(Received for publication, October 4, 1995; and in revised form, November 22, 1995)
From the
Factor VIIIa, the cofactor for the factor IXa-dependent
conversion of factor X to factor Xa, is proteolytically inactivated by
activated protein C (APC). APC cleaves at two sites in factor VIIIa,
Arg, near the C terminus of the A1 subunit; and
Arg
, bisecting the A2 subunit (Fay, P., Smudzin, T., and
Walker, F.(1991) J. Biol. Chem. 266, 20139-20145).
Factor VIIIa increased the fluorescence anisotropy of
fluorescein-Phe-Phe-Arg factor IXa (Fl-FFR-FIXa; K
= 42.4 nM), whereas cleavage of factor VIIIa
by APC eliminated this property. Isolation of the APC-cleaved
A1/A3-C1-C2 dimer (A1
/A3-C1-C2), and the fragments
derived from cleaved A2 subunit (A2
/A2
),
permitted dissection of the roles of individual cleavages in cofactor
inactivation. Intact A1/A3-C1-C2 dimer increased Fl-FFR-FIXa anisotropy
and bound factor X in a solid phase assay, while these activities were
absent in the A1
/A3-C1-C2. However, the residues removed
by this cleavage, Met
-Arg
, did not
directly participate in these functions since neither a synthetic
peptide to this sequence nor an anti-peptide polyclonal antibody
blocked these activities using intact dimer. CD spectral analysis of
the intact and truncated dimers indicated reduced
and/or
content in the latter. The A1/A3-C1-C2 dimer plus A2 subunit
reconstitutes cofactor activity and produced a factor VIIIa-like effect
on the anisotropy of Fl-FFR-FIXa. However, when A2 was replaced by the
A2
/A2
fragments, the resulting fluorescence
signal was equivalent to that observed with the dimer alone. These
results indicate that APC inactivates the cofactor at two levels within
the intrinsic factor Xase complex. Cleavage of either subunit modulates
the factor IXa active site, suggesting an essential synergy of
interactive sites in factor VIIIa. Furthermore, cleavage of the A1 site
alters the conformation of a factor X binding site within that subunit,
thereby reducing the affinity of cofactor for substrate.
Factor VIII, the plasma protein deficient or defective in individuals with hemophilia A, is synthesized as a 300-kDa precursor protein (1, 2) with domain structure A1-A2-B-A3-C1-C2(3) . Factor VIII is processed to a series of divalent metal ion-linked heterodimers (4, 5, 6) by cleavage at the A3-B junction, generating a heavy chain minimally represented by the A1-A2 domains which may possess all or part of the B domain and a light chain consisting of the A3-C1-C2 domains.
Factor VIII functions as the
cofactor for factor IXa in the intrinsic factor Xase ()complex, where it increases the k
for conversion of factor X to factor Xa by several orders of
magnitude(7) . Factor VIII must first be activated, through
limited proteolysis by thrombin, to the active cofactor form, factor
VIIIa. Thrombin cleaves the factor VIII at two sites within the heavy
chain, Arg
and Arg
(8) . The former
liberates the B domain or its fragments, while the latter bisects the
A1 and A2 domains. Thrombin also cleaves near the N terminus of the
light chain, at Arg
(8) . Thus factor VIIIa is a
heterotrimer of A1, A2, and A3-C1-C2 subunits. (
)The A1
subunit and the A3 domain retain the metal ion linkage and this stable
dimer (A1/A3-C1-C2) (9, 10) is associated with the A2
subunit mainly through electrostatic forces(11) . The factor
VIIIa subunits can be isolated separately and are inactive. However,
factor VIIIa activity can be reconstituted upon combining the isolated
A1/A3-C1-C2 dimer and the A2 subunit (11, 12, 13) .
Activated protein C, a
potent anticoagulant, proteolytically inactivates the cofactors,
factors VIIIa and Va, in a surface-dependent reaction (see (14) for review). The protease binds the light chain (-derived)
subunit of the cofactors (15, 16) at a region
localized to the C-terminal end of the A3 domain (A3-C1
junction)(17) . Inactivation results from cleavage of the heavy
chain (-derived) subunits. Activated protein C cleaves factor VIIIa
rapidly at Arg(9) , which bisects the A2 subunit,
and slowly within the A1 subunit at
Arg
(8, 9) , which releases a highly
acidic region from the C terminus. The cleavage of the A2 subunit most
closely correlates with factor VIIIa inactivation(9) .
The functions of the activated protein C-catalyzed cleavages are not well understood. In this report, we examine the mechanisms for loss of cofactor activity following proteolytic inactivation of factor VIIIa. Analyses using activated protein C-cleaved factor VIIIa fragments allow dissection of the effect of each cleavage event relative to factor VIIIa function. Results of this study show that cleavage of both the A1 and A2 subunits affects the orientation of cofactor with the active site of factor IXa, whereas cleavage at A1 results in a reduced affinity of cofactor for substrate factor X.
Figure 1:
Fluorescence anisotropy of
Fl-FFR-FIXa in the presence of cleaved or native factor VIIIa.
Anisotropy of Fl-FFR-FIXa was measured as described under
``Materials and Methods.'' Reactions containing Fl-FFR-FIXa
(20 nM) and PSPCPE (50 µg/ml) in buffer A were titrated
with either native (open circles) or cleaved (closed
circles) factor VIIIa at the indicated concentrations. Data for
the intact factor VIIIa were fitted to a quadratic
equation(43) . Fitted constants are: K = 42.4 ± 18.7; number of sites (n)
= 0.91 ± 1.2.
Figure 2:
SDS-polyacrylamide gel electrophoresis of
purified, activated protein C cleaved-factor VIIIa subunits.
A2/A2
and A1
/A3-C1-C2 were
isolated following Mono S chromatography and are shown in gel lanes
1 and 2, respectively. The gel was stained with silver
nitrate.
To examine effects of A1 cleavage, the A1/A3-C1-C2 or
A1/A3-C1-C2 dimer was titrated into a sample containing
Fl-FFR-FIXa (20 nM) and PSPCPE vesicles (50 µg/ml).
Results (Fig. 3) show that the A1/A3-C1-C2 dimer produced a
saturable increase in the anisotropy of Fl-FFR-FIXa with an incremental
increase of 0.063 at 200 nM dimer. When
A1
/A3-C1-C2 was used in place of the native dimer, there
was minimal change (
0.02) in the anisotropy of the Fl-FFR-FIXa.
This affect was similar to that observed using isolated factor VIII
light chain, which contains the high affinity binding site for factor
IXa (27) . Thus, it appears the activated protein C cleavage at
the A1 site eliminated the contribution of the A1 subunit to modulate
the active site of factor IXa. However, the acidic region of the A1
subunit (residues 337-372), which is removed following activated
protein C cleavage at Arg
(9) , did not appear to
be directly involved in this interaction, since neither high
concentrations (50 µM) of synthetic peptide
FVIII
corresponding to this region, nor a
rabbit antibody made against this peptide(19) , affected the
ability of the intact A1/A3-C1-C2 to modulate the anisotropy of the
FL-FFR-FIXa (data not shown). Therefore, cleavage of the acidic
terminal region of A1 may result in some conformational change that
renders this subunit unable to modulate the Fl-FFR-FIXa active site.
Figure 3:
Fluorescence anisotropy of Fl-FFR-FIXa in
the presence of A1/A3-C1-C2, A1/A3-C1-C2, or factor VIII
light chain. Reactions containing Fl-FFR-FIXa (20 nM) and
PSPCPE (50 µg/ml) in buffer A were titrated with A1/A3-C1-C2 (open circles), A1
/A3-C1-C2 (closed
circles), or light chain (open squares) at the indicated
concentrations.
An alternate possibility is that the cleavage at the A1 site
resulted in a marked reduction in the affinity of
A1/A3-C1-C2 for Fl-FFR-FIXa. This potential was remote
based upon a similar fluorescence effect observed with the isolated
factor VIII light chain. However, factor VIIIa reconstitution assays
were performed to assess the possibility of disparate binding following
cleavage at this site. Factor VIIIa can be reconstituted from isolated
subunits(11, 12, 13) , and active
site-modified factor IXa stably enhances this reconstitution at
physiologic pH(28) . Since A1
/A3-C1-C2 plus A2
subunit cannot reconstitute factor VIIIa activity(9) , the
capacity of this cleaved dimer to inhibit the factor IXa-dependent
enhancement of factor VIIIa reconstitution from native subunits was
determined. Factor VIIIa subunits, A2 and A1/A3-C1-C2 (20 nM each), were reacted with various concentrations of
A1
/A3-C1-C2 in the absence or presence of Fl-FFR-FIXa (20
nM) (Fig. 4). The A1
/A3-C1-C2 dimer did
not affect the reconstitution of subunits in the absence of
Fl-FFR-FIXa, consistent with the requirement for the acidic region of
A1 for association with A2 subunit(9) . However, the cleaved
dimer inhibited the Fl-FFR-FIXa-dependent enhancement of factor VIIIa
reconstitution with 50% inhibition observed at
70 nM A1
/A3-C1-C2. This result suggested a somewhat weaker
affinity (
3-fold) of factor IXa for A1
/A3-C1-C2
compared with native A1/A3-C1-C2. Similar results to those obtained
with the cleaved dimer were observed using isolated factor VIII light
chain (data not shown). Therefore, cleavage at the A1 site does not
appear to significantly affect the binding of A1
/A3-C1-C2
to Fl-FFR-FIXa, but does eliminate its ability to induce an A1
subunit-dependent change in the Fl-FFR-FIXa active site.
Figure 4:
Effect of A1/A3-C1-C2 on
factor VIIIa reconstitution. A1/A3-C1-C2 (20 nM) and A2
subunit (20 nM) were incubated with the indicated
concentrations of A1
/A3-C1-C2 in buffer A containing 0.5
mg/ml BSA and 100 µg/ml inosithin in the presence (closed
circles) and absence (open circles) of Fl-FFR-FIXa (20
nM) for 50 min at room temperature. Factor VIIIa activity was
determined using a one-stage clotting
assay.
Figure 5:
Effect of A2 cleavage on the factor
VIIIa-dependent fluorescence anisotropy of Fl-FFR-FIXa. Reactions
containing Fl-FFR-FIXa (20 nM), A1/A3-C1-C2 (50 nM),
and PSPCPE (50 µg/ml) in buffer A were titrated with either the
intact A2 subunit (open circles) or A2/A2
(closed circles) at the indicated
concentrations.
Figure 6:
Effect of A1/A3-C1-C2 cleavage on the
factor X-dependent change in Fl-FFR-FIXa anisotropy. Reactions
containing Fl-FFR-FIXa (20 nM), PSPCPE (50 µg/ml), and
either A1/A3-C1-C2 (100 nM; open circles) or
A1/A3-C1-C2 (100 nM; closed circles) in
buffer A were titrated with factor X at the indicated
concentrations.
To determine whether the cleaved dimer
retained the capacity to bind factor X, a microtiter plate binding
assay was developed. Briefly, factor VIII-derived subunits were
incubated in wells that had been coated with a capture antibody, ESH-8.
Various concentrations of factor X were then added to the wells. The
amount of factor X bound to the immobilized factor VIII-derived protein
was determined following reaction with RVV-X (to convert bound zymogen
to the active protease) and subsequent addition of the factor Xa
chromogenic substrate, S-2765. Results (Fig. 7) showed a linear
response only with the intact dimer. No significant binding was
observed with the cleaved dimer or either light chain form. These
results suggested that the intact A1 subunit possesses an interactive
site for factor X. In a similar experiment, reaction of intact dimer
with saturating levels of the anti-FVIII polyclonal IgG (as judged by enzyme-linked immunosorbent assay)
prior to addition of factor X did not effect the binding of substrate
to A1/A3-C1-C2 (data not shown). This result suggested that the binding
site for factor X is not contained within the acidic C-terminal region
of A1, since this region would be blocked by antibody. Rather, the
presence of these residues may be required to maintain the conformation
of the factor X binding site.
Figure 7:
Binding of factor X to factor VIII-derived
proteins. Factor X was titrated into microtiter wells containing
immobilized A1/A3-C-C2 (closed squares),
A1/A3-C1-C2 (closed circles), factor VIII light
chain (open squares), and A3-C1-C2 (open circles).
Binding is presented as nM factor Xa formed following
conversion of the bound zymogen to active serine protease with RVV-X
and assay with S-2765.
Figure 8:
CD analysis of A1/A3-C1-C2
and A1/A3-C1-C2 dimers. The samples were prepared as described under
``Materials and Methods.'' Ellipticity is expressed in mean
residue ellipticity, degree
cm
dmol
for the truncated (curve A) and native (curve
B) dimer forms.
In this report, we examined the mechanism by which activated
protein C-catalyzed proteolysis of factor VIIIa abolishes its cofactor
activity in the intrinsic factor Xase. Cleavage of factor VIIIa does
not appear to significantly perturb the affinity of the cofactor for
enzyme. However, based on fluorescence anisotropy measurements using
Fl-FFR-FIXa, these cleavages alter the interaction of factor VIIIa
relative to the factor IXa active site. Furthermore, while A1/A3-C1-C2
dimer binds factor X in a solid phase binding assay, the
A1/A3-C1-C2 dimer exhibits a marked reduction in this
activity. Thus, proteolysis by activated protein C results in
inhibition of cofactor function at two levels, altered orientation with
enzyme and reduced affinity for substrate.
Activated protein C
catalyzes cleavage at two sites in factor VIIIa (9) . From time
course studies, Arg in the A2 subunit is cleaved
initially to yield two fragments of similar size representing residues
Ser
-Arg
(A2
) and
Gly
-Arg
(A2
). This
cleavage closely correlates with the loss of cofactor activity. While
cleavage at Arg
in the A1 subunit of the A1/A3-C1-C2
dimer lags behind cleavage at the A2 site, initial cleavage within A2
is not required for this event to occur. Thus the proteolytic events
are not stringently ordered. Cleavage at the A1 site liberates a
C-terminal acidic region and markedly weakens the affinity of the
A2/dimer interaction(9) . Analysis using a synthetic peptide
corresponding to this region
(Met
-Arg
) indicated that this segment
contains an A2 interactive site(19) . Since proteolysis of A2
requires its association with the A1/A3-C1-C2 dimer on the phospholipid
surface, cleavage at Arg
in the A1 subunit prior to
cleavage of A2 in some factor VIIIa molecules results in the
persistence of low levels of intact A2, as a result of it having
dissociated from the dimer.
While no activated protein C-catalyzed cleavage of the factor VIII light chain-derived subunit, A3-C1-C2, is observed(9) , this subunit is essential for efficient catalysis since it contains a binding site for the protease(16) . This site was mapped to the C-terminal end of the A3 domain in factor VIII as well as in factor V(17) . The A3-C1-C2 subunit also contains a high affinity site for factor IXa which probably lies N-terminal to the activated protein C site(27) . Both proteases apparently compete for binding to factor VIIIa. Using active site-modified proteases, we showed that activated protein C blocked the factor IXa-dependent enhancement of factor VIIIa reconstitution from isolated subunits, whereas factor IXa blocked the factor VIIIa-dependent fluorescence enhancement of dansyl Glu-Gly-Arg-modified activated protein C(30) . Mesters et al.(31) found that a peptide derived from activated protein C (residues 142-155) bound factor Va and inhibited both protease-catalyzed inactivation and factor Va-dependent clotting activity of factor Xa. This competition likely reflects the capacity of factors IXa and Xa to protect their respective cofactors from activated protein C(30, 32, 33, 34) .
Factor Va is
cleaved by activated protein C at multiple sites homologous to those in
factor VIIIa. Three sites are cleaved in the human factor Va heavy
chain, Arg, Arg
, and
Arg
(35) . Recent studies using a mutant factor V
where Arg
is replaced by Gln (36) indicated that
cleavage at the Arg
site does not contribute to
inactivation but rather is required for efficient cleavage at the
remaining sites. Furthermore, activated protein C cleaves the light
chain of bovine factor Va (at residue Arg
; see (37) ) yielding fragments of 30 and 48 kDa(38) . Thus,
subtle differences exist in both effect and location of proteolysis
following interaction of protease with the two cofactors.
Earlier studies examining the mechanism of activated protein C-catalyzed inactivation of bovine factor Va indicated that proteolysis affected cofactor binding to both factor Xa and prothrombin. Guinto and Esmon (39) showed that isolated factor Va heavy chain bound the immobilized prothrombin, whereas both chains were required for binding the factor Xa. However, following reactions with activated protein C, both of these functions were lost. Lucklow et al.(40) observed that the affinity of factor Va heavy chain for prethrombin 1 was reduced by at least 100-fold following reaction with activated protein C. Loss of factor Va affinity for prothrombin following reaction with activated protein C likely results from a conformational change rather than cleavage at a binding site based upon the observation that prothrombin failed to protect factor Va from proteolytic inactivation(32, 33) .
Isolation of
factor VIIIa products following reaction with activated protein C and
reconstitution studies have permitted examination of the contribution
of each cleavage to cofactor inactivation. Activated protein C cleavage
at Arg in the A1 subunit is functionally equivalent to
cleavage by factor IXa, which also attacks that site(41) . This
event results in weakened affinity for the A2 subunit, presumably due
to loss of the A2 interactive site contained within residues
337-372(19) . Furthermore, in this study we show that
this truncation of the A1 subunit markedly reduced the
A1/A3-C1-C2-dependent increase in anisotropy of Fl-FFR-FIXa. This
latter effect is likely the result of conformation changes in the A1
and/or A3-C1-C2 subunits yielding altered orientation of cofactor with
enzyme.
Results in the present study show minimal affect on affinity
for factor IXa following cleavage of factor VIIIa. The
A1/A3-C1-C2 dimer effectively inhibited the factor
IXa-dependent enhancement of factor VIIIa reconstitution. A somewhat
higher concentration of cleaved dimer was required to yield 50%
inhibition of factor IXa-dependent cofactor reconstitution from intact
dimer plus A2 subunit. This result may reflect a slightly reduced
affinity of dimer for factor IXa following cleavage at that site.
Alternatively, inhibitory activity may be reduced due to partial
denaturation during its purification (as suggested by its broad elution
peak). One reason for the apparent disparity with the analogous factor
Va-factor Xa interaction described above is that bovine factor Va light
chain is cleaved by activated protein C, whereas factor VIIIa A3-C1-C2
subunit is not. Since this subunit contains a high affinity site for
factor IXa(27) , cleavage within the homologous chain of factor
Va could perturb its binding factor Xa.
Factor VIIIa A1 subunit
likely contains a binding site for factor X. Factor X bound to the
immobilized A1/A3-C1-C2 dimer in a dose-dependent manner, whereas no
appreciable factor X binding was observed using
A1/A3-C1-C2. This result is consistent with the role of
factor Va heavy chain (contiguous A1-A2 domains) in binding the
substrates prothrombin (39) and prethrombin 1 (40) , as
well as loss of this function following reaction with activated protein
C. At present we cannot exclude direct participation of A3-C1-C2 in the
factor X interaction, since this activity may be perturbed by its
association with the antibody. The acidic C-terminal region of A1
probably does not directly contribute to binding, since prior reaction
of A1/A3-C1-C2 dimer with a polyclonal antibody to this segment had no
effect on subsequent reaction with factor X. Thus, loss of substrate
binding activity following A1 cleavage may result from a conformational
change triggered by loss of the acidic region. This conclusion is
supported by CD analysis showing reduced
-helix and/or
-sheet
content in the cleaved dimer. One possible explanation for the altered
conformation is that acidic residues in the C-terminal region of A1
form salt links with other residues in the A1/A3-C1-C2 dimer which
stabilize certain structural elements. Therefore, this segment in A1
subunit appears critical for cofactor activity in that it contributes
to the retention of A2 subunit (19) as well as modulates the
active conformation of the cofactor.
Although the A1/A3-C1-C2 dimer
causes incremental increases in the anisotropy of the FIXa active site
and the FX-dependent increase in this parameter, both values are
further increased when A2 subunit is present to reconstitute factor
VIIIa. However, purified A2/A2
fragments, in
the presence of A1/A3-C1-C2, neither reconstitute factor VIIIa activity
as judged by clotting assay nor modulate the A2-dependent incremental
increases in the anisotropy parameters. Abrogation of these
A2-dependent activities following cleavage at Arg
is
consistent with the sequence in and around the scissile bond
representing a factor IXa interactive site. Recent results from our
laboratory have shown that factor IXa selectively blocks activated
protein C-catalyzed cleavage at Arg
(30) .
Furthermore, synthetic peptides spanning this region inhibit factor
Xase activity and the capacity of factor IXa to stabilize factor
VIIIa(42) , as well as block the A2-dependent component of the
increase in the anisotropy of Fl-FFR-FIXa by factor VIIIa(43) .
Since this cleavage temporally precedes cleavage at the A1 site, these
results confirm the role of A2 cleavage as a primary cause for factor
VIIIa inactivation.