIII-LC region
Gln
-Asp
(23) . It seems
conceivable that cleavage of FVIII-LC at Arg
provides a
relatively insignificant pathway of FVIII activation, since it may be
restricted to situations where FVIII is dissociated from vWF.
Its
tight association with vWF (K
10
M) (44) prevents FVIII from
interacting with components that bind with lower affinity, such as FIXa (23) and phospholipids(45) . Thus, disruption of the
FVIII-vWF complex is required for cofactor function. It has been well
established that dissociation is accomplished by FVIII-LC cleavage at
position
Arg
(13, 22, 46, 47) .
Our rigorous approach of FVIII subunit reassembly and cofactor function
analysis allowed us to establish a second role for FVIII-LC cleavage.
Employing vWF-free conditions, we found that cleavage of FVIII-LC alone
at either Arg
or Arg
activates FVIII to a
substantial extent (Fig. 7). This implies that cleavage of
FVIII-HC, although determining the final extent of FVIIIa activity (Fig. 7B), is not absolutely required for FVIII
activation. Moreover, this finding provides an explanation for the
observation that dysfunctional FVIII with a substitution at the
Arg
-Ser
cleavage site in FVIII-HC has some
residual FVIII activity and is associated with mild hemophilia A (47, 48) .
One conclusion of our study is that
FVIII activity is completely lacking when FVIII is composed of
uncleaved subunits (Fig. 7A). Apparently, the
amino-terminal portion Glu-Arg
of
the light chain of FVIII is an activation peptide that needs to be
cleaved off for exposure of cofactor activity. Within this fragment,
the sequence Glu
-Arg
serves a dual
role in regulating FVIII activity. First, this sulfated, acidic
sequence promotes the high affinity interaction between FVIII and
vWF(28, 36, 49) . On the other hand, the same
region seems to inhibit some interaction involved in FX activation.
Since our data seem to exclude the possibility that FVIII-LC cleavage
affects FIXa binding (Fig. 5), we propose that the region
Glu
-Arg
interferes in binding of
FVIII to other components of the FX-activating complex, such as FX or
phospholipids. In contrast, the region
Ser
-Arg
has no apparent role in the
cofactor function of FVIII. Although this region does contribute to
FVIII heterodimer assembly (Table 1), the physiological
implications of this phenomenon remain unclear.