(Received for publication, July 6, 1995)
From the
Inactivation of membrane-bound factor Va by activated protein C
(APC) proceeds via a biphasic reaction that consists of a rapid and a
slow phase, which are associated with cleavages at Arg and Arg
of the heavy chain of factor Va,
respectively. We have investigated the effects of protein S and factor
Xa on APC-catalyzed factor Va inactivation. Protein S accelerates
factor Va inactivation by selectively promoting the slow cleavage at
Arg
(20-fold). Factor Xa protects factor Va from
inactivation by APC by selectively blocking cleavage at
Arg
. Inactivation of factor Va
, which was
isolated from the plasma of a homozygous APC-resistant patient and
which lacks the Arg
cleavage site, was also stimulated by
protein S but was not affected by factor Xa. This confirms that the
target sites of protein S and factor Xa involve Arg
and
Arg
, respectively. Factor Xa completely blocked
APC-catalyzed cleavage at Arg
in normal factor Va (1
nM) with a half-maximal effect (K
) at 1.9 nM factor Xa.
Expression of cofactor activity of factor Va in prothrombin activation
required much lower factor Xa concentrations (K
= 0.08 nM). When
the ability of factor Xa to protect factor Va from inactivation by APC
was determined at low factor Va concentrations during prothrombin
activation much lower amounts of factor Xa were required (K
= 0.03 nM). This
indicates 1) that factor Va is optimally protected from inactivation by
APC by incorporation into the prothrombinase complex during ongoing
prothrombin activation, and 2) that the formation of a catalytically
active prothrombinase complex and protection of factor Va from
inactivation by APC likely involves the same interaction of factor Xa
with factor Va. In accordance with the proposed mechanisms of action of
protein S and factor Xa, we observed that the large differences between
the rates of APC-catalyzed inactivation of normal factor Va and factor
Va
were almost annihilated in the presence of factor Xa
and protein S. This observation may explain why, in the absence of
other risk factors, APC resistance only results in a weak prothrombotic
condition.
Activated protein C (APC) ()plays an important role
in the down-regulation of thrombin formation (1) . The
anticoagulant action of APC is associated with its ability to degrade
the activated cofactors of the coagulation cascade, factors Va (2, 3) and VIIIa(4, 5) , by limited
proteolysis. The mechanism of proteolytic inactivation of factor Va by
APC is rather well documented. Human factor Va loses its cofactor
activity in prothrombin activation after peptide bond cleavages in the
heavy chain(6) . Sequential cleavage at Arg
and
Arg
of the heavy chain appears to be the major pathway of
APC-catalyzed factor Va
inactivation(6, 7, 8) .
Optimal expression of anticoagulant activity of APC requires the presence of negatively charged phospholipids (3, 9, 10) and the protein cofactor, protein S(10, 11, 12) . The mechanism of action of protein S in factor Va inactivation is as yet not fully understood. In reaction systems with purified coagulation factors, protein S is a weak stimulator of APC-catalyzed factor Va inactivation(10, 12) . It has been suggested (12) that under physiological conditions protein S may act by annihilating the ability of factor Xa to protect factor Va from inactivation by APC(3, 9, 13, 14) .
The importance of protein C and protein S in the regulation of
hemostatic plug formation is demonstrated by the association between
thrombosis and protein C or protein S
deficiency(15, 16, 17) . Recently, it has
been reported (18, 19, 20, 21) that
a hereditary defect that results in a poor anticoagulant response to
APC (APC resistance) also is a basis for familial thrombophilia. APC
resistance is associated with a single point mutation in the factor V
gene that results in the replacement of Arg by
Gln(22, 23, 24, 25) . This
replacement affects the peptide bond in factor Va that has to be
cleaved by APC in order to obtain a rapid loss of factor Va cofactor
activity(6, 7, 8) .
In the present study
we have determined the effects of protein S and factor Xa on apparent
second order rate constants for APC-catalyzed inactivation of normal
factor Va and of factor Va that was purified from the plasma of a
patient who is homozygous for the mutation Arg
Gln. The loss of functional activity of factor Va was correlated with
the effects of protein S and factor Xa on the cleavage of
APC-susceptible peptide bonds in the heavy chain of factor Va by
immunoblot analysis.
in which factor Va is a reaction intermediate with
partial cofactor activity, factor Va
is a form of factor Va
that has completely lost its cofactor activity, k
is the second order rate constant for cleavage at
Arg
, k
is the second order rate
constant for cleavage at Arg
in factor Va
,
and k`
is the second order rate constant for
cleavage at Arg
in native factor Va.
Under first order conditions, i.e. conditions at which the inactivation rate is directly proportional to the concentrations factor Va and APC, the loss of cofactor activity is described by (43) ,
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. Time courses of
inactivation of normal factor Va were fitted with a fixed k`
, that was obtained from kinetic analysis of
factor Va
inactivation (cf. (8) ).
In some experiments (factor Va inactivation in the presence of high
factor Xa concentrations), there was a slow loss of factor Va cofactor
activity in the absence of APC. In these cases the rate constant for
the loss of cofactor activity in the absence of APC was determined in a
separate experiment and added to k`.
Figure 1:
APC
cleavage sites in the heavy chain of factor Va. The peptide bonds
located at Arg, Arg
, and Arg
in the heavy chain of human factor Va are susceptible to
proteolytic cleavage by APC. The molecular weights of possible cleavage
products correspond to those given in other
publications(6, 7) .
Figure 2:
Effect of protein S on APC-catalyzed
inactivation of membrane-bound factor Va. Purified human factor Va (1
nM) was incubated with 0.2 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 in the absence
(
) or presence of 490 nM protein S (
). At the
indicated time points, factor Va activity was determined as described
under ``Experimental Procedures.'' The rate constants and the
activity of the reaction intermediate were obtained from the best fit
of the data (solid lines) to given under
``Experimental Procedures'' using nonlinear least-squares
regression analysis.
The proposed mode of action of protein S was
confirmed by immunoblot analysis of heavy chain degradation during
factor Va inactivation (Fig. 3). When factor Va was inactivated
by APC in the absence of protein S, there was considerable accumulation
of a transient reaction product with M =
75,000 and formation of a M
= 26,000/28,000
doublet, which are generated as the result of rapid cleavage at
Arg
(Fig. 3A). The reaction intermediate
with M
= 75,000 was slowly converted into
products with M
= 45,000 and M
= 30,000, which is indicative for
cleavage at Arg
. The reaction product with M
= 30,000 was hardly visible on the gel
since it stains poorly with our polyclonal antibody against the heavy
chain.
Figure 3:
Immunoblot analysis of APC-catalyzed
inactivation of membrane-bound factor Va with and without protein S.
Purified human factor Va (7 nM) was incubated with 1.1 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 0.2 mg/ml BSA at 37 °C in the
absence (panel A) or presence of 1 µM protein S (panel B). 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 left of panels A and B. The
percentages of cofactor activity at the indicated time points were 100,
73, 56, 45, 30, 25, 23, and 0% (A) and 100, 32, 12, 6, 3, 2,
1, and 0% (B). Further experimental details are described
under ``Experimental
Procedures''.
Immunoblot analysis of a time course of factor Va
inactivation in the presence of protein S showed much less accumulation
of the M = 75,000 intermediate (Fig. 3B). The fact that formation of the fragments
with M
= 26,000/28,000 occurred at the same
rate as without protein S and that the fragments with M
= 60,000/62,000 and M
= 45,000
were already visible at early time points (<0.5 min) supports our
conclusion that protein S does not affect cleavage at Arg
but promotes cleavage at Arg
. This results in 1) an
increased rate of factor Va inactivation by direct cleavage at
Arg
and 2) accelerated processing of the partially active
reaction intermediate that is formed when the peptide bond at
Arg
is cleaved first.
Figure 4:
Effect of factor Xa and
1,5-DNS-GGACK-factor Xa on APC-catalyzed inactivation of membrane-bound
factor Va. Purified human factor Va (1 nM) was incubated with
0.2 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 in the absence (
) or presence of 20 nM factor Xa
(
) or 20 nM 1,5-DNS-GGACK-factor Xa (
). At the
indicated time points, factor Va activity was determined as described
under ``Experimental Procedures.'' The rate constants and the
activity of the reaction intermediate were obtained from the best fit
of the data (solid lines) to given under
``Experimental Procedures'' using nonlinear least-squares
regression analysis.
The selective inhibition of the cleavage at Arg was
confirmed by immunoblot analysis of APC-catalyzed inactivation of
membrane-bound factor Va in the absence (Fig. 5A) and
presence (Fig. 5B) of factor Xa. Comparison of the
immunoblots shows that factor Xa prevented formation of the M
= 75,000 heavy chain degradation product,
which is indicative for inhibition of the cleavage at Arg
and which shows that APC converts factor Va directly, but slowly,
into products with M
= 62,000/60,000 and
45,000 by cleavage at Arg
.
Figure 5:
Immunoblot analysis of APC-catalyzed
inactivation of membrane-bound factor Va in the absence or presence of
1,5-DNS-GGACK-factor Xa. Purified human factor Va (7 nM) was
incubated with 1.4 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 0.2 mg/ml BSA
at 37 °C in the absence (A) or presence of 20 nM 1,5-DNS-GGACK-factor Xa (B). 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 left of panels A and B. The percentages of cofactor activity at the indicated time
points were 100, 70, 51, 40, 25, 21, 18, and 0% (A) and 100,
92, 84, 70, 59, 49, 41 and 0% (B). Further details are
described under ``Experimental
Procedures.''
Figure 6:
Effect of protein S and factor Xa on
APC-catalyzed inactivation of membrane-bound factor Va.
Purified human factor Va
(1 nM) was incubated
with 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 in the absence (
) or presence of 490 nM protein
S (
) or 20 nM factor Xa (
). At the indicated time
points, factor Va activity was determined as described under
``Experimental Procedures.'' The rate constants of
inactivation were obtained from the best fit of the data (solid
lines) to given under ``Experimental
Procedures'' using nonlinear least-squares regression
analysis.
If, as argued above, protein S
accelerates inactivation by promoting cleavage at Arg and
factor Xa blocks inactivation of normal factor Va by protection of
cleavage at Arg
, it is to be expected that protein S will
considerably enhance the inactivation of this abnormal factor Va
variant and that factor Xa will have no effect on APC-catalyzed
inactivation of factor Va
. The data presented in Fig. 6indeed show that protein S accelerated the rate of
inactivation 14-fold (k
= 2.2
10
M
s
) and
that the inactivation of factor Va
was not affected by
factor Xa.
In Table 1we have summarized the rate constants
for APC-catalyzed cleavage at Arg and Arg
of the heavy chain of membrane-bound factor Va and factor
Va
that were determined in the absence and presence of
protein S or factor Xa.
Figure 7:
Effects of varying protein S and factor Xa
concentrations on rate constants of APC-catalyzed cleavage at
Arg and at Arg
in membrane-bound factor Va.
The rate constants k
and k
were obtained from time courses of
inactivation of 1 nM factor Va by 0.2 nM APC on 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 (37 °C) in the presence of
varying concentrations protein S and/or factor Xa as described at Fig. 2and Fig. 4. A, k
as
function of the concentration protein S. B, k
as function of the concentration factor
Xa.
We have also determined the ability of factor Xa to protect factor
Va from APC inactivation at varying factor Xa concentrations (Fig. 7B). Factor Xa could completely block cleavage at
Arg without having an effect on the slow phase of factor
Va inactivation (k
). Half-maximal protection of
cleavage at Arg
was observed at 1.9 nM factor
Xa.
It appeared that protein S and factor Xa can exert their effects
on APC-catalyzed factor Va inactivation independent of each other. At a
concentration of factor Xa that completely blocked cleavage at
Arg (20 nM), protein S was still able to
accelerate factor Va inactivation 12-fold (Fig. 8). The fact
that the time course of inactivation determined in the presence of
factor Xa and protein S significantly differs from that obtained in the
presence of protein S alone indicates that the factor Xa protection of
cleavage at Arg
was not annihilated by protein S.
Figure 8:
Combined
effects of protein S and factor Xa on APC-catalyzed inactivation of
membrane-bound factor Va. Purified human factor Va (1 nM) was
incubated with 0.2 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 in the presence of 20 nM factor Xa (
), 490
nM protein S (
), or 490 nM protein S plus 20
nM factor Xa (
). At the indicated time points, factor
Va activity was determined as described under ``Experimental
Procedures.''
Collectively these data support our conclusion that the stimulatory
effect of protein S and the protective effect of factor Xa are
associated with the different steps in the pathway of APC-catalyzed
factor Va inactivation, i.e. are confined to the peptide bond
cleavages at Arg and Arg
, respectively.
Figure 9:
APC-catalyzed inactivation of
membrane-bound factor Va and factor Va in the presence
of protein S and Factor Xa. 1 nM purified human factor Va or
factor Va
was incubated with 0.2 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 in the absence or
presence of 200 nM protein S plus 20 nM factor Xa. At
the indicated time points, factor Va activity was determined as
described in the ``Experimental Procedures''.
, factor
Va
;
, normal factor Va;
, factor
Va
plus protein S and factor Xa;
, normal factor
Va plus protein S and factor Xa.
Therefore, we have determined
in the same experiment the amounts of factor Xa required to protect
factor Va from APC cleavage and to express the cofactor activity of
factor Va in prothrombin activation (Fig. 10). Under these
conditions (low concentration of factor Va and prothrombin present),
similar amounts of factor Xa were required to protect factor Va from
inactivation by APC and to express factor Va cofactor activity in
prothrombin activation (K =
0.03 nM for protection, K
= 0.08 nM for the expression of cofactor
activity). These data indicate that factor Va is optimally protected
from inactivation by APC when it is incorporated into the
prothrombinase complex during ongoing catalysis and that complex
formation between factor Xa and factor Va at the same time expresses
the cofactor activity of factor Va and protects it from inactivation by
APC.
Figure 10:
Effects of factor Xa on factor Va
activity in prothrombin activation and on the protection of factor Va
from inactivation by APC. The rate constant k was obtained from time courses of inactivation of 6 pM factor Va by 0.2 nM APC determined in the presence of 25
µM phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol), 1
µM prothrombin, and varying concentrations of factor Xa in
25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM
CaCl
, and 5 mg/ml BSA (37 °C). Plotted is the value of k
as function of the concentration of factor Xa
(
). The factor Xa dependence of expression of factor Va cofactor
activity (
) was determined in the same reaction mixtures by
measuring the rate of thrombin formation before APC was
added.
Recently it has been reported that APC-catalyzed inactivation
of factor Va proceeds via a biphasic
reaction(6, 7, 8) . The first step of the
inactivation process is a rapid cleavage at Arg in the
heavy chain domain of factor Va that results in the formation of a
reaction intermediate that exhibits partial cofactor activity in
prothrombin activation. This reaction intermediate is subsequently
fully inactivated by slow cleavage at Arg
. Here we report
that protein S stimulates the inactivation of membrane-bound factor Va
20-fold by specific acceleration of the cleavage at Arg
and that factor Xa protects factor Va from inactivation by APC by
blocking the cleavage site at Arg
. However, factor Xa
does not completely inhibit factor Va inactivation by APC. At
saturating factor Xa concentrations, factor Va inactivation still
proceeds with a slow rate that is associated with APC-catalyzed
cleavage at Arg
. This means that the latter cleavage is
apparently not affected by factor Xa.
These conclusions are
supported by immunoblot analysis and by experiments with factor
Va obtained from a homozygous APC-resistant patient.
Immunoblots of time courses of inactivation of membrane-bound factor Va
by APC, in which the heavy chain and its degradation products were
visualized with a polyclonal antibody directed against the heavy chain,
showed transient generation of a reaction product with M
= 75,000 which is formed after rapid cleavage of the
peptide bond at Arg
by APC (Fig. 3A).
With protein S present there is much less accumulation of the M
= 75,000 fragment and increased formation
of products with M
of 60,000/62,000, and 45,000 (Fig. 3B). This is indicative for a protein S-dependent
increase of the rate of cleavage at Arg
, which either
results in increased direct factor Va inactivation or in accelerated
processing of the M
= 75,000 intermediate.
In the presence of factor Xa, there is also no formation of the M = 75,000 reaction intermediate (Fig. 5B). In this situation there is a slow formation
of products with M
= 62,000/60,000 and
45,000, which shows that factor Xa blocks cleavage at Arg
and that inactivation of factor Va proceeds via slow cleavage at
Arg
.
Similar information is obtained from kinetic
analysis of inactivation of factor Va. This abnormal
factor Va molecule lacks the cleavage site at Arg
due to
a substitution of Arg
by
Gln(22, 23, 24, 25) . In line with
the proposed effects of protein S and factor Xa on APC-catalyzed
peptide bond cleavages in the heavy chain of factor Va, we observed
that protein S drastically stimulates inactivation of factor
Va
(Fig. 6), which has been reported to be
primarily associated with cleavage at
Arg
(7, 8) . We further show that factor
Xa does not protect factor Va
from inactivation by APC.
This observation is explained by the fact that factor Va
lacks Arg
, which is the target site for factor Xa.
It is the first time that a profound effect is reported for protein
S in a reaction system with purified coagulation factors. In earlier
publications(10, 12) , modest effects were described
for protein S. The reason for this apparent discrepancy is that in the
earlier studies the effect of protein S was determined on initial rates
of APC-catalyzed factor Va inactivation that is a phase of the reaction
that is primarily associated with cleavage at Arg and
which, according to our data, is hardly affected by protein S. Here we
show that protein S actually acts at the slower step of the
inactivation process, that is the cleavage of the peptide bond at
Arg
. The presence of protein S thus ensures rapid
inactivation of factor Va by accelerating direct cleavage at
Arg
or by promoting the conversion of a partially active
reaction intermediate (factor Va cleaved at Arg
) into a
product with no detectable cofactor activity. Given the known clinical
consequences of protein S deficiencies, it is likely that the above
described contribution of protein S to factor Va inactivation is of
prime physiological importance.
Titration experiments at low APC and
factor Va concentrations on 25 µM DOPS/DOPC phospholipid
vesicles show that protein S stimulates APC-catalyzed inactivation of
factor Va with a K of 200 nM and that
protection by factor Xa is characterized by a K
of 1.9 nM. We have
further shown that protein S and factor Xa can exert their effects on
APC-catalyzed factor Va inactivation independent of each other. At a
concentration of factor Xa that completely blocks cleavage at
Arg
, protein S was still able to accelerate factor Va
inactivation 12-fold. These data support our conclusion that the
stimulatory effect of protein S and the protective effect of factor Xa
on inactivation of factor Va by APC result from different interactions
with factor Va and are confined to steps that involve the peptide bond
cleavages at Arg
and Arg
, respectively.
In terms of physiological relevance for regulation of factor Va
activity, factor Xa actually has two (procoagulant) functions: 1)
factor Xa protects factor Va from inactivation by APC and 2) factor Xa
brings the cofactor activity of factor Va to expression by formation of
the prothrombin-activating complex. It appeared that much higher
concentrations of factor Xa were required for protection of factor Va
from inactivation by APC (K = 1.9 nM) than for incorporation of factor Va
into a catalytically active prothrombinase complex (K
= 0.08 nM).
However, prothrombinase complex formation is usually determined at low
factor Va concentrations in reaction mixtures that contain prothrombin
as additional component. We observed that much lower amounts of factor
Xa (K
= 0.03 nM)
were required to protect factor Va from inactivation by APC when it was
incorporated into a prothrombinase complex that was catalyzing
prothrombin activation. These data demonstrate: 1) that factor Va is
most effectively protected from inactivation by APC when incorporated
into the prothrombinase complex during ongoing prothrombin activation
and 2) that protection of factor Va from APC cleavage and
prothrombinase complex formation likely involve the same interaction of
factor Xa with factor Va.
Recently, it was recognized that APC
resistance alone is only a mild risk factor for
thrombosis(46, 47, 48) . Considering the
large differences in inactivation rates of factor Va and factor
Va by APC, this is a somewhat puzzling observation. In
accordance with the proposed effects of protein S and factor Xa, we
observed that the large differences in the rates of APC-catalyzed
inactivation of factor Va and Va
were almost
annihilated when factor Va and factor Va
were
inactivated by APC in the presence of factor Xa and protein S (Fig. 9). Since it is likely that in vivo regulation of
factor Va activity by APC occurs in the presence of protein S and
factor Xa, our observations may explain why in the absence of other
genetic or environmental risk factors APC resistance only results in a
relatively weak prothrombotic condition.