(Received for publication, May 11, 1995)
From the
Factor V was purified from the plasma of an activated protein C
(APC)-resistant patient who is homozygous for the mutation Arg
Gln (factor V
). Factor V
was converted by thrombin into factor Va which was further
purified yielding a factor Va preparation that had the same cofactor
activity in prothrombin activation as normal factor Va. Inactivation of
low concentrations of normal factor Va (<5 nM) by 0.15
nM APC in the presence of phospholipid vesicles proceeded via
a biphasic reaction that consisted of a rapid phase (k = 4.3
10
M
s
), yielding a
reaction intermediate with reduced cofactor activity that was fully
inactivated during the subsequent slow phase (k = 2.3
10
M
s
). Inactivation
of factor Va
proceeded via a monophasic reaction (k = 1.7
10
M
s
). Immunoblot
analysis showed that APC-catalyzed inactivation of factor Va occurred
via peptide bond cleavages in the heavy chain. The rapid phase of
inactivation of normal factor Va was associated with cleavage at
Arg
and full inactivation of factor Va required
subsequent cleavage at Arg
. The slow monophasic
inactivation of factor Va
correlated with cleavage at
Arg
. Cleavage at Arg
in normal factor Va
resulted in accumulation of a reaction intermediate that exhibited 40%
cofactor activity in prothrombin activation mixtures that contained a
high factor Xa concentration (5 nM). Compared with native
factor Va, the reaction intermediate retained virtually no cofactor
activity at low factor Xa concentrations (0.3 nM). This
demonstrates that factor Va that is cleaved at Arg
is
impaired in its ability to interact with factor Xa. Michaelis-Menten
kinetic analysis showed that cleavage at Arg
in
membrane-bound factor Va was characterized by a low K
for factor Va (20 nM) and k
= 0.96 s
. For
cleavage at Arg
in factor Va
the kinetic
parameters were K
= 196 nM and k
= 0.37 s
.
This means that differences between APC-catalyzed inactivation of
factors Va and Va
become much less pronounced at high
factor Va concentrations. When factor Va
was
inactivated by APC in the absence of phospholipids, cleavage at
Arg
of the heavy chain also contributed to factor Va
inactivation. Comparison of rate constants for APC-catalyzed cleavage
at Arg
, Arg
, and Arg
in the
absence and presence of phospholipids indicated that phospholipids
accelerated these cleavages to a different extent. This indicates that
the binding of factor Va to phospholipids changes the accessibility of
the cleavage sites and/or the sequence of peptide bond cleavage by APC.
Human blood coagulation factor Va is a heterodimeric
glycoprotein (1) that consists of a heavy chain (M = 105,000) associated via a single
Ca
ion with a light chain (M
= 74,000 or 72,000, cf. Ref 2). Factor Va is
formed during hemostasis from the inactive procofactor V after limited
proteolysis. Factor Va is an essential nonenzymatic cofactor of the
prothrombin-activating complex, which further comprises the serine
protease factor Xa, calcium ions, and a procoagulant membrane surface.
Depending on the reaction conditions factor Va accelerates prothrombin
activation 10
-10
-fold (3, 4, 5) .
Proteolytic inactivation of
factor Va by activated protein C (APC), ()is one of the key
reactions in the regulation of thrombin formation. APC-catalyzed
cleavage of factor Va is stimulated by the presence of negatively
charged membrane surfaces (6, 7, 8) and by
the protein cofactor, protein S (8, 9, 10) .
The loss of cofactor activity of factor Va is associated with peptide
bond cleavages in the heavy chain of factor Va at Arg
,
Arg
, and Arg
(11, 12) . The
physiologic importance of the down-regulation of factor Va activity by
APC is underscored by the observation of recurrent thromboembolic
events in individuals that are deficient in either protein C or protein
S(13, 14, 15) .
Since a first publication
by Dahlbäck et al.(16) , several
laboratories have associated the occurrence of familial thrombophilia
in a large group of patients with a poor anticoagulant response to APC
(APC resistance). APC resistance is at least 10 times more common than
all other known genetic thrombosis risk factors together and has an
allelic frequency of 2% in the Dutch population (17) . The
molecular defect in APC-resistant patients was recently demonstrated (18, 19, 20, 21) to be linked to a
single-point mutation in the factor V gene that causes an amino acid
substitution (Arg
Gln) at a site that is cleaved
during APC-catalyzed inactivation of factor Va(11) .
In this
paper we present a kinetic analysis of APC-catalyzed inactivation of
human factor Va that was either obtained from normal plasma or from the
plasma of a patient who is homozygous for the mutation (Arg
Gln). Parallel experiments were performed in which changes
of functional activity of factor Va during inactivation by APC were
correlated with the cleavage of peptide bonds in the factor Va
molecule. This study was undertaken in order to elucidate the
consequences of the molecular defect in factor V for the regulation of in vivo prothrombin activation.
The kinetic parameters (K and V
) for factor Xa-catalyzed prothrombin
activation were determined by measuring the rate of thrombin formation
at varying prothrombin concentrations in the presence of a fixed
phospholipid concentration, a limiting amount of factor Xa, and a
saturating concentration of factor Va as described in the previous
paragraph. The kinetic parameters were obtained by fitting the data to
the Michaelis-Menten equation using non-linear least squares regression
analysis.
in which factor Va is a reaction intermediate with
partial cofactor activity, factor Va
is a reaction product
that has no cofactor activity, k
is the
second-order rate constant for cleavage of peptide bond 1, k
is the second-order rate constant for cleavage
of peptide bond 2 in factor Va
, and k
is the second-order rate constant for cleavage of peptide bond 2
in native factor Va.
Under first order conditions, i.e. conditions at which the inactivation rate is directly proportional to the factor Va and APC concentrations, the loss of cofactor activity is described by (38) :
in which Va is the cofactor activity
determined at time t, Va
is the cofactor activity
determined before APC is added, B is the cofactor activity of factor
Va
(expressed as fraction of the cofactor activity of
native factor Va), and k
, k
,
and k
are the rate constants defined above. The
rate constants and the cofactor activity of factor Va
were obtained by fitting the data to using
non-linear least squares regression analysis.
In the case of normal
factor Va inactivation, k appeared to be
associated with cleavage of the peptide bond at Arg
.
Therefore, time courses of normal factor Va inactivation were fitted
with a fixed k
, obtained from kinetic analysis of
factor Va
inactivation.
In some experiments (factor
Va inactivation in free solution) there was a slow loss of factor Va
cofactor activity in the absence of APC. In these cases the spontaneous
loss of cofactor activity (0.4%/min) was added to k.
Figure 1:
Schematic representation of APC
cleavage sites in the heavy chain of factor Va. The heavy chain of
human factor Va contains three peptide bonds that can be cleaved by APC
and that are located at Arg, Arg
, and
Arg
. The molecular weights of possible cleavage products
correspond to those given in other publications (11, 12) .
Factor Va and
factor Va had similar cofactor activities in
prothrombin activation. Kinetic analysis showed that prothrombin was
activated in the presence of 25 µM phospholipid vesicles
(DOPS/DOPC, 10/90, mol/mol) with the following kinetic parameters: K
for prothrombin = 0.13
µM, k
= 8570
min
and K
of Xa-Va complex
formation = 0.083 nM for prothrombinase complexes with
normal factor Va and K
for prothrombin
= 0.12 µM, k
= 8450
min
and K
of Xa-Va complex
formation = 0.105 nM for prothrombinase complexes with
factor Va
(data not shown).
This means that the
factor Va assay that we used, which measures the cofactor activity of
factor Va in prothrombin activation, allows quantitative comparison of
factor Va and factor Va.
Figure 2:
Inactivation of factor Va and factor
Va by APC in the presence of phospholipids. 0.3 nM purified human factor Va (
) or factor Va
(
) was incubated with (A) 0.15 nM APC or (B) 1.5 nM APC and 25 µM phospholipid
vesicles (DOPS/DOPC, 10/90, mol/mol) in 25 mM Hepes (pH 7.5),
175 mM NaCl, 3 mM CaCl
, and 5 mg/ml BSA
at 37 °C. At the indicated time points factor Va activity was
determined as described under ``Experimental Procedures.''
The solid lines represent exponential curves obtained after
fitting the data using given under ``Experimental
Procedures.'' The rate constants and activity of the reaction
intermediate were determined by nonlinear least-squares regression of
the data.
The
time courses of factor Va and Va inactivation (% factor
Va activity versus time) were not affected by varying the
concentrations of factor Va or Va
between 0 and 5
nM. Since the rates of factor Va inactivation were also
directly proportional to the amount of APC present (0.05-5
nM) we concluded that factor Va and Va
inactivation were first-order in both factor
Va(Va
) and APC. This allows calculation of apparent
second-order rate constants for factor Va inactivation from time
courses such as shown in Fig. 2.
On the basis of literature
data (11, 12) and experiments presented below it can
be postulated that the biphasic time courses of inactivation of normal
factor Va presented in Fig. 2result from rapid cleavage at
Arg yielding a reaction intermediate that possesses
partial cofactor activity in prothrombin activation. This reaction
intermediate is fully inactivated by subsequent slow cleavage at
Arg
. Inactivation of factor Va
likely
proceeds via a monophasic reaction that is the result of slow cleavage
at Arg
.
The time courses of inactivation could indeed
be fitted (Fig. 2, solid lines) using an equation that
describes the time-dependent loss of cofactor activity when factor Va
is inactivated via the random ordered two-step reaction model described
under ``Experimental Procedures.'' The fits of the particular
experiment presented in Fig. 2yielded the following
second-order rate constants: k = 4.7
10
M
s
, k
= 1.7
10
M
s
, and 40% cofactor activity for the reaction
intermediate (factor Va cleaved at Arg
) in the case of
normal factor Va inactivation and k
= 1.4
10
M
s
and a reaction product that has virtually lost its cofactor
activity in the case of factor Va
inactivation.
Figure 3:
Immunoblot analysis of factor Va and
factor Va inactivation by APC in the presence of
phospholipids. A, 6 nM purified human factor Va and 1
nM APC; or B, 15 nM factor Va
and 5 nM APC were incubated with 25 µM phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol) in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM
CaCl
, and 0.1 mg/ml BSA at 37 °C. In the case of normal
factor Va (A) the APC concentration was increased to 110
nM after 5 min. At the indicated time intervals samples from
the inactivation mixture were subjected to SDS-PAGE. After transfer to
Immobilon-P membranes, the heavy chain and derived fragments were
visualized with a polyclonal antibody directed against the heavy chain
of factor Va. The positions of the molecular weight markers are
indicated at the left of panels A and B. The
percentages cofactor activity at the indicated time points were (A) 100, 63, 46, 40, 25, 19, 17, and 0% and (B) 100,
80, 67, 43, 12, 3, 2, and 1%. Further experimental details are
described under ``Experimental
Procedures.''
During the initial phase of inactivation
of normal factor Va, reaction products with M of
75,000 and 26,000/28,000 were formed, which is indicative of cleavage
at Arg
(Fig. 3A). Prolonged incubation of
factor Va with a high APC concentration resulted in the disappearance
of the M
= 75,000 reaction intermediate
(cleavage at Arg
) and formation of reaction products with M
of 45,000 and 30,000, respectively. The fragment
with M
= 30,000 stains poorly with our
polyclonal antibody and was hardly visible on the gel. During the final
stage of the inactivation reaction the peptide bond at Arg
was also cleaved, as is indicated by the disappearance of the M
= 26,000/28,000 doublet and the formation
of low molecular weight products migrating with the dye front in the
gel.
The immunoblot of the time course of APC-catalyzed inactivation
of membrane-bound factor Va (Fig. 3B)
differs from that of normal factor Va. Inactivation of factor
Va
correlated with the appearance of only two reaction
products with M
= 60,000/62,000 and M
= 45,000, which corresponds with cleavage
at Arg
. The immunoblot further demonstrates that cleavage
at Arg
did not significantly contribute to factor
Va
inactivation since no additional bands became
visible in the heavy chain or M
=
54,000/56,000 region of the immunoblot. This would have been expected
if cleavage at Arg
had occurred.
The immunoblot
analysis supports the model for APC-catalyzed proteolysis of factor Va
presented in the previous section. The rapid phase of the time course
of inactivation of normal factor Va is associated with cleavage at
Arg and the subsequent slow phase is due to cleavage at
Arg
. Inactivation of factor Va
proceeds
via a monophasic reaction which is explained by cleavage at
Arg
.
Figure 4:
Inactivation of factor Va and factor
Va by APC in the absence of phospholipids. 19 nM purified human factor Va (
) or factor Va
(
) was incubated with 390 nM APC in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM
CaCl
, and 5 mg/ml BSA at 37 °C. At the indicated time
points factor Va activity was determined as described under
``Experimental Procedures.'' The solid lines represent exponential curves obtained after fitting the data using given under ``Experimental Procedures.'' The
rate constants and activity of the reaction intermediate were
determined by nonlinear least-squares regression analysis of the
data.
To correlate the loss of cofactor activity with
peptide bond cleavages in the heavy chain we analyzed the product
generation pattern by immunoblot analysis of samples taken from the
inactivation mixture at selected time intervals (Fig. 5).
Compared with the peptide bond cleavage pattern of membrane-bound
factor Va a striking difference was observed. In the absence of
phospholipids cleavage at Arg occurred at appreciable
rates in factor Va
(Fig. 5B). This is
concluded from the time-dependent appearance of an additional band in
the heavy chain region of the gel.
Figure 5:
Immunoblot analysis of factor Va and
factor Va inactivation by APC in the absence of
phospholipids. A, 40 nM purified human factor Va and
15 nM APC, or B, 40 nM factor Va
and 500 nM APC were incubated in 25 mM Hepes
(pH 7.5), 175 mM NaCl, 3 mM CaCl
, and 0.2
mg/ml BSA at 37 °C. In the case of normal factor Va (A)
the APC concentration was increased to 500 nM APC after 35
min. At the indicated time intervals samples from the inactivation
mixture were subjected to SDS-PAGE. After transfer to Immobilon-P
membranes, the heavy chain and derived fragments were visualized with a
polyclonal antibody directed against the heavy chain of factor Va. The
positions of the molecular weight markers are indicated at the left of panels A and B. The percentages cofactor activity at
the indicated time points were (A) 100, 88, 71, 59, 39, 24 and
1% and (B) 100, 95, 85, 69, 46, 34, 18, and 6%. Further
experimental details are described under ``Experimental
Procedures.''
Thus, it appears that in the case
of factor Va inactivation proteolysis at Arg
and Arg
contributed to factor Va inactivation and
that cleavage at Arg
preceded cleavage at
Arg
. Fitting the time course of APC-catalyzed factor
Va
inactivation (Fig. 4, solid lines)
to the equation for a biphasic reaction with a partially active
intermediate yields rate constants of 8.0
10
M
s
and 1.0
10
M
s
for k
and k
,
respectively, and 70% cofactor activity for the reaction intermediate
that has been cleaved at Arg
.
Immunoblot analysis (Fig. 5A) indicates that during the time courses of
normal factor Va inactivation (Fig. 4) only peptide bond
cleavages at Arg and Arg
significantly
contributed to the loss of factor Va cofactor activity. Inspection of
the immunoblot that represents inactivation of normal factor Va reveals
that cleavage at Arg
had a minor contribution to the loss
of factor Va cofactor activity. As a first approximation we have,
therefore, also fitted this time course with a biphasic exponential.
This fit (Fig. 4, solid line) yielded rate constants of
1.3
10
M
s
and 4.0
10
M
s
for cleavage at Arg
and
Arg
, respectively, and a reaction intermediate with 40%
cofactor activity. It should be emphasized that the values of the
latter rate constants may be slightly overestimated due to the
assumption that cleavage at Arg
did not contribute to
inactivation.
The data
presented in Table 1show that phospholipids stimulated the
APC-catalyzed peptide bond cleavages in factor Va to a different
extent. Phospholipids appeared to have no effect on k. Furthermore, it is clear that cleavage at
Arg
in free factor Va
occurred at a lower
rate than in normal factor Va. For membrane-bound factor Va
and factor Va this difference was annulated. These observations
are indicative for specific effects of phospholipids and/or prior
cleavage at Arg
on the cleavages at Arg
and
Arg
(see ``Discussion'').
The Lineweaver-Burk plots presented
in Fig. 6show that cleavage at Arg was
characterized by a K
of 20 nM and a k
of 0.96 s
.
Inactivation of factor Va
, which corresponds with
cleavage at Arg
, had a high K
(196 nM) and a k
of 0.37
s
. Theoretically the k
/K
obtained by
Michaelis-Menten analysis should equal the second-order rate constant
calculated from a time course of inactivation determined at low factor
Va concentrations (Fig. 2). Indeed the k
/K
values
determined for cleavage at Arg
(4.8
10
M
s
) and
Arg
(1.9
10
M
s
) closely resemble the rate constants
obtained at pseudo-first order reaction conditions (Table 1).
This shows that the kinetic parameters, K
and k
, are indeed associated with the
indicated peptide bond cleavages. The data further demonstrate that the
differences between the rate constants of cleavage at Arg
and Arg
are mainly due to differences between the K
values for these peptide bond
cleavages.
Figure 6:
Lineweaver-Burk plots for inactivation of
factor Va and factor Va by APC in the presence of
phospholipids. Initial rates of factor Va inactivation were determined
at varying concentrations of factor Va (
) or factor Va
(
) that were incubated with 0.28 nM APC (
)
or 1 nM APC (
) and 25 µM phospholipid
vesicles (DOPS/DOPC, 10/90, mol/mol) in 25 mM Hepes (pH 7.5),
175 mM NaCl, 3 mM CaCl
, and 5 mg/ml BSA
at 37 °C. After different time intervals the factor Va activity was
determined as described under ``Experimental Procedures'' and
initial rates of factor Va inactivation were expressed as nM factor Va inactivated/min/nM APC (assuming that normal
factor Va is converted into an intermediate with 40% cofactor
activity). The solid lines represent a fit of the data
according the Michaelis-Menten equation with K
= 20 nM and V
= 57.6 nM factor Va inactivated/min/nM APC (k
= 0.96 s
)
for factor Va (
) and K
=
196 nM and V
= 22.2 nM factor Va
inactivated/min/nM APC (k
= 0.37 s
) for
factor Va
(
).
The actual cofactor activity of factor Va is dependent on its
interaction with procoagulant membranes, factor Xa, prothrombin, and on
its ability to increase the catalytic activity (k) of factor
Xa(2, 3, 4, 5, 40) . To
examine which of these functions was impaired in factor Va that is
cleaved at Arg
, we have characterized the functional
properties of the inactivation intermediate. To this end normal factor
Va was inactivated by APC in the absence of phospholipids (cf. Fig. 4) and after completion of the first phase of inactivation
(Arg
cleavage) the reaction mixture was diluted and
chromatographed on a Mono S column. The reaction intermediate bound to
Mono S and eluted in a NH
Cl gradient as an overlapping
doublet between 700 and 800 mM NH
Cl. These column
fractions contained almost pure inactivation intermediate (Fig. 7B).
Figure 7:
Cofactor activities of factor Va and
factor Va cleaved at Arg. Purified human factor Va (40
nM) was incubated with 15 nM APC in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM
CaCl
, and 5 mg/ml BSA at 37 °C. After 45 min the
inactivation mixture was diluted 10-fold and applied to a Mono S
column. Bound protein was eluted with 20 ml of a linear
NH
Cl gradient (50-1000 mM) in 25 mM Hepes (pH 7.5). Immunoblot analysis of factor Va (A) and
the fraction eluting between 700 and 800 mM NH
Cl (B) is shown in the inset. Aliquots taken from the
inactivation mixture after 0 min (native factor Va) and from the column
fraction were used to determine rates of prothrombin activation at (A) 1.1 ng/ml factor Va (
) or (B) 1.2 ng/ml
factor Va cleaved at Arg
(
) in a reaction mixture
containing 25 µM phospholipid vesicles (DOPS/DOPC, 10/90,
mol/mol), 0.5 µM prothrombin, and varying concentrations
of factor Xa in 25 mM Hepes (pH 7.5), 175 mM NaCl, 2
mM CaCl
, and 5 mg/ml BSA at 37 °C. Rates of
prothrombin activation were determined as described under
``Experimental Procedures'' and were corrected for thrombin
formed in the absence of factor Va. The solid lines represent hyperbolas obtained after fitting the data with K
= 0.088 nM and V
= 50.2 nmol of IIa/min/µg of factor
Va (k
= 151 s
) for
factor Va (
) and K
= 3.9 nM and V
= 28.7 nmol of IIa/min/µg
of protein (k
= 72 s
)
for the column fraction (factor Va cleaved at Arg
)
(
). Inset, double reciprocal plots of the same
data.
To compare the abilities of factor Va and
the purified inactivation intermediate to assemble with factor Xa into
a membrane-bound prothrombinase complex we determined initial
steady-state rates of prothrombin activation at limiting amounts of
factor Va or factor Va cleaved at Arg as a function of
the factor Xa concentration (Fig. 7, A and B).
Low amounts of factor Xa were required for full expression of
prothrombinase activity in the case of normal factor Va (K
= 0.088 nM). With factor Va
that was cleaved at Arg
much higher factor Xa
concentrations (K
= 3.9 nM) were
needed to obtain maximal prothrombinase activity. The V
of prothrombin activation obtained with the
purified inactivation intermediate was lower than that determined for
native factor Va. The V
values could be
converted into k
values on the basis of the
protein contents of the titrated fractions. This yielded k
values of 153 and 96 s
for
native and cleaved factor Va, respectively.
The interaction of the
membrane-bound factor Xa-Va complex with prothrombin was not affected
by cleavage at Arg since the K
values for prothrombin were virtually the same for
prothrombinase complexes with normal and cleaved factor Va (data not
shown).
From these data we conclude that APC-catalyzed cleavage at
Arg of the heavy chain of factor Va results in the
formation of a reaction product that has lost part of its cofactor
activity mainly because of a reduction of its affinity for factor Xa.
We have also shown (Fig. 7) that the relative
cofactor activities of native factor Va and factor Va cleaved at
Arg strongly depend on the factor Xa concentration
present in the factor Va assay. It was demonstrated that factor Va that
is cleaved at Arg
will exhibit much lower cofactor
activity than native factor Va when assayed at factor Xa concentrations
below K
(3.9 nM).
These observations
predict that the observed time courses of factor Va or factor
Va inactivation by APC will depend: 1) on the factor Va
concentration in the inactivation mixture and 2) on the assay
conditions at which the loss of cofactor activity of factor Va is
followed. Compared with factor Va
, normal factor Va
will be more rapidly inactivated at low than at high factor Va
concentrations. The apparent loss of cofactor activity of normal factor
Va will be even more rapid and approach complete loss of activity much
faster when samples from the inactivation mixture are assayed at a
factor Xa concentration at which the inactivation intermediate has no
activity, i.e. [factor Xa] 3.9 nM (K
).
These predictions were confirmed by
an experiment in which time courses of factor Va inactivation were
determined at low (Fig. 8A) or high concentrations of
factor Va or Va (Fig. 8B) and in which
samples from the same inactivation mixture were assayed for the loss of
cofactor activity in assay mixtures that contained a low (Fig. 8A) or a high factor Xa concentration (Fig. 8B). As predicted, the differences in
APC-catalyzed loss of cofactor activity of normal factor Va and factor
Va
were most pronounced when factor Va was inactivated
at a low concentration and assayed in a prothrombin activation mixture
that contained a low factor Xa concentration (Fig. 8A).
Differences in time courses of inactivation of factor Va and factor
Va
were indeed greatly reduced when high factor Va
concentrations were inactivated by APC and the loss of cofactor
activity was monitored in prothrombin activation mixtures that
contained a high factor Xa concentration (Fig. 8B).
Figure 8:
The
effect of assay conditions and factor Va concentration on the loss of
cofactor activity during factor Va inactivation by APC. Purified human
factor Va () or factor Va
(
) was incubated
with APC and phospholipid vesicles in 25 mM Hepes (pH 7.5),
175 mM NaCl, 3 mM CaCl
, and 5 mg/ml BSA
at 37 °C. At the indicated time points factor Va activity was
determined as described under ``Experimental Procedures'' in
assay mixtures that contained 10 nM or 0.4 nM factor
Xa. A, inactivation mixture: 0.8 nM factor Va
(
) or 0.8 nM factor Va
(
), 0.15
nM APC, 25 µM phospholipid vesicles (DOPS/DOPC,
10/90, mol/mol); cofactor assay mixture: 0.4 nM factor Xa. B, inactivation mixture: 210 nM factor Va (
)
or 210 nM factor Va
(
), 2.7 nM APC, 25 µM phospholipid vesicles (DOPS/DOPC, 10/90,
mol/mol); cofactor assay mixture: 10 nM factor
Xa.
Proteolytic inactivation of membrane-bound factor Va by APC
requires cleavage of two peptide bonds located at Arg and
Arg
in the heavy chain domain of factor Va (Fig. 1). At low factor Va concentrations cleavage at
Arg
occurs at a rate that is approximately 20-fold higher
than the rate of cleavage at Arg
. This results in the
accumulation of a reaction intermediate that is cleaved at Arg
and that exhibits partial cofactor activity in prothrombin
activation (Fig. 2). Cleavage at Arg
, which may
occur both in the reaction intermediate and in native factor Va (see
below), results in a complete loss of cofactor activity.
The
cofactor activity of the factor Va derivative cleaved at Arg strongly depends on the factor Xa concentration in the
prothrombin activation mixture in which factor Va is assayed. Cofactor
activity of the reaction intermediate in prothrombin activation is only
observed at high factor Xa concentrations. Compared with native factor
Va, full expression of cofactor activity of factor Va that is cleaved
at Arg
requires a 45-fold higher factor Xa concentration.
This indicates that cleavage at Arg
in the heavy chain of
factor Va affects its interaction with factor Xa and results in a
considerable loss of affinity for factor Xa.
Factor Va obtained from factor V purified from the plasma of a homozygous
APC-resistant patient is slowly inactivated by APC (cf. Ref
12). This factor Va molecule lacks the cleavage site at
Arg
, due to replacement of Arg by
Gln(18, 19, 20, 21) . Time courses
of inactivation of low concentrations of membrane-bound factor
Va
could be fitted with an equation for a single
exponential curve. Immunoblotting experiments (Fig. 3B)
show preferential cleavage at Arg
during the inactivation
of membrane-bound factor Va
. These data indicate that
APC inactivates factor Va
by a single cleavage at
position Arg
which results in the formation of a reaction
product that has virtually lost its cofactor activity in prothrombin
activation.
Under the conditions of our experiments (low factor Va
concentrations) cleavage at Arg has a negligible
contribution to the loss of cofactor activity in the case of
APC-catalyzed inactivation of membrane-bound factor Va or factor
Va
. This is concluded from the fact that cleavage at
Arg
(i.e. disappearance of the M
= 26,000/28,000 fragment or appearance of doublet bands in
the heavy chain or the M
= 54,000/56,000
region of immunoblots) is only observed after time intervals at which
more than 90% of the cofactor activity of factor Va is already lost.
These data suggest that inactivation of membrane-bound factor Va and
factor Va by APC proceeds via the pathways depicted
below.
In this scheme factor Va exhibits partial cofactor
activity and factor Va
is a reaction product that has no
detectable cofactor activity. We propose that APC-catalyzed cleavage at
Arg
and Arg
in membrane-bound factor Va
occurs in a random fashion and that cleavage at Arg
is
not affected by prior cleavage at Arg
. This is inferred
from the fact that the rate constants for cleavage at Arg
calculated from time courses of inactivation of normal and
APC-resistant factor Va are approximately the same (k
is 2.3
10
M
s
and 1.7
10
M
s
for normal Va
and factor Va
, respectively).
Michaelis-Menten
analysis provided additional information regarding the mechanism that
accounts for the observed difference between k and k
. The kinetic parameters determined
for cleavage at Arg
in normal factor Va were: K
for factor Va = 20 nM and k
= 0.96 s
.
The peptide bond at Arg
in factor Va
was
cleaved with K
= 196 nM and k
= 0.37 s
.
The fact that the calculated catalytic efficiencies (k
/K
) have the
same values as the second-order rate constants calculated for cleavage
at Arg
in normal factor Va and for cleavage at
Arg
in factor Va
verifies that the
Michaelis-Menten kinetic parameters are indeed associated with the
indicated peptide bond cleavages. These data suggest that the
differences in rate constants for cleavage at Arg
and
Arg
mainly result from differences in the K
and explain why at high factor Va
concentrations (>K
) differences
between time courses of factor Va and factor Va
inactivation are much less pronounced (Fig. 8B).
APC can also fully inactivate factor Va and factor Va in the absence of a membrane surface (Fig. 4). Under these
conditions cleavage at Arg
also contributes to the loss
of factor Va cofactor activity. This is particularly observed for
factor Va
, in which the peptide bond at Arg
is even cleaved at a 8-fold higher rate than the site at
Arg
. Cleavage at Arg
does not require prior
cleavage at Arg
or Arg
since immunoblot
analysis shows the appearance of a doublet band in the M
= 105,000 region of the gel, which indicates that cleavage
at position Arg
can already occur in the intact heavy
chain.
Comparison of rate constants obtained for free and
membrane-bound factor Va shows that phospholipids greatly accelerate
APC-catalyzed factor Va inactivation. Part of this stimulation will
presumably be due to the fact that both the substrate (factor Va) and
the enzyme (APC) bind to the membrane surface, a condition that
promotes their interaction and reaction in a way similar to that of
phospholipid-dependent coagulation factor
activations(4, 5, 8, 40, 41, 42) .
However, in the case of APC-catalyzed factor Va inactivation,
phospholipids do not accelerate the peptide bond cleavages to the same
extent (Table 1). Cleavage at Arg and Arg
in normal factor Va is stimulated 300-600-fold, whereas
cleavage at Arg
in factor Va
is enhanced
1800-fold by phospholipids. This is indicative of a specific effect of
phospholipids on APC-catalyzed cleavage at Arg
(cf. (11) and (12) ). We observed that k
is not significantly increased in
membrane-bound factor Va. This demonstrates that after binding of
factor Va to phospholipids the peptide bond at position Arg
is hardly accessible to cleavage by membrane-bound APC.
Taken
together our conclusions seem to contradict earlier observations of
Kalafatis et al.(11, 12) who reported that
APC-catalyzed cleavages in the heavy chain of membrane-bound factor Va
occur in an ordered, sequential fashion in which cleavage at
Arg promotes cleavage at Arg
and
Arg
. They also suggested that cleavage at Arg
does not affect the cofactor activity of normal factor Va and
that cleavage at position Arg
is required for full
inactivation of factor Va. These apparent discrepancies may be
explained by our observations that: 1) the relative rates of peptide
bond cleavages in the heavy chain of factor Va depend on the factor Va
concentration present in the inactivation mixture (Fig. 6) and
2) that the residual cofactor activity of partially active reaction
intermediates strongly depends on the reaction conditions at which the
functional activity of factor Va and its degradation products is
assayed ( Fig. 7and Fig. 8). In this respect it is
essential to mention that Kalafatis et al.(11, 12) collected their data at high factor Va
concentrations (>200 nM) and used a factor Va assay system
with high concentrations of coagulation factors, while our conclusions
are based on experiments at low factor Va concentrations (<10
nM) and assessment of cofactor activity of factor Va in
prothrombin activation mixtures that contained varying concentrations
of coagulation factors.
To emphasize the importance of reaction and
assay conditions, we demonstrated that the apparent loss of factor Va
cofactor activity during in activation by APC indeed strongly depends
on the concentration at which factor Va is inactivated and on the
prothrombinase conditions at which the loss of cofactor activity is
monitored (Fig.8). Time courses of factor Va and factor Va inactivation at a high factor Va concentration, in which the loss
of cofactor activity of factor Va is followed in prothombin activation
mixtures that contain a high factor Xa concentration, show relatively
little difference (Fig. 8B and (12) ). However,
when factor Va is inactivated at a low concentration and when its
cofactor activity is assayed in a prothrombin activation mixture that
contains a low amount of factor Xa, the differences between factor Va
and factor Va
are profound (Fig. 8A).
This observation may have significant physiological consequences. If
thrombotic events result from ongoing coagulation at low concentrations
of factor Xa and factor Va (the plasma factor V concentration is
25 nM), down-regulation of the cofactor activity of
factor Va by APC will be most efficient in the case of normal factor Va
and will be maximally impaired in case of factor Va
.
This underscores the increased risk for venous thromboembolism in
individuals whose plasmas are APC-resistant as a consequence of a
factor V phenotype in which Arg
is substituted by Gln.