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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3839-3846
Inhibition of Activated Protein C Anticoagulant Activity by Prothrombin
By
Mikhail D. Smirnov,
Omid Safa,
Naomi L. Esmon, and
Charles T. Esmon
From the Cardiovascular Biology Research Program, Oklahoma Medical
Research Foundation, Oklahoma City; the Departments of Pathology and
Biochemistry and Molecular Biology, University of Oklahoma Health
Sciences Center, Oklahoma City; and the Howard Hughes Medical
Institute, Oklahoma City, OK.
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ABSTRACT |
In this study, we test the hypothesis that prothrombin levels may
modulate activated protein C (APC) anticoagulant activity. Prothrombin
in purified systems or plasma dramatically inhibited the ability of APC
to inactivate factor Va and to anticoagulate plasma. This was not due
solely to competition for binding to the membrane surface, as
prothrombin also inhibited factor Va inactivation by APC in the absence
of a membrane surface. Compared with normal factor Va, inactivation of
factor Va Leiden by APC was much less sensitive to prothrombin
inhibition. This may account for the observation that the Leiden
mutation has less of an effect on plasma-based clotting assays than
would be predicted from the purified system. Reduction of protein C
levels to 20% of normal constitutes a significant risk of thrombosis,
yet these levels are observed in neonates and patients on oral
anticoagulant therapy. In both situations, the correspondingly low
prothrombin levels would result in an increased effectiveness of the
remaining functional APC of  5-fold. Thus, while the protein C
activation system is impaired by the reduction in protein C levels, the
APC that is formed is a more effective anticoagulant, allowing protein
C levels to be reduced without significant thrombotic risk. In
situations where prothrombin is high and protein C levels are low, as
in early stages of oral anticoagulant therapy, the reduction in protein C would result only in impaired function of the anticoagulant system,
possibly explaining the tendency for warfarin-induced skin necrosis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IN THE LAST DECADE, mutations of
components within the protein C anticoagulant pathway and a prothrombin
dimorphism in the 3' untranslated region of the gene (G20210A)
have emerged as dominant contributors to thrombotic
risk.1-4 Why defects in the protein C system are associated
with thrombotic risk is relatively well understood, as failure of this
system to operate optimally results in more stable factor Va and
prothrombin activation complexes, almost certainly resulting in the
observed hypercoagulable state. The protein C concentration in plasma
(65 nmol/L)5 is well below the concentration required
for optimal activation by the physiological activation complex (0.7
µmol/L).6 In contrast, prothrombin circulates at the
highest level of any of the vitamin K-dependent coagulation zymogens.
Its circulating concentration (1.4 µmol/L) is about 5 times the
Km for prothrombin activation by the physiological
activation complex,7 leading to the prediction that changes
in prothrombin level should have little effect on the hemostatic
balance.8 In addition to the risk associated with the
prothrombin dimorphism, another feature observed experimentally is that
the decrease in prothrombin level is the best predictor of
antithrombotic efficacy of oral anticoagulants.9
Furthermore, optimal clinical anticoagulation seems to correlate best
with prothrombin levels and specifically when these levels are between about 12% to 24% of normal.10,11 These observations seem
at variance with the biochemistry.
Another unanswered question is why oral anticoagulants work so
effectively given that these agents reduce not only the clotting factor
levels, but also reduce the levels of protein C and protein S
comparably.12,13 Patients with 10% to 20% protein C or
protein S are at risk of thrombosis,3 and the combination
would be expected to constitute a more severe risk.14 In
contrast, reductions to 20% or so of the coagulation zymogens
constitutes little risk of hemorrhage.
Of the defects in the protein C pathway associated with venous
thrombosis, activated protein C (APC) resistance due to a polymorphism in factor Va (factor V Leiden) is by far the most common.3 In purified systems, the inactivation of factor Va Leiden is much slower than normal factor Va (about 20 to 30 times), but in plasma, the
presence of factor V Leiden has only about a 2-fold to 4-fold effect on
the APC anticoagulant activity of APC when the coagulation cascade is
triggered at the level of factor Xa.15,16 Based on the
reduced rates of inhibition observed in the purified system, one would
predict a more pronounced effect in plasma.
One possible explanation for these unanswered questions is that the
prothrombin concentration potently impacts the ability of the protein C
pathway to function. Indeed, prothrombin inhibition of APC function has
been observed.17 In these studies, the investigators concluded that inhibition is mediated by blocking protein S function. An alternative mechanism could involve prothrombin protection of factor
Va from APC proteolysis. Factor Va is known to bind to
prothrombin,18 and the cleavage of factor Va by APC results in the loss of prothrombin binding capacity.18,19 Given
these interactions of prothrombin with factor Va and the unanswered questions related to the mechanisms of prothrombin regulation of
hemostasis, we examined the ability of prothrombin to modulate APC
inactivation of factor Va in purified systems and to influence the
ability of APC to anticoagulate plasma. We find that the prothrombin concentration is a very strong determinant of APC anticoagulant activity and inhibition of APC inactivation of factor Va can occur on
both membrane surfaces and in solution and effectively retards factor
Va inactivation by APC in the absence of protein S.
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MATERIALS AND METHODS |
Proteins and reagents.
Human prothrombin,20 APC,21 Gla domainless APC
(GD-APC),22 protein S,23 factor
Xa,24 and the factor X activator from Russell's viper
venom (X-CP)25 were prepared as described previously. Human
factor Va was purified by immunoaffinity chromatography from normal
plasma26 or Factor V Leiden plasma27 as
described. Human factor IX was a kind gift of Centeon
L.L.C. (King of Prussia, PA). An APC chimera in which the
protein C Gla domain is replaced with that of prothrombin (APC/PTGla)
was prepared as described.26 Bovine serum albumin (BSA),
ovalbumin, gelatin, 3-(N-morpholino) propanesulfonic acid
(MOPS), Tris-HCl, and salts were from Sigma (St Louis,
MO). The chromogenic substrates Spectrozyme TH and Spectrozyme PCa were from American Diagnostica (Greenwich, CT). The
phospholipids used in these studies were
1-palmitoyl-2-oleoyl-sn-glycero-3 phosphatidylserine (PS),
1-palmitoyl-2-oleoyl-sn-glycero-3 phosphatidylcholine (PC), and
1,2-dilinoleoyl-sn-glycero-3-phosphatidylethanolamine (PE) and were
purchased from Avanti Polar Lipids, Inc (Alabaster, AL).
1-Palmitoyl-2-[1-14C-oleoyl]PC was from DuPont
NEN (Boston, MA). Factor V-deficient human plasma was
prepared by the method of Bloom et al.28
Measurement of APC activity in the purified system.
Factor Va inactivation was analyzed with a 3-stage assay essentially as
described (see Smirnov and Esmon29 for experimental details). All reactions were run at room temperature in 96-well polyvinyl chloride plates (Costar, Cambridge, MA). All
reagents were diluted in 150 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.4, 0.02% azide (TBS) containing 1 mg/mL gelatin, and 5 mg/mL BSA.
Briefly, factor Va (0.2 nmol/L unless noted) was incubated with APC or the chimera APC/PTGla in the first stage for 30 minutes with
phospholipid in the presence or absence of protein S. The
concentrations of reactants are indicated in the figure legends. In the
second stage, after inhibition of APC with
p-(amidinophenyl)-methanesulphonyl fluoride, residual factor Va
activity was monitored by its ability to enhance prothrombin activation
in the presence of excess factor Xa (1 nmol/L), prothrombin (1.4 µmol/L), and 5 mmol/L CaCl2. After 5 minutes of
activation, the reaction was stopped by the addition of EDTA and the
resultant thrombin was measured in the third stage using a chromogenic
assay. Remaining factor Va activity was calculated by dividing the rate
of thrombin formation in the presence of factor Va treated with APC in
stage 1 by the rate of thrombin formation with untreated factor Va. The
validity of this assay with respect to enzyme concentration and time of
reaction was presented elsewhere.26 All experiments were
repeated at least 2 times on separate days with comparable results.
Clotting assays.
Clotting was initiated at the level of factor X activation by use of
X-CP. All reagents were diluted in TBS containing 1 mg/mL gelatin.
Assays were performed at room temperature in 96-well plates. To
dilutions of APC (20 µL) were added 10 µL of phospholipid, 10 µL
of 0.25 nmol/L X-CP, and 20 µL of normal pooled plasma. Final
concentrations of reagents are indicated in the figure legends. The
entire mixture was incubated for 1 minute. Clotting was initiated by
the addition of 25 µL of 20 mmol/L CaCl2. The clotting
time was determined on a Vmax Kinetic Microplate Reader
(Molecular Devices, Sunnyvale, CA) as the time at which the
A405 increased 0.02 above the plasma background level
before initiation of clot formation. This value corresponds to 10%
of the increase in optical density of fully clotted plasma. The
progress curves were examined for all reactions to insure that this cut
off accurately reflected the clotting process. All experiments were
repeated at least 2 times on separate days with comparable results.
Electrophoresis.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was performed with 6% to 20% gradient acrylamide gels with the
Laemmli buffer system.30 For Western blots, gels were transferred to polyvinylidene difluoride membranes (PVDF; Millipore, Bedford, MA) in a semidry apparatus (Bio-Rad, Hercules,
CA). Membranes were blocked with 2% nonfat dry milk in
TBS and reacted with an affinity-purified goat antibovine factor Va
heavy chain immunoglobulin preparation. Biotinylated rabbit antigoat
IgG antibody was added, followed by streptavidin conjugated alkaline
phosphatase followed with Attophos reagent (Vistra fluorescence Western
Kit, Amersham, Arlington Heights, IL). The final image
was processed on a Storm 860 phosphoimager (Molecular Devices).
Preparation of phospholipid vesicles.
Extruded vesicles were prepared as described29 to insure
comparable size between phospholipid types. A 100-nm Nucleopore membrane was used. PS/PC vesicles were 20%PS:80%PC and PE/PS/PC vesicles were 40%PE:20%PS:40%PC. Lipids were mixed in the weight proportions indicated, dried under argon, and lyophilized overnight to
remove organic solvents. 14C-PC (Amersham) was included as
tracer for the determination of lipid concentrations. The lipids were
then reconstituted under argon in TBS to 2 mg total lipid/mL. After
extrusion, the vesicles were used immediately or stored at +20°C
under argon. Storage did not alter vesicle activity.
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RESULTS |
Recent studies of the APC/PTGla chimera showed that the chimera was a
much more potent anticoagulant than wild-type APC in plasma, but that
the two enzymes had very similar activity in certain purified
systems.26 These results suggested the possibility that
there might be a plasma component that interfered with the activity of
the native APC. Because prothrombin interacts with factor Va and is the
most plentiful of the vitamin K-dependent zymogens, we examined the
ability of prothrombin to interfere with APC inactivation of factor Va
(Fig 1A). It is apparent that the presence
of physiological concentrations of prothrombin shifts the APC
concentration dependence of factor Va inactivation far to the right
(ie, it requires much higher APC concentrations to inhibit equivalent
amounts of factor Va in the presence of prothrombin than in its
absence). This effect was apparent on phospholipid vesicles with or
without phosphatidylethanolamine, a lipid that increases APC activity
significantly.8,29 The presence of protein S had little
influence on the ability of prothrombin to inhibit factor Va
inactivation by APC (Fig 1B). In contrast, the APC/PTGla chimera that
exhibits much greater plasma anticoagulant activity was relatively
insensitive to prothrombin inhibition (Fig 1C). This suggests that
prothrombin might be a physiologically important inhibitor of factor Va
inactivation and that this inhibition is not dependent on protein S. The presence of PE had little effect on prothrombin inhibition of the
chimera. Because the chimera and wild-type APC bind to phospholipids
with similar affinity,26 it is unlikely that the mechanism
of prothrombin inhibition of factor Va inactivation is due entirely to
competition for the phospholipid surface.
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| Fig 1.
Prothrombin inhibits APC inactivation of factor Va
more effectively than it inhibits APC/PTGla. Factor Va (0.2 nmol/L) was
incubated for 30 minutes with APC (A and B) or APC/PTGla (C) at the
concentrations indicated. Either PS/PC vesicles or PE/PS/PC vesicles
were present at a final concentration of 20 µg/mL. Residual factor Va
activity was assessed as described in Materials and Methods. (A)
Compared with the absence of prothrombin ( ) on PS/PC
vesicles, physiological concentrations of prothrombin (1.4 µmol/L)
shifted the APC concentration dependence of factor Va inactivation
about 10-fold ( ). Compared with the absence of
prothrombin ( ) on PE/PS/PC vesicles, physiological prothrombin
concentrations shifted this concentration dependence about 30-fold
( ). (B) The above experimental conditions were used except that
protein S (72 nmol/L) was present. The symbols are the same as in (A).
(C) With APC/PTGla, addition of physiological concentrations of
prothrombin had little effect on the enzyme concentration dependence of
factor Va inactivation. The experiments were performed in the absence
of protein S, as it does not influence chimera activity. The symbols
are as described in (A).
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To determine if prothrombin modulates the hemostatic balance in plasma,
we analyzed the impact of changing prothrombin concentration on the
clotting time of plasma in the presence and absence of APC. Two
phospholipid concentrations were used to test whether observed effects
were due to membrane competition. In the absence of APC, at the lower
concentration of phospholipids containing PE, increasing concentrations
of prothrombin inhibited clotting slightly, probably due to competition
with factor Va and factor Xa for the membrane surface31
(Fig 2A, "+"). In the presence of
APC, as the prothrombin concentration increased, the ability of APC to
inhibit clotting was depressed. At the lower concentration of
phospholipid in the absence of PE, increasing prothrombin
concentrations had less effect on the clotting time in the absence of
APC (Fig 2B). Again, however, prothrombin potently inhibited the APC
anticoagulant effect at all concentrations of APC examined. At higher
lipid concentrations where increasing prothrombin concentration
decreased the clotting time slightly, increasing prothrombin
concentration again potently inhibited APC anticoagulant responses in
the presence (Fig 2C) or absence (Fig 2D) of PE. Note that at 25%
normal prothrombin levels, roughly the levels achieved during oral
anticoagulant therapy,10 APC is at least 5 times more
effective than when prothrombin is at physiological concentrations. As
was the case in purified systems, the chimera was considerably less
sensitive to inhibition by increasing prothrombin levels (Fig 2E and
F), suggesting that the decreased sensitivity to prothrombin inhibition is a major mechanism by which the chimera exhibits increased plasma anticoagulant activity. This decreased sensitivity was observed in vesicles that did (Fig 2E) or did not (Fig 2F) contain
PE.
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| Fig 2.
Prothrombin inhibits the anticoagulant activity of APC in
plasma. To study the influence of prothrombin on APC and APC/PTGla
anticoagulant activity in plasma, we initiated clotting at the level of
factor X activation using 0.25 nmol/L X-CP. Plasma was diluted to 25%,
with the concomitant reduction in prothrombin concentration (to 35 µmol/L final) by the other assay constituents. Prothrombin in TBS was
then added to give the final concentrations indicated. The arrow on the
x-axis indicates the approximate physiological concentration of
prothrombin (1.4 µmol/L). Either PE/PS/PC vesicles (left panels) or
PS/PC vesicles (right panels) were used. The PE/PS/PC concentration was
adjusted to give approximately the same clotting time in the absence of
added prothrombin or APC as the PS/PC vesicles. The PE/PS/PC
concentrations were 8 µg/mL in (A), 100 µg/mL in (C), and 8 µg/mL
in (E). The PS/PC concentrations were 30 µg/mL in (B), 100 µg/mL in
(D), and 30 µg/mL in (F). The prothrombin concentration dependence of
the anticoagulant response to APC (A through D) or APC/PTGla (E and F)
was then determined. Because of its increased potency as an
anticoagulant, the concentrations of the chimera were reduced relative
to APC. Final APC or APC/PTGla concentrations are indicated on the
figure (nmol/L).
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Plasma from factor V Leiden patients is poorly anticoagulated by APC.
In factor Xa-initiated clotting, the APC concentration required to
achieve equivalent anticoagulant responses is only about 4 to 5 times
higher than normal plasma. In contrast, in purified systems in the
absence of prothrombin, factor Va Leiden requires almost 30 times more
APC to achieve comparable inactivation relative to wild-type factor Va.
To determine if prothrombin might be responsible for some of the
differences in the plasma and purified systems, we added increasing
concentrations of prothrombin to factor Va Leiden and normal factor Va
and then measured their sensitivity to APC
(Fig 3). The concentration of APC required to achieve 50% factor Va inactivation increased rapidly with
increasing prothrombin concentrations. The APC concentration dependence
of factor Va Leiden inhibition was increased also, but to a much lesser
extent. As a result, the very large difference in inactivation rates
observed between normal and factor Va Leiden in the absence of
prothrombin became much smaller as the prothrombin concentration approached physiological levels. This phenomenon may explain part of
the reason that factor Va Leiden appears much more resistant to APC in
purified systems than in plasma. In addition, some of this difference
between the purified system with APC alone and plasma is mediated by
protein S.
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| Fig 3.
The influence of prothrombin on the inactivation of
factor Va and factor Va Leiden. Factor Va or factor Va Leiden (0.2 nmol/L) in TBS, 5 mmol/L CaCl2, 5 mg/mL BSA, pH 7.4 were
incubated for 30 minutes at room temperature with APC at different
concentrations in the presence of the prothrombin concentrations
indicated. The reactions contained 40 µg/mL PE/PS/PC with or without
72 nmol/L protein S. The APC concentration required for 50% factor Va
inactivation (y-axis) was calculated from the APC concentration
dependence curve of factor Va inactivation under each condition. The
reaction mixtures were: , factor Va, APC, and protein S; , factor
Va, APC, and no protein S;  , factor Va Leiden, APC,
and protein S;  , factor Va Leiden, APC, and no protein S.
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Although changes in prothrombin concentration in the physiological
range clearly reduce the ability of APC to inactivate factor Va in a
concentration-dependent fashion, the mechanism is unclear. One
possibility is that prothrombin is simply competing with APC for
binding to the membrane surface and because it is the most plentiful of
the vitamin K-dependent factors, changes in the physiological range
would have the greatest effect on factor Va inactivation. The
observation that APC inactivation of factor Va Leiden was less
sensitive to the presence of prothrombin than factor Va (Fig 3) and the
differences in prothrombin inhibition of factor Va inactivation by the
chimera (Figs 1C, 2E, and 2F) both argue against this conclusion.
Multiple approaches were taken to evaluate this possibility. In the
first, factor Va inactivation by APC was monitored in the absence of
phospholipid. Although phospholipid accelerates factor Va inactivation,
it is not required for the initial cleavage of factor Va at
Arg506,32 a cleavage that reduces factor Va activity in
plasma-based assays. In this phospholipid-free system, there was a
prothrombin concentration-dependent decrease in the rate of factor Va
inactivation (Fig 4). The prothrombin
concentrations required for inhibition are approximately in the range
of the binding constant observed for a prothrombin derivative
interacting with the prothrombin binding subunit of factor Va (10 to 15 µmol/L) in solution.18 The much lower concentration of
prothrombin required in the membrane system presumably reflects its
increased local prothrombin concentration.31 In addition,
the presence of factor Va has been shown to increase the affinity of
prothrombin for the membrane surface approximately
50-fold,33 further facilitating complex assembly between
factor Va and prothrombin.
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| Fig 4.
Factor Va inactivation by APC is inhibited by prothrombin
in the absence of phospholipid. Factor Va (100 nmol/L) was incubated at
37°C in 0.15 mol/L NaCl, 5 mmol/L CaCl2, 20 mmol/L
HEPES, pH 7.5, 0.2 mg/mL BSA with 10 nmol/L APC in the absence or
presence of different prothrombin concentrations. The reaction was
stopped at the times indicated by addition of 10 mmol/L benzamidine
HCl. The residual factor Va was measured with a 1-stage clotting assay
using factor V-deficient plasma. The prothrombin concentrations
present were: , no prothrombin;  , 1.4 µmol/L prothrombin;  ,
5 µmol/L prothrombin; and , 10 µmol/L prothrombin. Bars
represent the standard deviation of 2 experiments, duplicate samples.
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To rule out the possibility that the observed inhibition was mediated
by contaminating phospholipid in one of the reagents, 0.1% Tween 20 was added to the reaction mixture. The detergent did not prevent
prothrombin inhibition of APC inactivation of factor Va (data not
shown). To rule out phospholipid contamination in the purified system
further, we used Gla domainless activated protein C, which cannot bind
to phospholipid. Prothrombin still inhibited the inactivation of factor
Va by this form of APC (Fig 5). Addition of
phospholipid to this inactivation mixture slowed factor Va inactivation
without altering the prothrombin inhibition appreciably (data not
shown).
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| Fig 5.
Factor Va inactivation by GD-APC is inhibited by
prothrombin in the absence of phospholipid. Factor Va (100 nmol/L) was
incubated at 37°C in 0.15 mol/L NaCl, 5 mmol/L CaCl2,
20 mmol/L HEPES, pH 7.5, 0.1% gelatin with 10 nmol/L GD-APC in the
absence or presence of different prothrombin concentrations. The
reaction was stopped at the times indicated by addition of 10 mmol/L
benzamidine HCl. The residual factor Va was measured with a 1-stage
clotting assay using factor V-deficient plasma. The prothrombin
concentrations present were: , no prothrombin; , 1.4 µmol/L
prothrombin; , 10 µmol/L prothrombin. Bars represent the standard
deviation of 2 experiments, duplicate samples.
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To determine the specificity of the prothrombin inhibition, factor IX,
which has been reported to bind phospholipid with an affinity similar
to prothrombin (2 v 0.6 µmol/L, respectively34), was used as a potential inhibitor and the dose dependence of the inhibition compared with that of prothrombin
(Fig 6). Both factors reduced the factor Va
inactivation. However, prothrombin was much more effective, consistent
with a portion of the inhibition being due to competition for
phospholipid surface. In plasma, human factor IX has no significant
effect on the inhibition of APC anticoagulant activity, but inhibits
coagulation slightly in either the presence or absence of APC
(Fig 7). Although not definitive
mechanistically in itself, when taken together with the inhibition of
factor Va inactivation by prothrombin in solution, these results
suggest that the factor Va-prothrombin complex is resistant to
inhibition by APC.
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| Fig 6.
Comparison of the ability of prothrombin and factor IX to
alter the APC concentration dependence of factor Va inactivation in
the purified system. The APC concentration dependence of factor
Va (0.2 nmol/L) inactivation was determined with varying
concentrations of prothrombin or factor IX as indicated. PE/PS/PC
vesicles (40 µg/mL) were used. The reactions were performed in TBS, 5 mmol/L CaCl2, 5 mg/mL BSA, pH 7.4 at room
temperature. Residual factor Va activity was determined using the
prothrombinase assay described in Materials and Methods (, with
added factor IX; , with added prothrombin).
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| Fig 7.
Factor IX does not inhibit the anticoagulant activity of
APC in plasma. The anticoagulant activity of APC in plasma was
determined in the presence of varying concentrations of prothrombin or
factor IX as indicated. Clotting was initiated with 0.25 nmol/L X-CP in
the presence of 8 µg/mL PE/PS/PC. Final plasma dilution was 25%,
with the concomitant reduction in prothrombin concentration to 0.35 µmol/L. Prothrombin (open symbols) or factor IX (closed symbols) was
added at the concentrations indicated in the presence (,  ) or
absence (, ) of 16 nmol/L APC.
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To further analyze the mechanism of inhibition, we examined the
cleavage products of factor Va in the presence and absence of
prothrombin. For this purpose, Western blots were run and probed with
affinity-purified polyclonal anti-factor Va heavy chain antibodies. Although prothrombin slowed cleavage, there was no obvious change in
the relative distribution of the cleavage products, suggesting that the
presence of prothrombin inhibits both the cleavage at Arg506 and Arg306
(Fig 8). A minor, unidentified cleavage product was present in the
original factor Va preparation.
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| Fig 8.
Proteolytic processing of factor Va by APC in the
presence and absence of prothrombin. A total of 500 nmol/L factor Va,
120 µg/mL PE/PS/PC with or without 1.4 µmol/L prothrombin was
incubated in 150 NaCl, 20 mmol/L HEPES, pH 7.4, 5 mmol/L
CaCl2, 0.2% BSA at 37°C for the times indicated. The
reaction was stopped by addition of 10 mmol/L benzamidine HCl. At this
point, prothrombin was added to the samples from the time course
performed in the absence of prothrombin. This was to insure that the
differences observed in the blots were not due to a prothrombin effect
on the electrophoretic pattern. Gradient 5% to 15% SDS-PAGE gels were
run, blotted, and probed with an affinity-purified, goat anti-factor
Va heavy chain antibody. The blot was developed as described in
Materials and Methods. Residual Va activity is indicated along the
bottom of the gel. Molecular weight marker locations are indicated on
the left of the figure. The locations of the factor Va heavy chain
(HC), the product of cleavage at Arg506 (506), and the product of
cleavage at Arg306 (306) recognized by this antiserum are indicated
along the right side of the figure.
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DISCUSSION |
The present studies indicate that the prothrombin-factor Va complex is
resistant to APC inactivation whether the complex is formed on the
membrane surface or in solution. The major evidence in favor of this
mechanism is that the concentrations of prothrombin required to inhibit
factor Va inactivation by APC in solution are very similar to the
Kd for the prothrombin-factor Va
interaction.18 Complex formation between factor Va and
prothrombin could slow factor Va inactivation by APC either through
steric hindrance or due to conformational changes in the factor Va. The
latter possibility is supported by the observation that formation of the factor Va-meizothrombin complex results in conformational changes
in meizothrombin,35 suggesting the possibility of
reciprocal changes in the factor Va.
Our data appear to conflict with the earlier report from Mitchell et
al,17 which indicated that prothrombin inhibition of factor
Va inactivation was due to inhibition of protein S function, whereas
our data clearly indicate that prothrombin inhibition of this reaction
occurs in the absence of protein S. It is possible that their plasma
was from an individual with factor V Leiden, a mutation not appreciated
at that time. Supporting this possibility, as seen in Fig 3, in the
absence of protein S, APC inactivation of factor Va Leiden is
relatively insensitive to prothrombin.
The present findings provide a rational explanation for the clinical
observation that prothrombin levels strongly influence thrombotic
tendency. In addition to the very modest impact of increasing
prothrombin concentration on the rate of thrombin formation, there is a
very potent inhibition on factor Va inactivation by APC. It is now well
established that heterozygous protein C deficiency is a significant
risk factor for venous thrombosis. The basis for this observation is
almost certainly due to the fact that protein C circulates at
concentrations well below the levels required for maximal activation
and in fact well below the Km6 for activation. Thus, as the level of protein C decreases, there is a proportionate decrease in APC generated in response to a given level of thrombin generation. Direct measurement of APC levels in plasma has shown a
correlation with the protein C concentration.36 The present findings suggest that elevated prothrombin levels in concert with decreased protein C levels should greatly increase thrombotic risk. To
our knowledge, this combination has not been studied systematically,
but at least one case report of ischemic stroke in a young individual
with prothrombin G20210A gene mutation and protein C deficiency has
been presented.37
On the other hand, the risk of thrombosis increases significantly when
individuals carry the factor V Leiden and prothrombin G20210A
dimorphisms.38-41 The observations that prothrombin is somewhat less effective in inhibiting factor Va Leiden inactivation by
APC than wild-type factor Va may make the risk of the combined inheritance of these dimorphisms less severe than might have been anticipated. Our data would suggest that an even more severe risk might
be associated with simultaneous protein C deficiency and prothrombin
G20210A dimorphism.
Perhaps more intriguing is the relationship between low prothrombin
levels and the efficacy of APC as an anticoagulant. The observation
that the efficacy of APC as an anticoagulant is much greater at
prothrombin concentrations near those observed in stably anticoagulated
patients than at normal levels provides an explanation for the
effectiveness of oral anticoagulants despite the fact that they
severely depress protein C levels. In this case, the efficacy of any
APC that is generated is apparently enhanced by almost the same degree
that the activation of protein C is inhibited. Specifically, at 20% of
the normal prothrombin and protein C concentrations, levels that are
common in stably anticoagulated individuals,10,12 the
protein C activation rate would drop about 5-fold. The efficacy reported here suggests a 5-fold increase in anticoagulant function, resulting in maintenance of the natural anticoagulant function despite
reduction in circulating APC. This phenomenon may be offset somewhat by
the reduction in protein S levels. These observations suggest further
that supplementation with relatively small amounts of protein C should
enhance the antithrombotic effects of coumadin anticoagulants more
substantially than previously appreciated. They further suggest that
for patients difficult to manage on oral anticoagulants, the hemostatic
balance might be better preserved with decreased warfarin doses and
relatively minor protein C supplementation.
| |
FOOTNOTES |
Submitted May 3, 1999; accepted August 2, 1999.
Supported in part by a project of a Specialized Center of Research
grant from the National Heart, Lung, and Blood Institute (to N.L.E.)
Grant No. P50 HL54502.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Charles T. Esmon, PhD, Oklahoma Medical
Research Foundation, 825 NE 13th St, Oklahoma City, OK 73104; e-mail:
Charles-Esmon{at}omrf.ouhsc.edu.
| |
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ABSTRACT
INTRODUCTION