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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2818-2829
Platelet-Derived Factor Va/VaLeiden Cofactor Activities
Are Sustained on the Surface of Activated Platelets Despite the
Presence of Activated Protein C
By
Rodney M. Camire,
Michael Kalafatis,
Paolo Simioni,
Antonio Girolami, and
Paula B. Tracy
From the Department of Biochemistry, University of Vermont, College
of Medicine, Burlington, VT; and the Institute of Medical Semeiotics,
University-Hospital of Padua Medical School, Padua, Italy.
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ABSTRACT |
We investigated the role of the thrombin-activated platelet in
modulating the rate and extent of activated protein C (APC)-catalyzed inactivation of platelet-derived factor Va and factor
VaLeiden. Platelet-derived factor Va and factor
VaLeiden were inactivated by APC at near identical rates;
however, complete inactivation of the cofactors was never achieved.
Greater residual cofactor activity remained when using
thrombin-activated platelets compared with that observed with synthetic
phospholipid vesicles and platelet-derived microparticles, suggesting
that thrombin-activated platelets protect the cofactors from
APC-catalyzed inactivation. This apparent protection was not due to (1)
an insufficient number of membrane binding sites for APC or factor Va;
(2) the destruction of these sites; or (3) the presence of a
platelet-associated APC inhibitor. Results from a plasma-based clotting
assay (with or without APC) with platelets or PCPS vesicles added to
induce clot formation indicated that, even in the presence of high
concentrations of APC, platelets offered protection of the cofactor by
delaying cleavage at Arg506. This resulted in incomplete
proteolysis of the heavy chain, suggesting that platelets can also
protect plasma-derived factor Va from APC-catalyzed inactivation.
However, additional experiments indicated that the plasma-derived
cofactor, bound to thrombin-activated platelets, was completely
inactivated by APC, suggesting that the plasma and platelet-derived
cofactor pools represent different substrates for APC. Collectively,
these results indicate that platelets sustain procoagulant events by
providing a membrane surface that delays cofactor inactivation and by
releasing a cofactor molecule that displays an APC resistant phenotype.
Thus, at sites of arterial injury, the factor VLeiden
mutation may not as readily predict arterial thrombosis, because the
normal and variant platelet-derived cofactors are equally resistant to
APC at the activated platelet surface.
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INTRODUCTION |
PLATELETS PLAY AN essential role in the
normal hemostatic process by adhering and aggregating at sites of
vascular injury, thus providing a physical barrier to control blood
loss. However, platelets also participate in the generation and
regulation of thrombin by providing a suitable membrane surface that
localizes, amplifies, and subsequently modulates procoagulant enzymatic
reactions.1 Activated human platelets promote the catalysis
of at least two procoagulant reactions: the conversion of factor X to
Xa by the intrinsic tenase complex and the conversion of prothrombin to thrombin by the prothrombinase complex.2-4 Prothrombinase
is a stoichiometric enzymatic complex consisting of the serine protease factor Xa and the nonenzymatic cofactor factor Va derived from platelet
or plasma stores,5 assembled on an appropriate cellular membrane surface in the presence of Ca2+
ions.1,4 Human platelets are also able to promote the
catalysis of at least one anticoagulant reaction, the activated protein C (APC)-catalyzed inactivation of factor Va,6,7 which
downregulates the assembly and function of prothrombinase. Deletion of
factor Va from the prothrombinase complex reduces the rate of thrombin generation by four orders of magnitude.8 Hence, significant variations in prothrombin activation can be accomplished via
proteolytic alterations in factor Va.
Recently, the mechanism of inactivation of human plasma-derived factor
Va by APC on phospholipid vesicles has been detailed. Proteolysis of
plasma-derived factor Va by APC occurs within the heavy chain of the
cofactor at position Arg506 followed by cleavages at
positions Arg306 and Arg679.9
Cleavage at Arg306 only occurs when the cofactor is
membrane-bound and is the major inactivating cleavage
site.9,10 Although cleavage at Arg506 is
membrane independent, proteolysis at this site is significantly enhanced in the presence of a membrane surface.9,10
In addition to the progress that has been made in unraveling the
mechanism of plasma-derived factor Va inactivation by APC, our
understanding of the protein C pathway and thrombophilia have expanded
greatly in recent years with the discovery of APC resistance. In 1993, Dahlback et al11,12 described a new mechanism of familial thrombophilia characterized by a poor anticoagulant response to APC
(APC resistance). The molecular defect in APC-resistant individuals was
identified in several laboratories as a single point mutation in the
factor V gene (G1691  A), predicting a single
amino acid substitution in the factor V protein (Arg506
Gln).13-17 However, in about 10% of the
APC-resistant patients, the Arg506 Gln mutation
is not present,17 but no alternative molecular basis for
this finding has yet been identified. Resistance to APC is the most
common identifiable defect among patients with venous thrombosis (20%
to 60% in patients with venous thrombosis)18,19 and is
also quite common in the general Caucasian population with an allelic
frequency of approximately 3% to 5%.18-21 The most common clinical manifestation of APC resistance is venous thrombosis; however,
few studies have established a correlation between the factor
VLeiden mutation and arterial thrombosis.22-24
The molecular basis of APC-resistance (factor VLeiden) is
due, at least in part, to the slower rate of inactivation (~10- to
20-fold, relative to wild-type) of the plasma-derived factor
VaLeiden heavy chain due to a change in the initial APC
cleavage site at position 506.25-28 However, the
contribution of platelet-derived factor Va and factor
VaLeiden to the APC resistance phenotype is not known. Our
laboratory has recently shown that the mechanism of inactivation of
platelet-derived factor Va by APC may be different from its plasma
counterpart. Initial cleavage of platelet-derived factor Va occurs at
either Arg506 or Arg306, and complete
inactivation of the platelet-derived cofactor could not be
achieved.29 In studies of platelet-derived factor Va from
an individual heterozygous for the factor VLeiden mutation,
platelet-derived factor VaLeiden was initially cleaved at
position Arg306 followed by cleavage at Arg679.
However, as observed with the normal platelet-derived cofactor, complete inactivation could not be achieved. Collectively, these results indicated that a platelet-derived factor Va molecule that does
not have the initial cleavage site at Arg506 (factor
VLeiden) or is in a structural conformation such that
cleavage by APC at Arg306 is preferred over
Arg506 may be observed to be APC-resistant.29
Elucidating how platelet-derived factor Va is inactivated by APC at the
platelet surface is critical to our understanding of how the
coagulation system is downregulated, especially because previous
studies have demonstrated that the platelet-derived cofactor plays an
essential role in maintaining normal hemostasis. This concept is
perhaps best shown in studies of a family (factor VQuebec)
whose afflicted members lack functional platelet-derived factor V, but
have normal plasma-derived factor V and exhibit a severe bleeding
diathesis.30 In addition, a patient with a neutralizing inhibitor to plasma-derived factor V, but not to platelet-derived factor V, shows no bleeding tendency.31 These studies
directly implicate platelet-bound, platelet-derived factor Va, whose
concentration within a platelet-rich thrombus may be significantly
greater (>100-fold) than plasma-derived factor Va31 in
the generation of thrombin at the site of vascular injury, thus
maintaining the normal hemostatic balance.
The current study was initiated to compare the rate and extent of the
APC-catalyzed inactivation of platelet-derived factor Va and factor
VaLeiden and to determine if thrombin-activated platelets
would modulate this reaction when compared with synthetic phospholipid
vesicles. The results of these studies should provide insight into how
the platelet-derived cofactors (normal or variant) and the intact thrombin-activated platelet contribute to the APC-resistant phenotype.
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MATERIALS AND METHODS |
Materials and reagents.
Tris[hydroxymethyl]aminomethane (Trizma-Base),
L- -phosphatidyl-L-serine [bovine brain] (PS),
L--phosphatidylcholine [egg yolk] (PC), ARG-GLY-ASP-SER (RGDS)
peptide, Tween-20, 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES), prostaglandin E1 (PGE1), heparin
(bovine lung), and glycine were purchased from Sigma (St Louis,
MO). Sodium chloride and calcium chloride dihydrate were
purchased from J.T. Baker (Phillipsburg, NJ). Pure nitrocellulose
membrane sheets (0.45 µm) were purchased from Bio-Rad (Hercules, CA).
The chemiluminescent substrate, Luminol, and Reflection autoradiography
film were purchased from DuPont, NEN Research Products (Boston, MA).
Crystallized bovine serum albumin was purchased from ICN
ImmunoBiologicals (Aurora, OH). The -thrombin inhibitor hirudin was
obtained from Genentech (South San Francisco, CA). The fluorescent
-thrombin inhibitor dansylarginine N-(3-ethyl-1,5-pentanediyl)amide
(DAPA)32 and human APC were gifts from Haematologic
Technologies Inc (Essex Junction, VT). Phospholipid vesicles composed
of 75% (% wt/wt) PC and 25% (% wt/wt) PS (PCPS) were prepared as
previously described.33 The concentration of the
phospholipid vesicles was determined by phosphorous
assay.34
Preparation of coagulation proteins.
All proteins were of human origin and purified from fresh-frozen
plasma. Plasma-derived factor V was isolated by immunoaffinity chromatography as described and was activated to factor Va with 1 to 2 NIH U/mL (10 to 20 nmol/L) of -thrombin for 10 minutes at
37°C.35,36 Factor X and prothrombin were purified by
the method of Bajaj et al.37 Factor X was activated with
the factor X activator purified from Russell's viper
venom.38 -Thrombin was prepared by activation of
prothrombin with taipan snake venom as described by Owen and
Jackson.39 All proteins used were greater than 95% pure as
judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) before and after disulfide bond reduction according to the
method of Laemmli.40 Molecular weights and extinction
coefficients (E1%280nm) of the various
proteins used were taken as follows: prothrombin 72,000, 14.237; thrombin 37,000, 17.441; factor V
330,000, 9.636; factor Xa 50,000, 11.637; and
APC 56,200, 14.5.42
Isolation of platelets.
Platelets were isolated from consenting normal or homozygous (as
determined by DNA analysis13) factor VLeiden
individuals. Briefly, 26 mL of blood was collected into a 30-mL syringe
(8 syringes, ~200 mL of blood) containing 4 mL of ACD (0.022 mol/L
citrate, 0.014 mol/L dextrose, final concentrations) and 5 µmol/L
PGE1 (final concentration). The blood in each syringe was
transferred to a 50-mL conical polypropylene centrifuge tube, everted
twice, split into two tubes, and centrifuged (190g for 15 minutes) at ambient temperature to obtain platelet-rich plasma (PRP).
The PRP (~7.5 mL/centrifuge tube) was removed as well as the buffy
coat into a second centrifuge tube, and this suspension was combined
with another tube to bring the volume to approximately 15 mL. The PRP
suspension was centrifuged at 1,100g for 15 minutes at ambient
temperature. The platelet-poor plasma (PPP) was removed and the
remaining platelet pellet was gently resuspended in a small volume
(~10 mL) of PPP. All platelets from the same donor were pooled (~30
to 40 mL) to generate a platelet concentrate. This platelet concentrate
was then shipped from Padua, Italy to Burlington, VT via express mail
(~72 hours). Upon receipt, the platelet suspensions were placed
immediately at 37°C and platelets were isolated as previously
described.29,43 Platelets were counted on a Coulter counter
(Coulter Electronics, Ltd, Hialeah, FL) and brought to a
final platelet concentration of 1 × 109/mL in 5 mmol/L HEPES-Tyrode's (0.14 mol/L NaCl, 2.7 mmol/L KCl, 12 mmol/L
NaHCO3, 0.42 mmol/L
NaH2PO4·H2O, 1 mmol/L
MgCl2, 2 mmol/L CaCl2, 5 mmol/L dextrose)
buffer, pH 7.4, for all experiments. Control studies performed on site
indicated that the time of shipment and the presence of
PGE1 had no influence on the rate or mechanism of
platelet-derived factor Va inactivation by APC on activated platelets
or PCPS vesicles.
APC-catalyzed inactivation and proteolysis of platelet-derived
factor Va and factor VaLeiden.
The inactivation of normal platelet-derived factor Va (n = 3) and
factor VaLeiden (n = 3) by APC was performed in the
presence of PCPS vesicles or thrombin-activated platelets. For
experiments in which activated platelets provided the membrane surface,
RGDS peptide (1 mmol/L) was added to platelets before activation to
prevent aggregation. Platelet-derived factor Va release and activation
was accomplished by platelet incubation (1 × 109/mL)
with 50 nmol/L -thrombin (5 NIH U/mL) for 5 minutes at ambient temperature, followed by the addition of 60 nmol/L hirudin. In experiments in which thrombin-activated platelets did not provide the
membrane surface, activated platelets were removed from
platelet-derived factor Va by gentle centrifugation (1,100g for
5 minutes) and PCPS vesicles (20 µmol/L) were then added. In
experiments such as those shown in Fig 4, normal human platelets were
removed by centrifugation (1,100g for 5 minutes) and the
resulting centrifugation-induced platelet microparticles were used as
the presumed membrane surface. In all experiments, the initial
concentration of platelet-derived factor Va was donor-dependent and
ranged from 0.81 to 2.7 nmol/L.
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| Fig 4.
APC-catalyzed inactivation of platelet-derived factor Va
bound to thrombin-activated platelets or centrifugation-induced
platelet microparticles. Platelets (1 × 109/mL) in the
presence of RGDS peptide (1 mmol/L) from a normal individual were
treated with 2 NIH U/mL (20 nmol/L) of -thrombin for 5 minutes to
both activate the platelet and release and activate platelet-derived
factor Va. Hirudin (30 nmol/L) was then added to inhibit thrombin. The
thrombin-activated platelets were either used as the required membrane
surface ( ; initial cofactor concentration, 2.5 nmol/L) or platelets
were centrifuged out of solution (1,100g for 5 minutes)
generating platelet microparticles that presumably provided an adequate
membrane surface ( ; initial cofactor concentration, 1.9 nmol/L). APC (0.25 nmol/L) was then added and at selected time
points residual cofactor activity was monitored as described in Fig 1.
The data points were normalized to the initial concentration of
released cofactor for each donor. Upon stabilization of the platelet-derived cofactor activity on thrombin-activated platelets (~120 minutes), 20 µmol/L PCPS vesicles were added (arrowhead) to
the platelet membrane/platelet-derived factor Va/APC mixture and a
substantial loss of platelet-derived cofactor activity was observed
after 30 minutes of incubation ( ).
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After platelet activation with -thrombin (50 nmol/L for 5 minutes),
the inactivation of platelet-derived factor Va was initiated immediately by APC addition (0.25 nmol/L). At selected time intervals, samples of the inactivation mixture were withdrawn and assayed for
residual cofactor activity in a prothrombinase assay using purified
protein components with saturating amounts of factor Xa (5 nmol/L) and
PCPS vesicles (20 µmol/L) as the membrane surface, as previously
described.8,29,36 At the same time intervals, samples were
withdrawn and prepared for SDS-PAGE followed by immunoblotting analyses
as described previously.29 The platelet-derived factor Va
antigen (~50 ng/lane) was probed with a mouse antihuman factor Va
heavy chain IgG monoclonal antibody (MoAb)
HFVaHC#625,29,44 or
HFVaHC#17,45 both of which recognize an
epitope between amino acids 307-506 in the factor Va heavy chain. The
secondary antibody used was a horse antimouse IgG coupled to
horseradish peroxidase (HRP; Southern Biotechnologies, Birmingham, AL).
Proteolysis of factor Va by APC in a plasma-based clotting assay.
Pooled normal human plasma was diluted (1:10) in a glass test tube with
20 mmol/L HEPES/0.15 mol/L NaCl, pH 7.4. Phospholipid vesicles (PCPS;
10 µmol/L) or freshly isolated washed normal human platelets (1 × 108/mL) were added. CaCl2 (5 mmol/L,
final) was then added to initiate clot formation, which was observed
visually.46 In some experiments, exogenous APC (2.0 nmol/L)
was added subsequent to clot formation. At selected time intervals,
samples of the reaction mixture (60 µL or ~60 ng/lane of FV/Va)
were analyzed by SDS-PAGE and Western blotting techniques with MoAb
HFVaHC#17, as described previously.45,46
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RESULTS |
Effects of PCPS vesicles or thrombin-activated platelets on the
APC-catalyzed inactivation of platelet-derived factor Va and factor
VaLeiden.
Previous results from our laboratory25 and other
laboratories26,28 have shown that there is an approximately
10- to 20-fold difference in the rate of the APC-catalyzed inactivation
between normal plasma-derived factor Va and factor VaLeiden
when bound to synthetic phospholipid vesicles. Studies were initiated here to directly compare the ability of APC to inactivate
platelet-derived factor Va and factor VaLeiden on both PCPS
vesicles (Fig 1) and thrombin-activated
platelets (Fig 2) to determine if the
platelet-derived cofactors exhibited a similar differential rate of
inactivation on these surfaces. Upon addition of APC, both the normal
(Fig 1A) and variant (Fig 1B) platelet-derived cofactors were rapidly
inactivated when bound to PCPS vesicles, with 60% to 70% of the
activity lost within 10 minutes; the inactivation profiles were very
similar. Even though it is difficult to compare rates of inactivation
directly between these platelet-derived cofactors because of the rapid rate of inactivation, our data suggest that the substantial
(~10-fold) difference in the rates of inactivation observed
previously with the plasma-derived cofactors bound to PCPS
vesicles25,26,28 was not mimicked by the platelet-derived
cofactors. Immunoblotting studies indicated that platelet-derived
factor VaLeiden was cleaved at Arg306 followed
by cleavage at Arg679 (appearance of the 60-kD and 54-kD
fragments, respectively; Fig 1B, inset) and normal platelet-derived
factor Va was cleaved at Arg506 followed by cleavage at
Arg306 (appearance of the 75-kD and 30-kD fragments,
respectively; Fig 1A, inset). Additionally, platelet-derived factor Va
was also initially cleaved at Arg306 followed by cleavage
at Arg506 (appearance of the 60-kD and 30-kD fragments,
respectively). Complete proteolysis of the heavy chain of either
platelet-derived cofactor could not be achieved even after  150
minutes of incubation with APC. Approximately 20% residual activity
remained for both cofactors, suggesting that a subpopulation of both
platelet-derived factor Va and factor VaLeiden may be
completely resistant to APC.
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| Fig 1.
APC-catalyzed inactivation of platelet-derived factor Va
and factor VaLeiden bound to phospholipid vesicles.
Platelets (1 × 109/mL) from normal (n = 3) or
homozygous factor VLeiden individuals (n = 3) were
treated with 5 NIH U/mL (50 nmol/L) of -thrombin for 5 minutes to
both activate the platelets and release and activate the
platelet-derived factor V. Hirudin (60 nmol/L) was then added to
inhibit thrombin. The activated platelets were immediately removed from
suspension by gentle centrifugation (1,100g for 5 minutes), and
PCPS vesicles (20 µmol/L) were added to the supernatant to provide an
appropriate alternate anticoagulant surface. APC (0.25 nmol/L) was
added and at selected time points residual cofactor activity was
monitored in a prothrombinase assay using purified protein components
with saturating amounts of factor Xa (5 nmol/L) and PCPS vesicles (20 µmol/L) as previously described.8,36 At the same time
intervals, samples of the reaction mixture were withdrawn and subjected
to SDS-PAGE using a 5% to 15% gradient gel. After transfer to
nitrocellulose, fragments were visualized using an MoAb
(HFVaHC#6), as described,25,29,44 that
recognizes an epitope on the heavy chain of factor Va between amino
acids 307-506. The line drawn through the inactivation profiles (A and B) represents the average of the three donors at each given time point
and does not represent an attempt to fit the data to a first-order rate
equation. The data points were normalized to the initial concentration
of released cofactor for each donor. In (A), each of the symbols
represents the time-dependent, APC-catalyzed inactivation of the
platelet-derived factor Va cofactor activity from three normal donors
(initial cofactor concentrations: 0.88, 0.91, and 2.70 nmol/L). The
inset represents the proteolytic fragments derived from the
inactivation on PCPS vesicles as visualized using immunoblotting techniques. Lane 1, platelet-derived factor Va, no APC; lanes 2 through
9, membrane-bound platelet-derived factor Va with APC for 1, 5, 10, 15, 30, 60, 90, and 120 minutes. In (B), each of the symbols represents the
time-dependent, APC-catalyzed inactivation of the platelet-derived
factor VaLeiden cofactor activity from three factor
VLeiden donors (initial cofactor concentrations: 0.81, 0.91, and 1.30 nmol/L). The inset represents the proteolytic fragments
derived from the inactivation on PCPS vesicles. Lane 1, platelet-derived factor VaLeiden, no APC; lanes 2 through
10, membrane-bound platelet-derived factor VaLeiden with
APC for 1, 5, 10, 15, 30, 60, 90, and 120 minutes. The position of the
molecular weight markers are indicated at the left of the insets.
Controls here, and in other experiments, indicated that, in the absence
of APC, platelet-derived factor Va and factor VaLeiden
retained full cofactor activity throughout the time course either in
the presence of PCPS vesicles or thrombin-activated platelets.
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| Fig 2.
APC-catalyzed inactivation of platelet-derived factor Va
and factor VaLeiden bound to thrombin-activated platelets.
Platelets (1 × 109/mL) in the presence of RGDS peptide (1 mmol/L) from normal (A; n = 3) and factor VLeiden
individuals (B; n = 3) were treated with 5 NIH U/mL (50 nmol/L) of
-thrombin for 5 minutes. Hirudin (60 nmol/L) was then added to
inhibit thrombin. The resulting thrombin-activated platelets were then
used as the required membrane surface for the APC-catalyzed inactivation of platelet-derived factor Va. APC (0.25 nmol/L) was added
and residual cofactor activity and proteolytic fragments derived from
APC-catalyzed inactivation were monitored as described in Fig 1. The
line drawn through the inactivation profiles (A and B) represents the
average of the three donors at each given time point and does not
represent an attempt to fit the data to a first-order rate equation.
The data points were normalized to the initial concentration of
released cofactor for each donor. In (A), each of the symbols
represents the time-dependent, APC-catalyzed inactivation of the
platelet-derived factor Va cofactor activity from three normal donors
(initial cofactor concentrations: 1.40, 1.90, and 2.30 nmol/L). The
inset represents the proteolytic fragments derived from the
inactivation on thrombin-activated platelets. Lane 1, platelet-derived
factor Va, no APC; lanes 2 through 9, platelet-bound platelet-derived
factor Va with APC for 1, 5, 10, 15, 30, 90, 120, and 180 minutes. In
(B), each of the symbols represents the platelet-derived factor
VaLeiden cofactor activity from three factor
VLeiden donors (initial cofactor concentrations: 0.91, 0.93, and 1.40 nmol/L). The inset represents the proteolytic fragments
derived from the inactivation on thrombin-activated platelets. Lane 1, platelet-derived factor VaLeiden, no APC; lanes 2 through
10, platelet-bound platelet-derived factor VaLeiden with
APC for, 1, 5, 10, 15, 30, 90, 120, 150, and 180 minutes. The
position of the molecular weight markers are indicated at the left of
the insets.
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When platelets derived from normal or homozygous factor
VLeiden individuals were thrombin-activated (50 nmol/L for
5 minutes) to provide both the cofactor and the required membrane
surface for APC inactivation, near identity in the rate and extent of
inactivation was observed for all platelet donors (Fig 2A and B). These
rates were markedly attenuated when compared with PCPS vesicles (Fig 1A
and B). Approximately 70% to 75% cofactor activity remained after 10 minutes of incubation with APC, with as much as 50% residual cofactor
activity persisting after 3 hours of incubation. Additional studies
with three normal donors indicated that complete inactivation of
platelet-bound, platelet-derived factor Va could not be achieved even
in the presence of high concentrations of APC (50 nmol/L), with as much
as 15% residual cofactor activity remaining after 1 hour of incubation
(data not shown).
A direct comparison of the initial (30 minutes) phase of the
APC-catalyzed inactivation of normal platelet-derived factor Va
(Fig 3, solid symbols; n = 3) and factor
VaLeiden (Fig 3, open symbols; n = 3) indicated that the
rates of inactivation between the normal and the variant cofactors on
thrombin-activated platelets could not be distinguished. Experiments
with 5 additional normal donors (Fig 3, inset) confirmed this
observation and also indicated that the moderate normal donor
variability (n = 4) observed previously29 is not consistent
with the results of the expanded normal donor pool (n = 12) in this
study. In fact, in our previous experiments, only one normal donor did
not give results represented in Fig 3 (inset).
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| Fig 3.
Comparison of the initial phase of the APC-catalyzed
inactivation of platelet-derived factor Va and factor
VaLeiden bound to thrombin-activated platelets. Data points
from Fig 2A (normal platelet-derived factor Va; solid symbols, solid
line) and 2B (platelet-derived factor VaLeiden; open
symbols, dashed line) were plotted such that the initial 30 minutes of
the reaction could be compared. The inset represents the initial (30 minutes) phase of the APC-catalyzed inactivation of platelet-derived
factor Va on thrombin-activated platelets from 5 normal donors. The
line drawn through the inactivation profiles for both graphs represents
the average of the donors at a given time point and does not represent
an attempt to fit the data to a first-order rate equation.
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Immunoblotting experiments indicated that the APC-catalyzed cleavage at
Arg506 in the normal platelet-derived cofactor (Fig 2A,
inset) was substantially delayed when thrombin-activated platelets were
used as the membrane surface as compared with that observed with PCPS
vesicles. Thrombin-activated platelets were also unable to promote
complete proteolysis of the platelet-derived factor Va heavy chain
(normal or variant), confirming that a subpopulation of
platelet-derived factor Va exists that is completely resistant to
APC-induced proteolysis.
The substantial amounts of cofactor activity (normal or variant)
remaining on thrombin-activated platelets relative to PCPS vesicles
suggest that platelets protect platelet-derived factor Va or factor
VaLeiden from inactivation by APC. We next investigated
whether platelet-derived microparticles shared this unique ability.
Platelet-derived microparticles (Fig 4, ) more closely mimicked PCPS
vesicles rather than the intact, thrombin-activated platelet (Fig 4,
) with respect to supporting the rate and extent of APC-catalyzed
inactivation of the cofactor. Platelet-derived microparticles supported
the rapid inactivation of platelet-derived factor Va with only 12%
cofactor activity remaining after 2 hours of incubation with APC, and
Western blotting analyses indicated that APC promoted the rapid
cleavage at Arg506 in a manner analogous to that observed
with PCPS vesicles (data not shown). In contrast, the inactivation of
platelet-derived factor Va (derived from the same donor) bound to
thrombin-activated platelets was approximately fivefold slower, such
that approximately 40% cofactor activity remained after 2 hours of
incubation. Western blotting analyses indicated that cleavage of the
platelet-bound cofactor at Arg506 was delayed and
substantial amounts of the heavy chain remained (data not shown),
consistent with results in Fig 2A (inset). Our results indicate that,
once intact thrombin-activated platelets are removed, the apparent
protection of platelet-derived factor Va from APC is lost and rapid
cleavage at Arg506 occurs, suggesting that this protective
effect resides with the intact thrombin-activated platelet and not
centrifugation-induced microparticles. We were also able to show that
the addition of PCPS vesicles to the thrombin-activated
platelet/platelet-derived factor Va/APC mixture led to the rapid, yet
again incomplete inactivation of platelet-derived factor Va
(Fig 4, arrowhead, ). Thus, the addition
of an anticoagulant surface could overcome the apparent protective
effect imparted by thrombin-activated platelets.
Effect of thrombin-activated platelets on plasma-derived factor Va
inactivation catalyzed by APC.
The apparent protective effect imparted by thrombin-activated platelets
could result from the presence of an APC inhibitor released by
platelets and/or the destruction of membrane binding sites for
APC or factor Va. The observation that the addition of PCPS vesicles to
an activated platelet/platelet factor Va/APC mixture (Fig 4, arrowhead)
resulted in further inactivation of the platelet-derived cofactor, even
though the cofactor activity had remained stable at approximately 45%
for 1 hour, indicated that the protective effect imparted by the
intact, activated platelets was not due to the presence of a
membrane-associated or released, slow-acting inhibitor of APC. To
verify this observation and to test the hypothesis that the
membrane-binding sites required to support either platelet-derived
factor Va or APC binding were being destroyed during the assay,
experiments such as those shown in Fig 5
were performed. Subsequent to thrombin-mediated platelet activation as
described previously to effect platelet-derived factor Va release and
activation and to provide an appropriate membrane surface for the
APC-catalyzed inactivation of the bound cofactor, APC was added and
approximately 60% of the platelet-derived factor Va cofactor activity
was lost within 10 minutes. However, approximately 40% of the cofactor
activity remained for more than 2 hours, at which time purified,
plasma-derived factor Va (3 nmol/L, Fig 5, arrowhead) was added to the
reaction mixture. A sharp increase in cofactor activity was observed,
consistent with the presence of additional cofactor. However, this
cofactor activity was lost such that, within 20 minutes, the cofactor
activity once again stabilized at approximately 40% of the original
activity, suggesting that the added plasma-derived factor Va was
completely inactivated by APC and that the cofactor activity that
remained was most likely due to the residual platelet-derived factor Va
that appeared to be resistant to APC-catalyzed inactivation. These
results indicate (1) that a sufficient number of membrane binding sites
remain on the activated platelet surface for effective inactivation; (2) that the APC is fully functional during the course of the assay
verifying the absence of a platelet-associated or released APC
inhibitor; and (3) again suggest that the platelet- and plasma-derived cofactors represent different substrates for APC.
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| Fig 5.
APC-catalyzed inactivation of plasma-derived factor Va
bound to thrombin-activated platelets. Platelets (1 × 109/mL) in the presence of RGDS peptide (1 mmol/L) from a
normal individual were treated with 2 NIH U/mL (20 nmol/L) of
-thrombin for 5 minutes to both activate the platelet and release
and activate platelet-derived factor Va. Hirudin (30 nmol/L) was added
to inhibit thrombin. APC (0.25 nmol/L) was then added to initiate the
reaction and residual cofactor activity was monitored as described in
Fig 1. () The inactivation of platelet-derived factor Va on
thrombin-activated platelets. After 2.5 hours, purified normal
plasma-derived factor Va (3.0 nmol/L) was added (arrowhead) to the
activated platelet/platelet factor Va/APC mixture (). After the
addition of plasma-derived factor Va, samples of the reaction mixture
were immediately assayed for cofactor activity. Values are expressed as
the initial rate (in micromoles per liter of IIa generation per minute)
of prothrombinase activity, which is directly proportional to the
amount of functional cofactor. No additional APC was added to the
reaction mixture.
|
|
Effect of thrombin-activated platelets on the APC-catalyzed
proteolysis of plasma-derived factor Va subsequent to clot formation.
Studies were performed to analyze and compare what effect platelets and
PCPS vesicles have on the APC-catalyzed proteolysis of plasma-derived
factor Va (predominately) in a plasma-based clotting
assay46 subsequent to clot formation. Addition of PCPS vesicles (10 µmol/L) and 5 mmol/L Ca2+ to platelet-free
plasma (Fig 6A) resulted in
clot formation by approximately 4 minutes, consistent with the loss of
the procofactor factor V (Mr = 330,000) and the appearance
of the 105-kD heavy chain. Subsequent to clot formation and in the
absence of added APC (Fig 6A), time-dependent proteolysis of the factor
Va heavy chain was observed with initial cleavage occurring at
Arg506 (appearance of 75-kD fragment) followed by cleavage
at Arg306 (appearance of 30-kD fragment). Because these
fragments are identical in molecular weight to fragments obtained using
purified plasma-derived factor Va and purified APC,9,25 we
can conclude that plasma-derived factor Va is cleaved by endogenous APC
that is generated during the assay. The addition of platelets (1 × 108/mL) and 5 mmol/L Ca2+ to
platelet-free plasma resulted in clot formation by approximately 4 minutes (Fig 6B). The near identical time to clot formation in both
experiments indicated that both plasma-based assay systems contained
equivalent amounts of procoagulant surface required for thrombin
formation. Subsequent to clot formation and in the absence of added APC
(Fig 6B), cleavage of the factor Va heavy chain appeared slightly
delayed, relative to that observed in the presence of PCPS vesicles,
which is most likely due to slow initial cleavage at
Arg506. However, once cleavage at Arg506
occurred, rapid cleavage at Arg306 followed, as seen by the
appearance of the 30-kD fragment. These results suggest that both
plasma-based clotting assay systems contained not only similar amounts
of procoagulant surface required for thrombin generation, but also
contained similar amounts of anticoagulant surface required for
APC-catalyzed inactivation of the formed factor Va.
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| Fig 6.
APC-catalyzed inactivation of plasma-derived
factor Va subsequent to clot formation in the presence of PCPS vesicles
or platelets. Pooled normal human plasma was diluted (1:10) in a glass
test tube with 20 mmol/L HEPES/0.15 mol/L NaCl, pH 7.4. Phospholipid vesicles (PCPS; 10 µmol/L; A and C) or washed normal human platelets (1 × 108/mL; B and D) were added. CaCl2 (5 mmol/L, final) was then added to initiate clot formation, which was
observed visually. In (C) and (D), exogenous APC (2.0 nmol/L) was added
subsequent to clot formation as indicated by the arrow above the blots.
At selected time intervals (indicated above each gel), samples of the
reaction mixture were analyzed by SDS-PAGE and Western blotting
techniques with MoAb -HFVaHC#17, as
described.45 The position of the molecular weight markers
are indicated at the left of each blot and residue numbers
corresponding to factor Va fragments are given at the right of each
blot. Fragments migrating at approximately 45 kD and approximately 40 kD, which represent amino acids 307-709 and 307-679, respectively,
normally migrate at approximately 60 kD and approximately 54 kD.
However, because we are working with dilute plasma, the mobility of
these fragments appears increased because of the high concentration of
albumin present.
|
|
APC addition (2 nmol/L), subsequent to clot formation supported by PCPS
vesicles (Fig 6C), resulted in the rapid and complete proteolysis of
the factor Va heavy chain within 5 minutes, with cleavage occurring at
Arg506 followed by rapid cleavage at Arg306.
These results indicate that the incomplete proteolysis of the factor Va
heavy chain observed in the previous experiment (Fig 6A) was a result
of the very low concentrations of APC generated during the assay,
because the addition of high concentrations of APC resulted in rapid
proteolysis of the factor Va heavy chain. In marked contrast to these
results, APC addition (2.0 nmol/L), subsequent to clot formation
supported by platelets (Fig 6D), resulted in the delayed proteolysis of
the plasma-derived factor Va heavy chain. Thus, even in the presence of
high concentrations of APC, platelets were ineffective in accelerating
the membrane-independent cleavage at Arg506, and as a
result significant amounts of the factor Va heavy chain remained even
after 150 minutes.
These combined results suggest (1) that activated platelets are less
effective in promoting the APC-catalyzed inactivation of plasma-derived
factor Va than PCPS vesicles; (2) that the activated platelet membrane
surface can protect plasma-derived factor Va, in part by delaying the
initial membrane-independent cleavage at Arg506, a
mechanism not mimicked by PCPS vesicles; and (3) that both plasma- and
platelet-derived factor Va will have sustained cofactor activity on the
surface of thrombin-activated platelets even in the presence of APC.
| |
DISCUSSION |
Results from this study indicate that platelet-derived factor Va and
factor VaLeiden are inactivated by APC at near identical
rates, which is in marked contrast to the substantial difference
observed (~10- to 20-fold) in the APC-catalyzed rate of inactivation
of plasma-derived factor Va and factor
VaLeiden.25,26,28 Our results also indicate
that complete inactivation of the platelet-derived cofactors could
never be accomplished, with greater residual cofactor activity
remaining on thrombin-activated platelets than that observed on
platelet-derived microparticles or PCPS vesicles, suggesting that the
thrombin-activated platelet protects the platelet-derived cofactor from
inactivation by APC. In addition, the plasma-derived cofactor showed
delayed inactivation on the platelet, indicating that activated
platelets can also protect plasma-derived factor Va from APC-catalyzed
inactivation. However, in contrast to the platelet-derived cofactor,
plasma-derived factor Va was completely inactivated on the
thrombin-activated platelet surface, indicating that the two cofactor
pools must represent different substrates for APC.
What makes these two cofactor pools different substrates for APC is
currently not known; however, differences in factor Va posttranslational modification events may provide some insight. Factor
V undergoes several posttranslational modification events, such as
sulfation,47,48 phosphorylation,49,50 and
glycosylation,51,52 which may positively or negatively
regulate its activity. For example, sulfation of factor V appears to be
important for full procoagulant activity and for efficient thrombin
cleavage and activation.48 Our group of investigators have
demonstrated that plasma-derived factor Va is phosphorylated on the
heavy and light chains by two platelet kinases.49,50 These
phosphorylation events, especially within the heavy chain, appear to
modulate its activity, because fully phosphorylated plasma-derived
factor Va is inactivated approximately threefold faster by APC than its native dephosphorylated counterpart.49,53 Interestingly,
platelet-derived factor Va only incorporates phosphate on the light
chain upon release from the platelet,50 and this
differential phosphorylation relative to plasma-derived factor Va may
explain in part why platelet-derived factor Va is a poorer substrate
for APC. Alternatively, recent studies indicate that removal of the
N-linked carbohydrate from the heavy chain of plasma-derived factor Va
increases its susceptibility to inactivation by APC.52
Thus, elucidation of the variations in the platelet-derived factor V
glycosylation pattern compared with plasma-derived factor V may help
explain why the platelet-derived cofactor pool appears more resistant
to APC.
Sustained cofactor activity at the activated platelet surface in the
presence of APC is not due solely to the release of a more resistant
cofactor. The observation that the APC-catalyzed rate of inactivation
of platelet-derived factor Va and factor VaLeiden on
thrombin-activated platelets is substantially attenuated compared with
that observed on PCPS vesicles and platelet-derived microparticles suggests that a component of the intact, thrombin-activated platelet is
involved in regulating this reaction. Support for this conclusion comes
from the observation that the rate of inactivation was increased approximately fivefold upon removal of intact platelets from
suspension, leaving platelet-derived microparticles. This increased
rate of inactivation was paralleled by enhanced cleavage at
Arg506, suggesting that activated platelets protect
platelet-derived factor Va in part by delaying cleavage at
Arg506. This concept is supported by the observations of
Tans et al,7 who reported that platelet (<1 × 107/mL) anticoagulant activity increased more than 10-fold
when reaction mixtures were stirred as compared with unstirred
mixtures, suggesting that the generation of platelet-derived
microparticles increased cofactor inactivation and overcame the
apparent protection afforded by the intact platelet.
The ability of activated platelets to protect factor Va from
APC-catalyzed inactivation is not due to either an insufficient number
of membrane binding sites for APC or the cofactor or to the destruction
of these sites, because the addition of plasma-derived factor Va to the
reaction mixture resulted in its delayed, yet complete, inactivation by
APC. This observation also eliminates the possibility that platelets
contain and secrete an APC inhibitor, an hypothesis articulated by Jane
et al,54 who observed that intact platelets require
substantially higher concentrations of APC to inactivate plasma-derived
factor Va compared with phospholipid vesicles. Rather, we would argue
that activated platelets express a membrane component(s) that can
protect both the plasma- and platelet-derived cofactors from
APC-catalyzed inactivation. This membrane component may represent a
specific platelet binding site for factor Va that has yet to be
identified.
Although the mechanism by which platelets protect their associated
factor Va from APC-catalyzed inactivation remains to be elucidated, our
data clearly indicate that both normal platelet-derived factor Va and
factor VaLeiden express an apparent APC-resistant phenotype
in that they are inactivated at near identical rates and retain
substantial cofactor activity, despite the presence of APC and
independent of the membrane surface to which they are bound. These
combined observations and the differences between the composition of an
arterial and venous thrombus may help explain at the molecular level
why there is a lack of association between factor VLeiden
and arterial thrombosis.22-24 The composition of a thrombus
is highly dependent on location and hemodynamic factors and differs greatly in arterial and venous thrombosis. Venous thrombi are formed
under conditions of hypercoagulability in areas of stasis and are
mainly composed of fibrin and red blood cells, but with relatively few
platelets. Hence, plasma-derived factor Va or factor VaLeiden would be the predominant cofactor involved in
thrombus formation. Because of increased resistance of plasma-derived
factor VaLeiden to APC, this mutation would predispose to
and be associated with venous thrombosis. In contrast, arterial thrombi
form in regions of high flow, subsequent to injury (eg, atherosclerotic
plaque rupture) and are composed mainly of platelets bound by fibrin strands. Because the main cellular procoagulant surface in arterial thrombi is the platelet, the predominant cofactor pool at the site of
arterial injury would be platelet-bound, platelet-derived factor Va or
factor VaLeiden. In fact, studies have indicated that the
concentration of platelet-derived factor Va within a platelet-rich
arterial thrombus is significantly increased (>100-fold) over
plasma-derived factor Va.31 Because platelet-derived factor
Va from both normal individuals and individuals homozygous for factor
VLeiden are equally resistant to APC, factor
VLeiden may not demonstrate a strong association with
arterial thrombosis.
The ability of normal platelets to contribute to an apparent
APC-resistant phenotype is supported by several
studies55-57 that have shown that the addition of platelets
into an APC-resistance assay (APTT ± APC) results in a decrease in
the APC-sensitivity ratio. This effect is enhanced if the platelet
samples have been freeze-thawed before use.55-57 Results
from our plasma-based clotting assay reconstituted with platelets
extend these studies and provide more insight into the mechanism of
this effect. APC addition to a plasma clot formed in the presence of
platelets resulted in substantially delayed factor Va heavy chain
proteolysis at Arg506 relative to observations made when
APC was added to a plasma clot formed in the presence of PCPS vesicles,
which were present at an equivalent concentration of
procoagulant/anticoagulant sites (Fig 6C and D). These observations
were interpreted to indicate that platelets also protect plasma-derived
factor Va from inactivation by APC. As a result of delayed cofactor
inactivation in the presence of platelets, thrombin generation would be
prolonged and an APC resistant phenotype would be observed compared
with assay systems that use synthetic vesicles or rabbit brain
phospholipid.
Delaying APC-catalyzed factor Va inactivation at the activated platelet
surface would lead to the sustained activity of prothrombinase, resulting in ongoing thrombin generation at a site of arterial injury.
One possible consequence of sustained thrombin generation is the
inhibition of fibrinolysis through the enhanced activation of
thrombin-activatable fibrinolysis inhibitor (TAFI). Studies from our
laboratory indicate that APC, which is profibrinolytic in the absence
of platelets,58-60 is virtually ineffective in augmenting fibrinolysis in the presence of platelets and TAFI.61 This
effect could be attributed specifically to the release of APC-resistant platelet-derived factor Va. Thus, in that setting, platelets played both a procoagulant and antifibrinolytic role, resulting in formation of a thrombus resistant to fibrinolysis. This observation is consistent with studies performed in experimental models of arterial
disease.62-65 One study demonstrated that recombinant tick
anticoagulant peptide (rTAP), a potent factor Xa inhibitor, when
administered conjunctively with recombinant tissue plasminogen
activator, can significantly accelerate thrombolytic reperfusion and
prevent acute reocclusion, whereas standard heparin appeared to be far
less effective.63 Therefore, the effects of rTAP in this
system directly implicate de novo ongoing thrombin formation as a major
source of procoagulant activity within a platelet-rich thrombus, with
the sustained thrombin formed most likely due to the APC-resistance of
the released and bound platelet-derived factor Va.
Although the studies reported here demonstrate that platelet-derived
factor Va and factor VaLeiden are not effectively
inactivated at the platelet surface by APC, in vivo studies with
primates suggest that, under arterial flow conditions,66,67
infused APC may be an effective antithrombotic by partially inhibiting
both platelet and fibrin thrombus formation as well as by enhancing the
thrombolytic effectiveness of infused urokinase.68
Thrombin, through activation of protein C, was shown to have similar
effects.69 Although these in vivo studies could be
interpreted to argue that APC is effective in preventing arterial
thrombosis, the mechanism(s) of this effect is not clear. APC may be
exerting this effect by acting as an anticoagulant through inactivation
of the procofactors factors V and VIII as well as the cofactors factors
Va and VIIIa. Alternatively, APC may be exerting a profibrinolytic
effect or reducing platelet activation. Direct conclusions regarding
the mechanism of APC antithrombotic effects may be difficult to make
because, to our knowledge, the concentration of baboon platelet-derived
factor Va and its susceptibility to APC inactivation have not been
investigated, parameters that are essential to consider when evaluating
the effects of APC on a system that involves platelets and the
regulation of thrombin generation at their surface.
Collectively, the results of our study underscore the role that
activated platelets play in sustaining procoagulant events. Platelets
fulfill this function in part by delaying and/or preventing inactivation of their membrane-bound factor Va and through the release
of a pool of factor Va molecules expressing an APC-resistant phenotype.
Such mechanisms will allow for the continuous assembly and function of
the prothrombinase complex via platelet-bound factor Va, which will
lead to the sustained generation of thrombin. Whereas these mechanisms
that platelets use to sustain procoagulant events are a vital part of
their physiological function in preventing hemorrhage subsequent to
vascular injury, these same mechanisms appear to contribute to the
formation and maintenance of thrombi formed in pathological arterial
disease states. APC may not be as effective an inhibitor/inactivator of
those processes. Therefore, effective strategies in attenuating ongoing
thrombin generation in arterial thrombosis will require inhibition of
the bound factor Xa or displacement of the prothrombinase complex from
the surface of the activated platelet.
| |
FOOTNOTES |
Submitted March 17, 1997;
accepted November 11, 1997.
Supported by Grant No. HL P01-46703, Project 4 (to P.B.T.), and Regione
del Veneto, Giunta Regionale-Ricerca Sanitaria Finalizzata No.
483/03/94, Venezia, Italia (to P.S.).
Presented in part in abstract form at 37th Annual Meeting of the
American Society of Hematology, December 1-5, 1995, Seattle, WA
(Blood 86:201a, 1995 [abstr, suppl 1]) and the XVIth
Congress of the International Society on Thrombosis and
Haemostasis, June 6-12, 1997, Florence, Italy (Thromb
Haemost June 1997 [abstr 2504, suppl]).
Address reprint requests to Paula B. Tracy, PhD, Department of
Biochemistry, Given C409, University of Vermont, College of Medicine,
Burlington, VT 05405-0001.
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.
| |
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Abstract
Introduction
Abstract