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INTRODUCTION |
Activated protein C
(APC)1 is a serine protease
that inhibits thrombin formation by limited proteolysis of the
nonenzymatic cofactors factor Va and factor VIIIa of the prothrombin
and the factor X-activating enzyme complex, respectively. Efficient
proteolysis of the cofactors requires the presence of membranes that
contain anionic phospholipids, calcium ions, and protein S. In the
presence of membranes that contain negatively charged phospholipids,
plasma factor Va is inactivated by APC-catalyzed cleavage of its heavy chain at Arg306 and Arg506 (1, 2). The cleavage
at Arg506 is relatively rapid and yields a reaction
intermediate that still retains partial cofactor activity in
prothrombin activation. The slower cleavage at position
Arg306 results in complete loss of cofactor activity (3).
The rapid cleavage at Arg506 is inhibited when factor Va is
in complex with factor Xa (4-8).
Recently, it was reported that in contrast to synthetic phospholipid
membranes, thrombin-activated platelets partially protect platelet-derived and plasma-derived factor Va from inactivation by APC.
Thrombin-activated platelets appeared to slow down the cleavage at
Arg506 (9). It was speculated that activated platelets
express a membrane component(s) in addition to anionic phospholipids
that specifically binds factor Va resulting in a factor Va molecule with an apparent APC resistant phenotype (10). This protection of
APC-catalyzed inactivation of factor Va was not observed in the
presence of microparticles or synthetic phospholipid vesicles (9). One
of the questions that remain to be answered is how platelets influence
APC-dependent factor Va inactivation once factor Va is
assembled in the prothrombinase complex at the plasma membrane of
activated platelets.
The purpose of the present study was to establish the kinetics of
APC-dependent inhibition of ongoing prothrombin activation at the plasma membrane of platelets adhered to immobilized collagen. To
account for transport limitations of reactants, the experiments were
conducted under well defined flow conditions on a rotating disc. Our
findings indicate no difference in the kinetics of
APC-dependent inactivation of prothrombinase at the
membrane of activated, collagen-adherent platelets compared with that
at the surface of a planar phospholipid membrane.
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EXPERIMENTAL PROCEDURES |
Materials--
Bovine serum albumin (BSA), bovine fibrinogen,
and apyrase were from Sigma. S2238, a chromogenic substrate for
thrombin, was obtained from Chromogenix (Mölndal, Sweden). Human
factor Xa, human prothrombin, and bovine factor Va were prepared and
quantified as described previously (11). Native type I collagen fibrils were extracted from bovine Achilles tendon in the absence of proteases using 0.5 M acetic acid and precipitated with 1.7 M NaCl as described (12). Human
-thrombin was prepared
as described previously (13). Human activated protein C (APC) was
purchased from Kordia (Leiden, The Netherlands).
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and
1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) were obtained
from Avanti Polar Lipids (Alabaster, AL). All other reagents used were of analytical grade.
Platelets--
Suspensions of washed human platelets were
prepared as described previously (14). Briefly, blood was drawn from
healthy volunteers who had not taken any anti-platelet medication in
the preceding 2 weeks. Platelet-rich plasma was prepared by
centrifugation. The platelets were then sedimented by centrifugation
and washed twice with HEPES buffer (10 mM HEPES, 136 mM NaCl, 5 mM glucose, 2.7 mM KCl,
2 mM MgCl2, 1 mg/ml BSA, and 0.1 units/ml
apyrase, pH 6.6). Finally, the platelets were resuspended in HEPES
buffer adjusted to pH 7.45 (buffer A). Platelets were counted on a
Coulter counter (Coulter, Miami, FL), and the suspensions were adjusted to 5 × 107 platelets/ml.
The Rotating Disc Device--
Rotating disc experiments were
performed in a device described previously (15). Briefly, a circular
glass coverslip with a diameter of 20 mm (Menzel Gläser,
Braunschweig, Germany), was rotated at 63 rad/s at the bottom of a
cylindrical reaction vessel containing reactants in 3 ml of buffer A. This angular velocity resulted in a wall shear rate of 3681 s
1 at the edge of the rotating disc. The
reaction vessel was pretreated for 1 h with 20 mg/ml BSA in buffer A.
Preparation of Discs with Collagen-adherent
Platelets--
Circular glass coverslips with a diameter of 20 mm were
cleaned with a 1:1 mixture of ethanol (96 volume %) and HCl (37 volume %) and subsequently rinsed with deionized water. The discs were coated
with collagen by incubating the coverslips for 3 h with 300 µl
of 0.5 mg/ml collagen type I in 0.5 M acetic acid. Coated discs were rinsed extensively with 40 mM phosphate buffer
(pH 7.4) containing 0.15 M NaCl and stored in this buffer
until used. Inspection of the discs by phase-contrast microscopy showed
a homogeneous distribution of the collagen fibrils over the glass surface. The collagen-coated discs were incubated for 15 min with buffer A, followed by a 40-min incubation at room temperature with 300 µl of a suspension of washed platelets. Nonadherent platelets were
removed by rinsing with buffer A.
Preparation of Phospholipid-coated Discs--
Spinning circular
glass coverslips (63 rad/s) were exposed for 20 min to 20 µM vesicles composed of 25 mol % DOPS and 75 mol % DOPC, prepared as described previously (16). Fluid phase vesicles were
removed by flushing for 5 min (10 ml/min) with buffer A. The
phospholipid-coated discs were then transferred to a reaction vessel
containing 3 ml of buffer A for further experimentation.
Thrombin Generation at Rotating Discs--
Discs with
collagen-adherent platelets or coated with a phospholipid membrane were
spun at 63 rad/s in 3 ml of buffer A containing 3 mM
CaCl2. Factor Xa and, when indicated, factor Va were added, and thrombin generation was started after 3 min by adding prothrombin. Timed samples (10 µl) were taken and transferred to cuvettes with 440 µl of Tris buffer (50 mM Tris-HCl, 175 mM
NaCl, 0.5 mg/ml BSA, pH 7.9) containing 20 mM EDTA.
Thrombin was assayed by adding 2.4 mM S2238 (50 µl) to
the cuvette. The change in optical density was monitored at 405 nm. The
thrombin concentrations in the samples were calculated from a standard
curve obtained with known amounts of the enzyme. All procedures were
performed at 37 °C.
Assay for Procoagulant Microvesicles--
Samples (10 µl) from
the reaction vessel were added to cuvettes containing factor Xa, factor
Va, and 3 mM CaCl2 in 137 µl of Tris buffer.
After a 3-min incubation, thrombin generation was started by adding 3 µl of prothrombin. The final concentrations were: 1 pM
factor Xa, 0.5 nM factor Va, and 200 nM
prothrombin. Thrombin generation was stopped after 5 min by the
addition of Tris-EDTA buffer and assayed as described. A reference
curve was constructed using different phospholipid vesicle (25 mol % DOPS, 75 mol % DOPC) concentrations and was linear up to 1 µM.
Kinetic Data Analysis of Time Courses of
APC-dependent Inactivation of Prothrombinase--
The rate
of thrombin generation at the surface of the rotating disc decreases in
time especially when prothrombin concentrations are used below the
apparent Km for prothrombin. Thrombin generation in
the presence of APC was therefore corrected for substrate depletion by
assuming an APC-independent pseudo first order rate constant of
inactivation, k1, utilizing the equation
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(Eq. 1)
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in which [thrombin]t is the thrombin concentration at
time t, and V0 the initial rate of
thrombin formation. Inhibition of prothrombin activation in the
presence of APC was analyzed according to
|
(Eq. 2)
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with ti the time at which APC was added to
the reaction and k2 the
APC-dependent pseudo first order rate constant of
prothrombin activation inhibition. Values for
V0, k1, and
k2 were estimated by a least square fit of
Equations 1 and 2 to the experimental data obtained from thrombin
generation experiments performed in the absence or presence of APC.
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RESULTS |
Thrombin Formation at the Plasma Membrane of Collagen-adherent
Platelets--
Platelet adhesion on immobilized collagen in the
presence of extracellular calcium is accompanied by the shedding of
microparticles and exposure of negatively charged phospholipids in the
outer leaflet of the plasma membrane (14). These microparticles may provide a procoagulant surface that supports prothrombin activation and
thus could complicate our study on the kinetics of
APC-dependent inactivation of prothrombin activation at the
surface of collagen-adherent platelets. Therefore, initial experiments
were conducted to establish the extent of microvesiculation and their
contribution to prothrombin activation and, when necessary, to redesign
the experiment in such a way that the contribution of microparticles to
prothrombin activation would be negligible.
Coverslips with collagen-adherent platelets were spun in buffer A, and
after 12 min CaCl2 (3 mM) was added. Samples
were taken from the reaction vessel and assayed for procoagulant
vesicles. Fig. 1 shows that immediately
after the addition of calcium the concentration of solution phase
procoagulant phospholipid increased, reaching a maximum after 30-40
min. To investigate the relative contributions of these microparticles
and the collagen-adherent platelets to prothrombin activation, factor
Xa (50 pM) followed 3 min later by prothrombin (100 nM) were added to the reaction vessel. Immediately after
the addition of prothrombin an aliquot (100 µl) was taken from the
reaction vessel, transferred to a test tube, and incubated at 37 °C.
Timed samples were taken from both the reaction vessel and the test
tube and assayed for thrombin activity. The rates of thrombin
generation were 2.2 and 1.3 nM/min in the reaction vessel
and test tube, respectively, demonstrating that microvesicles and
adherent platelet contributed about equally to thrombin generation
(Fig. 2). In the second step of this
experiment the reaction vessel was flushed for 5 min (10 ml/min) with
buffer A containing 3 mM CaCl2 to remove
microparticles. After the re-addition of factor Xa (50 pM)
followed by prothrombin (100 nM) no thrombin generation
could be detected in the fluid phase. This finding indicated
that procoagulant microparticles were absent and that thrombin-generating activity was now solely confined to the spinning surface with collagen-adherent platelets (Fig. 2). The rate of thrombin
generation (0.3 nM/min) was, however, lower than the rate
of thrombin generation at the spinning surface before the removal of
microparticles (0.9 nM/min). The combined results of three
similar experiments showed that the rinsing step decreased the
surface-associated thrombin production by 59 ± 10% (mean ± S.D.). This loss of activity was most likely due to a loss of platelet-associated factor Va activity because addition of
plasma-derived factor Va (1 nM) resulted in an increase in
the rate of thrombin generation from 0.3 to 1.8 nM/min. All
further experiments were performed with discs containing
collagen-adherent platelets that were first spun for 30 min at 63 rad/s
in buffer A containing 3 mM CaCl2 and then
flushed with the same buffer for 5 min at 10 ml/min to remove
microparticles prior to thrombin generation.

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Fig. 1.
Extracellular calcium-induced shedding of
procoagulant microvesicles. A collagen-coated circular glass
coverslip with adherent platelets was spun at 63 rad/s in calcium-free
buffer A (pH 7.4). At the indicated time (arrow) 3 mM CaCl2 was added. Timed samples were taken
and assayed for procoagulant activity as described under
"Experimental Procedures."
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Fig. 2.
Platelet surface and microvesicle-associated
thrombin generation. A collagen-coated coverslip with adherent
platelets was spun at 63 rad/s for 30 min in buffer A containing 3 mM CaCl2. Factor Xa (50 pM) was
added followed by prothrombin (100 nM) to start thrombin
generation. Immediately after the addition of prothrombin an aliquot
(100 µl) was taken and incubated at 37 °C. Timed samples were
taken from both the rotating disc reaction vessel and the removed
aliquot to determine, respectively, the rate of total thrombin
generation ( ) and the rate of fluid phase thrombin generation ( ).
After 10 min the reaction vessel was flushed with 3 mM
CaCl2-containing buffer A for 5 min at 10 ml/min. Factor Xa
(50 pM) and prothrombin (100 nM) were re-added.
An aliquot (100 µl) was taken from the reaction vessel and further
incubated at 37 °C. Timed samples from the reaction vessel ( ) as
well as from the incubation mixture ( ) were assayed for
thrombin.
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Optimization of Thrombin Generation at the Plasma Membrane of
Collagen-adherent Platelets--
Fig. 3
shows the initial rates of thrombin generation at a fixed prothrombin
concentration (100 nM) as a function of the factor Xa
concentration. The apparent dissociation constant,
Kd, of surface-bound factor Xa is described by the
simple single site binding isotherm, Vobs = Vmax [Xa]/([Xa] + Kd),
with Vobs the initial rate of thrombin
formation, [Xa] the factor Xa concentration, and
Vmax the initial rate of thrombin generation at
saturating factor Xa concentration. The value for the apparent Kd estimated by fitting this equation to the data
from two similar experiments is 3.5 ± 0.9 pM
(estimated value ± 1 S.E.). Further experiments were
performed at a saturating concentration of factor Xa (50 pM).

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Fig. 3.
Rate of thrombin formation at
collagen-adherent platelets as a function of factor Xa
concentration. A disc with collagen-adherent platelets was
spun in buffer A containing 3 mM CaCl2 and 100 nM prothombin. The initial rates of thrombin formation were
measured for increasing concentrations of factor Xa. The solid
line is the result of the fit procedure described under
"Results."
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Fig. 4 shows the prothrombin dependence
of thrombin generation at the surface of collagen adherent platelets in
the presence of 50 pM factor Xa. The data could be
described adequately by the Michaelis-Menten equation,
Vobs = Vmax
[prothrombin]/([prothrombin] + Km,app), in which
Vmax is the initial rate of thrombin formation
at a saturating prothrombin concentration, [prothrombin] the
prothrombin concentration in free solution, and
Km(app) is the apparent Michaelis constant. The
solid line in Fig. 4 represents the best fit of this equation to the
experimental data. The combined result of two similar experiments
yielded a Km(app) of 42 ± 5 nM
(estimated value ± 1 S.E.).

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Fig. 4.
Rate of thrombin formation at
collagen-adherent platelets as a function of the prothrombin
concentration. A disc with collagen-adherent platelets was
spun in buffer A containing 3 mM CaCl2 and 50 pM factor Xa. The initial rates of thrombin formation were
measured for increasing concentrations of prothrombin. The solid
line is the result of the fit procedure described under
"Results."
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APC-dependent Inhibition of Thrombin Formation at the
Plasma Membrane of Collagen-adherent Platelets during Ongoing
Prothrombin Activation--
The results of a typical prothrombinase
inactivation experiment at the surface of collagen-adherent platelets
are shown in Fig. 5. The first 8 min of
the experiment was performed in the absence of APC to enable the
determination of the initial rate of thrombin formation,
V0. Upon the addition of APC, the rate of
thrombin formation rapidly decreased. To visualize the
concentration-dependent effect of APC, a considerable
interdisc variation in the rate of thrombin generation (0.5-1.2
nM/min) was corrected by setting the initial rates in the
absence of APC to the same value. The total time courses of thrombin
generation in the absence or presence of APC were analyzed by a least
squares fit according to Equations 1 and 2 as described under
"Experimental Procedures." The first order rate constant of
inhibition as a function of the APC concentration is shown as an
insert on Fig. 5. The APC-dependent inactivation of prothrombinase obtained from these data was 3.3 × 106 M
1
s
1.

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Fig. 5.
APC-dependent inactivation of
prothrombinase activity associated with collagen-adherent
platelets. Discs with collagen-adherent platelets were spun in
buffer A containing 3 mM CaCl2, 50 pM factor Xa, and 100 nM prothrombin. Timed
samples were removed and assayed for thrombin. At the indicated time
(arrow) a small aliquot of buffer ( ) or 0.25 nM ( ), 0.5 nM ( ), or 1 nM APC
( ) was added. The initial rates of thrombin formation in the absence
of APC were set to the same value. The solid lines represent
the best fit of Equations 1 and 2 to the data. The first order rate
constants of inhibition thus obtained are shown as a function of the
APC concentration in the insert.
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To assess the influence of the prothrombin concentration on the
inhibition of the prothrombinase activity, comparable inhibition experiments were performed at prothrombin concentrations ranging from
20 to 500 nM. The results presented in Table
I show that varying the prothrombin
concentration did not influence the pseudo first order rate constant of
inactivation of platelet-associated prothrombinase activity.
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Table I
Effect of prothrombin concentration on APC-catalyzed inhibition of
prothrombinase activity
Discs with collagen-adherent platelets were spun in buffer A containing
3 mM CaCl2, 50 pM factor Xa, and the
indicated prothrombin concentrations. After 3 min, 0.5 nM
APC was added, and the pseudo first order rate constants of inhibition
by APC were calculated by fitting the thrombin generation data as
described under "Experimental Procedures."
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The inhibition of prothrombinase activity by APC at a platelet surface
was compared with that at a rotating planar phospholipid membrane
composed of 25 mol % DOPS and 75 mol % DOPC. The experimental conditions for thrombin generation and inhibition were the same as
described for collagen-adherent platelets. However, in addition to
factor Xa (50 pM) and prothrombin (100 nM),
plasma factor Va (10 pM) also was added. Typical thrombin
generation curves in the absence or presence of APC are shown in Fig.
6. Thrombin generation was analyzed by a
least squares fit of Equations 1 and 2 to the data. A plot of the first
order rate constants of inactivation as a function of the APC
concentration is shown as an insert on Fig. 6. Linear
regression to these data yielded a second order rate constant of
inhibition of prothrombin activation of 2.5 × 106
M
1
s
1.

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Fig. 6.
APC-dependent inactivation of
prothrombinase activity associated with a synthetic phospholipid
membrane. Rotating discs with planar phospholipid membranes
composed of 25 mol % DOPS and 75 mol % DOPC were spun in buffer A
containing CaCl2 (3 mM), factor Xa (100 pM), and factor Va (10 pM). After 5 min
thrombin generation was started by the addition of prothrombin (100 nM). At the indicated time (arrow) a small
aliquot of buffer ( ) or 0.5 nM ( ), 1 nM
( ), or 2 nM APC ( ) was added. Timed samples were
removed and assayed for thrombin. The solid lines represent
the best fit of Equations 1 and 2 to these data. The first order rate
constants of inhibition thus obtained are shown as a function of the
APC concentration in the insert.
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DISCUSSION |
It is generally believed that upon vessel wall injury the adhesion
of platelets to exposed collagen stimulates thrombus formation. The
interaction between platelets and immobilized collagen induces the
release of the content of the
-granula, exposure of anionic phospholipids, and shedding of microvesicles. As a result, highly reactive procoagulant platelets and microvesicles are generated in
which factor Va from
-granula (17, 18) and anionic phospholipids (12) provide the essential accessory factors for the
prothrombin-converting enzyme factor Xa.
This study focuses on the role of APC as an inhibitor of ongoing
thrombin generation at the surface of collagen-adherent platelets. To
account for the transport-limited supply of substrate at these surfaces
(19, 20) and to approach the in vivo situation of thrombin
formation under flow conditions, activation and inactivation experiments were performed utilizing saturating factor Xa and prothrombin concentrations in a previously described rotating disc
device (15, 20-22).
Thrombin Generation at the Surfaces of Collagen-adherent
Platelets--
Initial experiments confirmed that platelet adhesion to
collagen in the presence of extracellular calcium resulted in the formation of microparticles. As a matter of fact, about 60% of total
thrombin generation could be attributed to prothrombinase associated
with these microparticles. Because this study was focused on ongoing
thrombin generation at adherent platelets, subsequent experiments were
performed after the microparticles were removed from the reaction system.
To further characterize the kinetics of thrombin generation at the
surfaces of collagen-adherent platelets, dependence on factor Xa and
prothrombin concentration was determined. The apparent Kd for factor Xa on collagen-adherent platelets was
3.5 pM. We note that this Kd value is
determined in the presence of a fixed prothrombin concentration (100 nM) but in the absence of both microvesicles and exogenous
factor Va. Much higher apparent Kd values for factor
Xa have been reported for thrombin-activated platelets in suspension
(Kd = 142 pM) (24) and for von
Willebrand factor-adherent platelets (Kd = 4 nM) (25), but the value found here is in close agreement
with the value of 1 pM reported for the interaction of
factor Xa with planar phospholipid surface composed of 25% PS, 75% PC
and containing preabsorbed factor Va (23).
The prothrombin concentration in the solution required to obtain a
half-maximal rate of thrombin generation was 42 nM. This apparent Km value is lower than the value reported
for vesicles in suspension (Km = 100 nM)
(26) but higher than the values of 5 and 7 nM for
phospholipid bilayers in a tubular flow system (11) and for prothrombin
activation experiments on rotating discs (20), respectively. However,
the values reported for the tubular flow reactor were obtained after
correction for prothrombin depletion near the catalytic surface. If the
same correction is made here, a Km value of 14 nM would be obtained. Interestingly, the plasma prothrombin
concentration is more than 100-fold higher, meaning that inhibitors
like antithrombin will have no chance to compete successfully with
prothrombin for the active site of prothrombinase (27, 28). It is,
therefore, unlikely that proteinase inhibitors like antithrombin can
regulate platelet-associated prothrombinase activity.
APC-dependent Inhibition of Ongoing Thrombin Generation
at Adherent Platelets--
It has been shown that platelets greatly
accelerate the rate of APC-dependent inactivation of
factor Va by providing a negatively charged phospholipid surface (29).
However, it has also been reported that platelets show an APC-resistant
phenotype. That is, despite the presence of APC, platelet-derived
factor Va activity is sustained on the surface of
thrombin-activated platelets (9-10, 30-31). The present report
demonstrates that APC inhibits platelet-associated prothrombinase
activity in a mono-exponential way with a second order rate constant of
inactivation of 3.3 × 106
M
1 s
1.
This value is in excellent agreement with the second order rate constant of inhibition (2.5 × 106
M
1 s
1)
found for prothrombinase associated with a planar synthetic phospholipid membrane composed of 25 mol % PS, 75 mol % PC. Moreover, the inhibition rates reported here for ongoing thrombin generation are
also very close to the reported (32) rate constant for APC-catalyzed cleavage at Arg306 in plasma-derived factor Va
(k = 6.5 × 106
M
1
s
1).
Thus, in the experimental setup of the present study, which mimics
physiologically relevant conditions, we observed complete inactivation
by APC of the prothrombin-converting activity of the factor Va-factor
Xa complex bound to collagen-adherent platelets. Moreover, no
differences were found between the first order rate constant of
inactivation of prothrombinase assembled at collagen-adherent platelets
and at a synthetic phospholipid membrane. In contrast, Camire et
al. (9) found different kinetics for the inactivation of factor Va
at platelets and synthetic phospholipid membranes, with a slower, and
most importantly, incomplete inactivation of factor Va at the membrane
of platelets. We note that these investigators used thrombin-activated
platelets and that the decline of factor Va cofactor activity was
assayed from timed samples as prothrombinase activity using a high
factor Xa concentration. Thrombin, however, has been demonstrated to be
a rather weak agonist in inducing exposure of negatively charged
phospholipids (PS) in the outer leaflet of the platelet plasma
membrane (14, 33). In addition, Camire et al. (9) prevented
thrombin-activated platelets from aggregation by using the RGDS peptide
and did not stir the platelet suspension. It was recently
reported that these conditions prevent thrombin-induced exposure of PS
(34). In view of the stimulating role of negatively charged
phospholipids on the kinetics of the cleavage of factor V at
Arg306 (32), it is tempting to speculate that under
their experimental conditions (9), insufficient platelets
provide the suitable surface to bind factor Va and to stimulate APC
activity. Although this notion would explain the finding that part of
the factor Va escaped inactivation, it is contradicted by their
observation that exogenous plasma-derived factor Va added to a
thrombin-activated platelet suspension was rapidly inactivated (9).
Recently, it was reported that prothrombin dramatically inhibits the
ability of APC to inactivate factor Va but scarcely inhibits the
inactivation of factor VaLeiden (35). Moreover, it was suggested that
prothrombin inhibits the cleavage at both Arg506 and
Arg306. In contrast, our experiments clearly demonstrate
that the rate of inactivation of platelet-bound prothrombinase by APC
is not slowed down even under conditions that result in full saturation of the prothrombinase complex with its substrate prothrombin
(cf. Fig. 4, Table I). At present we have no explanation for
these deviating results.
In summary, our data show that APC is an efficient inhibitor of
platelet-dependent thrombin generation. The half-life of
prothrombinase in the presence of 1 nM APC and under the
conditions of the experiment is 5 min. Whereas it has been reported
that platelet-bound factor Va is resistant to APC, our results clearly
indicate that platelet-bound factor Va, as part of the prothrombinase
complex, is inactivated by APC with a rate that is comparable with that
found on a membrane of synthetic phospholipids. Sustained
platelet-derived factor Va cofactor activity therefore could be less
critical than proposed (9, 10).