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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1694-1702
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Surface expression and functional characterization of  -granule
factor V in human platelets: effects of ionophore A23187, thrombin,
collagen, and convulxin
L. Alberio,
O. Safa,
K. J. Clemetson,
C.
T. Esmon, and
G. L. Dale
From the W. K. Warren Medical Research Institute and Department of
Medicine, University of Oklahoma Health Sciences Center; the
Cardiovascular Biology Research Program, Oklahoma Medical Research
Foundation, and Howard Hughes Medical Institute, Oklahoma City, OK; and
Theodor Kocher Institute, University of Berne, Switzerland.
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Abstract |
Factor V (FV) present in platelet  -granules has a significant but
incompletely understood role in hemostasis. This report demonstrates
that a fraction of platelets express very high levels of surface-bound,
-granule FV on simultaneous activation with 2 agonists, thrombin and
convulxin, an activator of the collagen receptor glycoprotein VI. This
subpopulation of activated platelets represents 30.7% ± 4.7% of
the total population and is referred to as convulxin and
thrombin-induced-FV (COAT-FV) platelets. COAT-FV platelets are also
observed on activation with thrombin plus collagen types I, V, or VI,
but not with type III. No single agonist examined was able to produce
COAT-FV platelets, although ionophore A23187 in conjunction with either
thrombin or convulxin did generate this population. COAT-FV platelets
bound annexin-V, indicating exposure of aminophospholipids and were
enriched in young platelets as identified by the binding of thiazole
orange. The functional significance of COAT-FV platelets was
investigated by demonstrating that factor Xa preferentially bound to
COAT-FV platelets, that COAT-FV platelets had more FV activity than
either thrombin or A23187-activated platelets, and that COAT-FV
platelets were capable of generating more prothrombinase activity than
any other physiologic agonist examined. Microparticle production by
dual stimulation with thrombin and convulxin was less than that
observed with A23187, indicating that microparticles were not
responsible for all the activities observed. These data demonstrate a
new procoagulant component produced from dual stimulation of platelets
with thrombin and collagen. COAT-FV platelets may explain the unique
role of -granule FV and the hemostatic effectiveness of young platelets.
(Blood. 2000;95:1694-1702)
© 2000 by The American Society of Hematology.
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Introduction |
Platelets activated at sites of vascular injury play 2 key roles in normal hemostasis. By adhering to the exposed
subendothelium and aggregating, they create a physical barrier that
limits blood loss.1 In addition, platelets accelerate
thrombin generation by providing a surface that promotes 2 procoagulant
reactions, conversion of factor X to Xa and production of thrombin from
prothrombin.2 These reactions are performed by homologous,
membrane-bound, Ca++-dependent complexes: the tenase
complex, consisting of the serine protease IXa and the nonenzymatic
cofactor factor VIIIa,3 and the prothrombinase complex,
composed of the serine protease factor Xa and the nonenzymatic cofactor
factor Va.3 Furthermore, activated platelets also control
at least 1 anticoagulant reaction, inactivation of factor Va by
activated protein C (APC).4
Approximately 20% of factor V (FV) contained in whole blood is found
in the  -granules of platelets5 and can be secreted after
platelet activation.6 There is clinical and experimental evidence suggesting that platelet-derived FV plays a critical role in
maintaining physiologic hemostasis. Factor V Quebec was originally
described as an autosomal dominant bleeding disorder characterized by
mild thrombocytopenia, fully functional plasma FV but defective
platelet FV,7 suggesting that platelet-derived FV might be
more important than plasma-derived FV. This concept is reinforced by
the description of 2 patients with acquired inhibitors of FV. A patient
with non-Hodgkin's lymphoma and gastrointestinal bleeding was found to
have a FV inhibitor directed against both plasma and platelet
FV,8 whereas a second patient with a neutralizing inhibitor
active only against plasma-derived FV presented no bleeding tendency,
despite surgical challenge.9 In addition, it has recently
been shown that platelets can protect platelet-derived but not
plasma-derived FV from proteolytic inactivation by APC.10 Together, these observations indicate that platelet-derived,
membrane-bound FV has a pivotal role in promoting and maintaining
hemostasis at sites of vascular damage.
The ability of platelets to sustain assembly and activity of the tenase
and prothrombinase complexes depends on the type of agonist
used,11,12 and this correlates with the agonists' ability to induce expression of negatively charged membrane
phospholipids.13 The most effective agonists are the
Ca++ ionophore A23187, the complement membrane attack
complex C5b-9, and the combined stimuli of collagen and
thrombin.11,12 Therefore, the most important physiologic
stimulus able to induce procoagulant activity at sites of endothelial
damage would be the combined action of thrombin and
collagen.11 In this report, we have investigated the
ability of thrombin plus collagen to elicit platelet-FV surface expression and the functional competence of this platelet-derived FV.
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Materials and methods |
Materials
Collagen type I (calf skin), type III (human), type V (human),
Sepharose CL-2B, FITC-goat-antimouse IgG (FITC-GAMG), and bovine serum
albumin (BSA) were from Sigma Chemical Co, St Louis, MO. Collagen type
VI (human) was obtained from Heyltex Corp, Houston, TX.
Phycoerythrin-labeled streptavidin (PE-SA) and
phycoerythrin-goat-antimouse IgG (PE-GAMG) were provided by Molecular
Probes, Eugene, OR. Thiazole orange (TO) was from Becton Dickinson, San
Jose, CA. Phycoerythrin-labeled annexin-V was from Pharmingen, San
Diego, CA, and FITC-labeled annexin-V was provided by
Boehringer-Mannheim Corp, Indianapolis, IN. Streptavidin TRI-COLOR was
from Caltag Laboratories, Burlingame, CA. Chromogenic substrate S-2238
for thrombin determination was from Chromogenix, Mölndal, Sweden.
Convulxin was purified as published.14 Monoclonal
antibodies (mAb) HFV-237, HVF-227, and HFV-271 against human factor V and HFXa-327 against human factor X were prepared as previously described.15 Prothrombin,16 FXa,17
FV,18 and FV coagulant protein (VCP)19 were
prepared as described. Monoclonal antibodies G5 recognizing P-selectin
and TAB recognizing glycoprotein IIb/IIIa20 were provided
by Dr R. P. McEver, Oklahoma University Health Sciences Center,
Oklahoma City.
Buffers
The following buffers were used: acid citrate dextrose (ACD), 38.1 mmol/L citric acid, 74.8 mmol/L Na3 citrate, 136 mmol/L glucose. Buffered saline-glucose-citrate (BSGC), 129 mmol/L NaCl, 13.6 mmol/L Na3 citrate, 11.1 mmol/L glucose, 1.6 mmol/L
KH2PO4, 8.6 mmol/L
NaH2PO4, pH adjusted with NaOH to either 6.5 or
7.3. Phosphate-buffered saline (PBS), 150 mmol/L NaCl, 10 mmol/L
NaH2PO4, pH 7.4. Saline, 150 mmol/L NaCl.
TBS-gelatin, 100 mmol/L NaCl, 20 mmol/L Tris, pH 7.4 with 0.1% (w/v) gelatin.
Characterization of monoclonals against factor V
The anti-FV mAbs used for analysis of activated platelets were
evaluated for their relative affinity toward FV and FVa. Polystyrene beads (6.4 µ) coated with phosphatidylcholine (PC),
phosphatidylserine (PS), and phosphatidylethanolamine (PE) 40:20:40, as
previously described,18 were provided by Dr Patricia Liaw,
Oklahoma Medical Research Foundation, Oklahoma City. Beads representing
90 nmol/L total phospholipid were incubated in 10 mmol/L HEPES, pH 7.5 with 140 mmol/L NaCl, 2 mmol/L CaCl2, and 1 mmol/L
MgCl2, 1 nmol/L FV or FVa, and 6 nmol/L of FITC-labeled
antibody (HFV-237, 271, or 227) at room temperature for 20 minutes.
Relative fluorescence associated with the beads was then determined by
flow cytometry.
Human platelets
Informed consent was obtained in accordance with local Institution
Review Board guidelines. Five milliliters of whole blood were drawn
with a 19-gauge needle from the antecubital vein into a plastic syringe
containing 0.5-mL ACD. Platelet-rich plasma (PRP) was prepared
immediately by 1:2 dilution of whole blood with room temperature (RT)
BSGC, pH 7.3, and centrifuged in 12 × 75 plastic tubes at
170g for 8 minutes at RT. Gel filtration of the platelets was
performed by layering 2 mL of PRP onto a 25 × 60 mm (30 mL)
column of Sepharose CL-2B equilibrated either with BSGC, pH 6.5 or 150 mmol/L NaCl. Gel-filtered platelets (GFP) were normalized to a
concentration of 4 × 107 platelets/mL in BSGC, pH
7.3 for flow-cytometric studies or to 5 × 107
platelets/mL in 10 mmol/L HEPES, pH 7.5, 140 mmol/L NaCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2 for functional studies.
Collagen preparations
Collagens were dissolved at 1 mg/mL in 85 mmol/L acetic acid
overnight at 4°C. Stock solutions were prepared with a 1:5 dilution in water to yield a final collagen concentration of 200 µg/mL in 17 mmol/L acetic acid and stored in glass tubes as previously described.21
Platelet activation for flow-cytometric studies
Reactions were performed in 12 × 75 mm polypropylene,
round-bottom, culture tubes. For a final concentration of 20 µg/mL, 10 µL of collagen stock solution was diluted with 17 mmol/L acetic acid up to 40 µL and kept on ice until needed. Other agonists (convulxin, thrombin, TRAP, ionophore A23187) were diluted in 1 mg/mL
BSA, 10 mmol/L HEPES pH 7.5, 140 mmol/L NaCl, up to 40 µL.
Immediately before the assay was initiated, 50 µL of RT 100 mmol/L
HEPES pH 7.5, 150 mmol/L NaCl (for collagen), or 10 mmol/L HEPES pH
7.5, 140 mmol/L NaCl (for other activators), each with 4 mmol/L
CaCl2, 2 mmol/L MgCl2, and the relevant
antibodies (see below). The reaction was initiated with 10 µL of GFP,
allowed to proceed for 10 minutes at 37°C, and then stopped with
200 µL of ice-cold 1.5% formalin in PBS (or as described below for
experiments avoiding platelet fixation). After 20 minutes fixation at
RT, 3.5 mL of 1 mg/mL BSA in PBS (BSA/PBS) were added, the platelets pelleted at 1500g for 15 minutes at RT, and the pellet
resuspended in 200 µL BSA/PBS with the appropriate detection system
(see below). After 30 minutes of labeling and a further washing step
when required, platelets were analyzed by flow cytometry.
Detection of surface expressed factor V
Anti-FV-mAb, either underivatized or biotin-conjugated, was present
with platelets during the activation. After fixation and washing as
described previously, underivatized mAb were detected with PE-GAMG,
whereas biotin-conjugated mAb were labeled with 5 µg/mL PE-SA. In a
separate set of experiments we verified that optimal binding of mAb to
exposed FV was obtained within 30 seconds at 37°C. In experiments
investigating the ability of reticulated platelets to express FV,
biotin-conjugated anti-HFV-237 was used. After fixation and washing,
the biotinylated antibody was labeled with PE-SA for 30 minutes.
Platelets were then diluted into 600 µL TO for flow cytometric
studies; compensation parameters were set to avoid cross-over
fluorescence between FL1 (TO) and FL2 (PE-SA).
Analysis of intracellular factor V
Quiescent platelets at 4 × 106/mL in BSGC, pH
7.3, were fixed with 1% (final) formalin in PBS for 20 minutes at RT
and washed with BSA/PBS as previously described. Platelets were
permeabilized with 0.2% (w/v; final) saponin and incubated with 10 µg/mL of the relevant, biotinylated anti-FV monoclonal. After washing
again, the biotinylated antibody was detected with 5 µg/mL PE-SA as above.
Detection of exposed negatively charged membrane phospholipids
Annexin-V was used as a probe for aminophospholipid
exposure.13 GFP were activated in the presence of
PE-labeled annexin-V as detailed previously. After a 10-minute
incubation at 37°C, the 100 µL reaction mix was diluted with 600 µL of 10 mmol/L HEPES pH 7.5, 140 mmol/L NaCl, 2 mmol/L
CaCl2, and promptly assayed by flow cytometry. In
experiments investigating the ability of reticulated platelets to
expose negatively charged aminophospholipids, PE-annexin-V and TO were
used. After 10 minutes incubation at 37°C, the reaction mix was
diluted in 600 µL of TO with 2 mmol/L CaCl2. Flow
cytometer parameters were set to avoid cross-over fluorescence between
FL1 (TO-staining) and FL2 (PE-annexin-V). In
particular, the following controls were performed with each experiment:
a sample of ionophore A23187-activated platelets labeled with
PE-annexin-V but without TO confirmed that FL2 fluorescence did not mimic TO-positive events in the FL1 window, and a
sample of unactivated platelets confirmed that TO-staining did not
mimic positive events in the FL2 window.
Dual labeling experiments with annexin-V and HFV-237 were performed
with slight modification of the parameters previously described.
Platelets were activated with 5 nmol/L thrombin plus 500 ng/mL
convulxin in the presence of PE-annexin-V, 0.5 µg/mL biotinylated
HFV-237 and 4.5 µg/mL underivatized HFV-237 for 10 minutes at
37°C. Streptavidin-TRI-COLOR (5 µg/mL) was then added for 5 minutes at room temperature. Samples were diluted as above and analyzed
for FL2 (PE) and FL3 (TRI-COLOR).
Detection of platelet-derived microparticles
Microparticles (MP) were distinguished according to size and ability
to bind either FITC-annexin-V13 or FITC-TAB.20
In experiments examining the time-dependent generation of MP on
platelet activation, FITC-TAB was used, and the generation of MP was
stopped by diluting the reaction mix in a buffer containing 5 mmol/L
EDTA, 10 mmol/L HEPES pH 7.5, 140 mmol/L NaCl. EDTA stopped MP
generation but did not affect TAB binding. MP were analyzed without
formalin fixation or washing. For flow cytometric analysis of MP,
forward scatter (FSC) was set on E01 (log scale), to allow a better
visualization of the MP, which were defined as particles smaller (less
FSC) than the parent platelet population. MP were expressed as
percentage of total TAB-positive events.
Flow cytometric analysis
Flow cytometry was performed on a Becton Dickinson FACSCalibur,
equipped with an argon-ion laser emitting at 488 nm (Becton Dickinson,
Mountain View, CA). Parameters were set on a log scale.
Prothrombinase assay
GFP were normalized to 5 × 107 platelets/mL in
10 mmol/L HEPES pH 7.5, 140 mmol/L NaCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2. Aliquots of 200 µL were activated with 20 µL of 1 mg/mL BSA in 10 mmol/L HEPES pH 7.5, 140 mmol/L NaCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2 containing either
no supplement (negative control) or the various agonists. After an
incubation time of 10 minutes at 37°C, prothrombin was added to a
final concentration of 1.4 µmol/L, and thrombin generation was
started by addition of FXa (final 1 nmol/L). Every 30 seconds, aliquots
of 20 µL were removed to 80 µL of ice cold buffer, containing 10 mmol/L EDTA, 10 mmol/L HEPES, 140 mmol/L NaCl, 0.5% (w/v) BSA, pH 7.5. Generated thrombin was assessed by adding the chromogenic substrate
S-2238 (20 µL of 2 mmol/L) and measuring the rate of hydrolysis in a
Vmax microplate reader at 405 nm (Molecular Devices). From the rate of
change in absorbance, thrombin concentrations were calculated by
comparison to a standard curve generated with purified thrombin.
In another set of experiments comparing the procoagulant activity of
platelets versus platelet-derived MP, 660 µL of GFP were activated
with various agonists as previously detailed. At different time points
after activation (2, 9, and 19 minutes) 2 aliquots of 100 µL were
removed. One was left untreated, the other was centrifuged at
13 800g for 1 minute.4 Prothrombin was then added
to the first aliquot (platelets plus MP) and to the supernatant of the
second aliquot (MP enriched), thrombin generation started by addition
of 1 nmol/L FXa, and the reaction stopped every 30 seconds up to 2 minutes as previously detailed.
Factor V activity assay
FV coagulant activity was assayed in a 1-stage clotting assay using
FV-deficient human plasma.22 Specifically, 200 µL of GFP
normalized to 5 × 107 platelets/mL in 10 mmol/L
HEPES pH 7.5, 140 mmol/L NaCl, 2 mmol/L CaCl2, and 1 mmol/L
MgCl2 were activated with 20 µL of 1 mg/mL BSA in 10 mmol/L HEPES, 140 mmol/L NaCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2, pH 7.5 containing the various agonists. After 2 minutes incubation at 37°C, a 50-µL aliquot was mixed with 50 µL TBS-gelatin buffer and 50 µL FV-deficient plasma; clot formation
was initiated with 50 µL thromboplastin and monitored at 37°C
using a coagulometer (Diagnostica Stago Model ST4). FV activity was
determined on the basis of a standard curve constructed with normal
plasma.22
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Results |
Characterization of monoclonals against factor V
Three anti-FV mAbs were characterized for their relative affinities
for FV versus FVa. Polystyrene beads coated with phospholipid (PC/PS/PE; 40:20:40) were prepared as previously
described.18 Beads representing 90 nmol/L total
phospholipid were incubated at room temperature with 1 nmol/L FV or FVa
along with 6 nmol/L of the relevant, FITC-labeled antibody. Relative
fluorescence associated with the beads was then determined by flow
cytometry. The data presented in Figure 1
indicate that anti-HFV-237, recognizing the FV light chain, bound both
FV and FVa. Anti-HFV-271, recognizing the heavy chain, also reacted
with both FV and FVa. Anti-HFV-227, recognizing the connecting region
of FV, reacted with FV but not FVa as expected.
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| Fig 1.
Characterization of anti-FV monoclonal antibodies.
Purified FV (solid line), FVa (shaded), and no protein (dotted) were
added to phospholipid-coated beads as described in "Materials and
methods." FITC-labeled antibody against FV light chain
(anti-HFV-237; Panel A), FV heavy chain (anti-HFV-271; Panel B), and FV
connecting region (anti-HFV-227; Panel C) were then added and
particle-bound fluorescence measured by flow cytometry.
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Platelet activation with thrombin and convulxin induces high levels
of factor V surface expression in a discrete fraction of platelets.
We examined the potential for thrombin and convulxin, a specific
agonist for the collagen receptor GPVI,14 to promote
surface expression of -granule FV. Figure
2 shows the ability of thrombin (Figure
2A), convulxin (Figure 2B), and ionophore A23187 (Figure 2C) as single
agonists to induce expression of FV on the platelet surface. These
single agonists were used at concentrations well above that required to
induce maximal -granule release as reported previously23
and confirmed here (data not shown). Figure 2D shows that dual
stimulation with both thrombin and convulxin results in a dramatically
different pattern of FV distribution. A fraction of the platelets
express very high levels of factor V (region M2), whereas the remainder
still express factor V but at lower levels than that observed with
convulxin alone (region M1 minus M2; referred to as low-level FV). We
shall identify the high level of surface FV expression shown in region
M2 as COAT-FV (convulxin and
thrombin-induced FV). Costimulation of
platelets with ionophore plus thrombin or ionophore plus convulxin also
generates a COAT-FV population, although with these nonphysiologic
agonist combinations essentially all platelets express high levels of
surface bound FV. Duplication of the experiments in Figure 2 with
antibody HFV-271 against FV heavy chain gives similar results for all
agonists (data not shown). It is noteworthy that the majority of
platelets stimulated with convulxin plus thrombin (Figure 2D) have a
level of FV below that for platelets activated with convulxin alone (Figure 2B). The basis for this observation is not clear but may be
explained as the mechanism for COAT-FV formation is elucidated. In
addition, we recognize that reactions using thrombin may result in
variable cleavage of platelet-derived FV to FVa or partially activated
FV.24 Because the extent of FV cleavage in these
experiments is uncertain, the term FV will be used here to describe all
platelet-bound forms of FV, FVa, and partially activated FV that may be
formed during these reactions.
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| Fig 2.
Factor V binding to activated platelets.
In a representative experiment, gel-filtered human platelets were
activated with 5 nmol/L thrombin (Panel A), 500 ng/mL convulxin (Panel
B), 2 µmol/L A23187 (Panel C), thrombin plus convulxin (same
concentrations; Panel D), thrombin plus A23187 (Panel E), or convulxin
plus ionophore (Panel F) as described in "Materials and methods."
Surface-bound FV was detected with biotinylated monoclonal antibody
HFV-237 against factor V and phycoerythrin-streptavidin
(FL2). In each panel, control platelets are indicated by
the line histogram, and stimulated cells are depicted with the shaded
histogram. Region M1 represents all cells binding FV, and region M2
represents cells binding very high levels of FV. Cells in region M2 are
referred to as COAT-FV (see text). Experiments performed with antibody
HFV-271 gave similar results to those for HFV-237 (data not shown).
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Figure 3A summarizes the ability of single
agonists to express -granule FV on the surface of activated
platelets. Although these individual agonists are capable of
stimulating FV expression on up to 80% of all platelets, none of them
generate COAT-FV. On the other hand, the combined action of thrombin
and convulxin results in a lower overall level of FV-positive events,
even though this dual stimulation results in COAT-FV
expression (Figure 3B). Furthermore, when thrombin is maintained at 5 nmol/L, there is a clear dose-response to convulxin for generation of
COAT-FV (Figure 3B). When the convulxin concentration is fixed at 500 ng/mL, there is a dose-dependent increase in COAT-FV formation between
0.1 and 1 nmol/L thrombin, whereas with higher thrombin concentrations, the percentage of COAT-FV remains constant (data not shown).
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| Fig 3.
Analysis of surface FV expression elicited by various
agonists.
Platelets were stimulated with various agonists at the concentrations
depicted on the abscissa. In Panel A single agonists were used similar
to experiments in Figures 1A and 1B; the percentage of cells with
surface FV is shown on the ordinate (mean ± 1 SD; n = 3-8). In
Panel B, dual agonist stimulation was performed with thrombin held
constant at 5 nmol/L and convulxin varied from 0.5 to 500 ng/mL. Two
parameters are reported: COAT-FV and low-level FV corresponding to
region M2 and region M1 minus M2, respectively, of Figure 2. Note that
in Panel B the percentage of cells with low-level binding remains
relatively constant, whereas the number of cells with COAT-FV increases
with increasing convulxin concentration (n = 3-12).
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We have also investigated whether COAT-FV is generated by the combined
action of thrombin and collagen. Table 1
summarizes the results obtained with collagen types I, III, V, and VI.
By using all collagens at 20 µg/mL, a concentration that induces maximal -granule degranulation,21 types I, V, and VI are
able to promote COAT-FV to varying degrees, whereas collagen type III only induces low-level FV expression.
Factor V is detectable in all platelets and the complete molecule is
expressed on the surface of activated platelets.
To investigate whether all the platelets contain FV, we permeabilized
formalin-fixed platelets with saponin and incubated them with the 3 mAbs characterized in Figure 1. Figure 4
demonstrates that all 3 mAbs recognize platelet FV and that all
platelets contain FV. These 3 mAbs were also used to show that the
entire factor V molecule is expressed on the surface of COAT-FV
platelets, because each mAb resulted in the same percentage of COAT-FV
cells on activation with thrombin plus convulxin: 30.7% ± 4.7%
(mean ± 1 SD; n = 10) for anti-HFV-237; 29.6% ± 4.9%
(n = 6) for anti-HFV-227; and 30.4% ± 8.9% (n = 6) for
anti-HFV-271.
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| Fig 4.
Detection of intracellular FV in quiescent platelets.
Control platelets were formalin-fixed and permeabilized with saponin as
described in "Materials and methods." Three different
biotinylated, anti-FV monoclonal antibodies were then used to stain
intracellular FV. Panel A is anti-HFV-237 that recognizes the FVa light
chain; Panel B represents anti-HFV-227 that detects the FV connecting
region (B domain); and Panel C is anti-HFV-271 that recognizes the
heavy chain of FVa. The line histograms represent nonspecific antibody,
and the shaded histograms represent the anti-FV monoclonals. All
platelets bind the 3 monoclonals indicating that all platelets contain
the entire FV molecule.
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The exposure of negatively charged membrane phospholipids parallels
the expression of COAT-FV but is not sufficient for its generation.
The generation of COAT-FV in response to thrombin and collagen (Table
1) or thrombin and convulxin (Figure 3B) is restricted to a
subpopulation of platelets similar to previous reports on the exposure
of aminophospholipids by activated platelets.13 We
therefore investigated whether these events might be associated. Exposure of negatively charged membrane phospholipids was monitored with fluorochrome conjugated annexin-V. Figure
5 shows representative flow cytometric dot
plots of annexin-V binding promoted by ionophore A23187 (Figure 5B) and
the combined action of thrombin plus convulxin (Figure 5C). Ionophore
resulted in essentially all platelets binding annexin-V, whereas
thrombin plus collagen generated only a subpopulation of
annexin-V-positive cells. Results obtained from several individuals are
summarized in Figure 6. Except for A23187,
single agonist stimulation of platelets resulted in very modest numbers
of platelets binding annexin-V (Figure 6A); however, even though
ionophore elicits a high level of annexin-V binding, it does not
promote significant COAT-FV expression (Figure 2C). On the other hand, dual stimulation of platelets with 5 nmol/L thrombin and increasing concentrations of convulxin elicited annexin-V binding (Figure 6A).
Interestingly, the percentage of platelets binding annexin-V in
response to the combined stimulus of thrombin plus convulxin is very
similar to the platelet fraction expressing COAT-FV (Figure 6B),
suggesting that the same subpopulation of platelets is positive for
surface FV and annexin-V. This is confirmed in Figure 6C in which dual
staining of thrombin plus convulxin activated platelets indicates that
the COAT-FV and annexin-V-positive populations are identical.
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| Fig 5.
Annexin-V binding to stimulated platelets.
Platelets were stimulated with either 2 µmol/L A23187 (Panel B) or 5 nmol/L thrombin plus 500 ng/mL convulxin (Panel C) in the presence of
phycoerythrin-labeled annexin-V (FL2). Panel A represents
resting platelets. Note that thrombin plus convulxin results in only a
fraction of the platelets binding annexin-V. Additional data are
summarized in Figure 6.
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| Fig 6.
Annexin-V binding to platelets stimulated with single or
dual agonists.
For Panel A, platelets were activated with the various agonists
indicated on the abscissa, and the binding of phycoerythrin-labeled
annexin-V was monitored. All convulxin concentrations represent ng/mL
and T indicates thrombin at 5 nmol/L. Bars represent mean ± 1 SD,
n = 6. Panel B demonstrates the percentage of platelets positive for
COAT-FV (abscissa) and annexin-V (ordinate) on stimulation with 5 nmol/L thrombin and the convulxin concentration depicted in the plot.
Data are extracted from Figures 3B and 6A. Panel C represents dual
labeling of platelets stimulated with convulxin plus thrombin as
described in "Materials and methods." The abscissa
(FL3) depicts biotin-HFV-237/streptavidin TRI-COLOR
binding, and the ordinate (FL2) represents PE-annexin-V
binding. Events in region R1 are positive for both annexin-V and FV.
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Both exposure of aminophospholipids and COAT-FV expression are
increased among reticulated platelets.
Because only a portion of platelets express both COAT-FV and negatively
charged membrane phospholipids in response to the combined action of
thrombin and convulxin, we investigated whether this might be related
to platelet age. To this purpose we used TO, a fluorescent dye that
binds to the remnant RNA still contained in reticulated platelets,
allowing identification of the youngest platelets in the
circulation.25-27 Figure 7
shows the exposure of negatively charged membrane phospholipids in
response to 5 nmol/L thrombin and 500 ng/mL convulxin for a
representative experiment. When TO-negative platelets are examined,
24.2% ± 7.0% (mean ± 1 SD; n = 6) of the cells bind
annexin-V versus 73.1% ± 4.5% (P < .001) for the
TO-positive platelets. Similarly, the percentage of COAT-FV expressing
cells is enriched among the reticulated platelets: only
21.6% ± 3.1% (n = 5) of TO-negative platelets express COAT-FV
in response to thrombin plus convulxin versus 65.6% ± 6.3% of
the TO-positive platelets (P < .001).
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| Fig 7.
Annexin-V binding to thiazole orange-positive and
-negative platelets stimulated with thrombin plus convulxin.
Platelets were stimulated with 5 nmol/L thrombin plus 500 ng/mL
convulxin and then stained with PE-annexin-V to label negatively
charged surface phospholipids and thiazole orange (TO) to identify
reticulated platelets. Panel A represents the TO+
platelets, Panel B depicts the entire population, and Panel C
represents the TO platelets.
|
|
Factor V peak surface expression is functionally relevant.
To assess the functional relevance of COAT-FV, we have used 3 different
approaches. First, we investigated whether COAT-FV is able to bind
plasma factor Xa (FXa). Exogenous FXa was added to platelets incubated
with thrombin plus convulxin. Double labeling with biotin conjugated
anti-HFV-237 and FITC-anti-HFX-327, a mAb directed against FX and FXa
that does not inhibit FXa plasma clotting activity, demonstrated that
all platelets expressing COAT-FV also maximally bind exogenous FXa
(Table 2). Second, we examined the ability
of variously activated platelets to affect the clotting time in
FV-deficient plasma (Table 3). The combined
activation by thrombin plus convulxin is a more potent inducer of
platelet FV-activity than is any single agonist examined
(P < .02); only when ionophore A23187 is potentiated by the
addition of thrombin does the FV activity approximate that observed
with thrombin plus convulxin. Third, we determined the ability of
different platelet agonists to promote platelet dependent-prothrombin
activation. Platelets were activated for 10 minutes with the agonists
indicated in Figure 8, prothrombin and
factor Xa were then added and the initial rate of prothrombin
activation was determined. Prothrombin activation was nearly linear for
the first 4 minutes (Figure 8A). Unstimulated platelets exhibited
little ability to support prothrombin activation. Convulxin stimulated
the platelet activity in a concentration-dependent fashion, and the
combination of thrombin plus convulxin generated more prothrombinase
activity than would be predicted for a simple summation of that for the
individual agonists (Figure 8B).
View larger version (2742K):
[in this window]
[in a new window]
| Fig 8.
Prothrombinase activity generated by single and dual
agonists.
Gel-filtered platelets were stimulated with various single- and
dual-agonist combinations for 10 minutes as described in "Materials
and methods." Platelets were then added to exogenous Factor Xa and
prothrombin, and the initial rate of prothrombin activation was
determined. Panel A depicts a representative experiment and
demonstrates that the initial rates of thrombin generation were linear
for up to 4 minutes. Agonist concentrations were 5 nmol/L thrombin, 500 ng/mL convulxin, and 2 µmol/L A23187. Panel B represents the
prothrombinase activity (nmol/L thrombin generated per minute) for
various agonists. Ionophore (A) was 2 µmol/L; thrombin (T), 5 nmol/L;
and convulxin (CVX) concentration was 500 ng/ml unless specifically
designated (ng/mL) otherwise. Bars represent mean ± 1 SD;
n = 3.
|
|
The role of microparticles (MP)
To define the relative contributions of platelets and
platelet-derived MP to the observed procoagulant activity, we assessed the ability of thrombin and convulxin to promote MP-generation and
compared this with the corresponding thrombin generation rates. Although surface expression of FV and the development of COAT-FV are
essentially complete 2 minutes after platelet activation (Figure 9A), the generation of MP is slower and is
still increasing at 20 minutes (Figure 9B). Also, ionophore A23187
induces a higher percentage of MP than the combined stimulus of
thrombin plus convulxin (Figure 9B). However, when prothrombinase
activity is examined as a function of platelet activation time,
thrombin plus convulxin is a more potent stimulus than ionophore A23187
shortly after platelet activation (3-5 minutes; Figure
10A), a time when MP generation is still
low (Figure 9B). After 10 to 12 minutes of platelet activation, both
stimuli are equivalent in promoting thrombin generation, and after 20 to 22 minutes of activation, the thrombin/convulxin combination is
again superior to that of A23187 alone (Figure 10A). These data suggest
that the generation of microparticles is not tightly coupled to
prothrombinase activity under these conditions. To further address this
question, we assessed the level of residual procoagulant activity after
separation of platelets and MP by centrifugation.4 At any
given time the relative contribution of MP to the measured procoagulant
activity is less than 20% for thrombin plus convulxin (Figure 10B);
whereas the contribution of MP for A23187-induced prothrombinase
activity reaches 40% after activation for 20 minutes (Figure 10B), a
time point at which this agonist is less potent than the combined
stimulus of thrombin plus convulxin in promoting the assembly of a
functional prothrombinase complex (Figure 10A).
View larger version (2421K):
[in this window]
[in a new window]
| Fig 9.
Time course of -granule FV surface expression and
microparticle generation.
A time course for the generation of total FV-positive (region M1,
Figure 2D) and COAT-FV (region M2, Figure 2D) cells after stimulation
with 5 nmol/L thrombin plus 500 ng/mL convulxin is shown in Panel A. Both populations are essentially stable after 3 minutes of activation
(mean ± 1 SD; n = 3). For Panel B, the percentage of MP after 2 to 20 minutes of stimulation was determined as detailed in
"Materials and methods." With 2 µmol/L A23187, there is a
time-dependent increase in the percentage of MP, whereas the absolute
number of MP and their time-dependent increase with 5 nmol/L thrombin
plus 500 ng/mL convulxin stimulation is considerably less.
|
|
View larger version (4134K):
[in this window]
[in a new window]
| Fig 10.
Contribution of microparticles to prothrombinase
activity.
Platelets were activated with either 2 µmol/L A23187 or 5 nmol/L
thrombin plus 500 ng/mL convulxin. At various times of platelet
activation, samples were briefly centrifuged to pellet intact platelets
and leave MP in the supernatant as described in "Materials and
methods." The prothrombinase activity of complete (Panel A) and
microparticle-enriched supernatant (Panel B) samples was then
determined. Prothrombinase rates for Panel A were normalized to the
value for A23187 at 10 minutes (mean ± 1 SD; n = 3). For Panel B,
the MP-related prothrombinase activity is presented as percentage of
activity for the corresponding unfractionated sample.
|
|
| |
Discussion |
In this report we demonstrate that the combined action of 2 physiologic agonists, thrombin and collagen, is able to promote high
levels of -granule factor V expression on the surface of a discrete
fraction of platelets; we have referred to this population as COAT-FV
platelets. Convulxin, a specific agonist for the collagen receptor
GPVI,14 will substitute for collagen in this reaction. We
also show that generation of COAT-FV parallels the exposure of
negatively charged membrane phospholipids, although aminophospholipid exposure is not sufficient to generate COAT-FV platelets. Similarly, -granule release is required but not sufficient for COAT-FV
formation, since we observe a dose-dependent increase in COAT-FV
formation (Figure 3B) with agonist concentrations well above that
necessary to induce P-selectin expression in more than 95% of all
platelets.23 In addition, platelets expressing FV, in
particular COAT-FV, are functionally relevant and quantitatively more
important under these conditions than platelet-derived MP in promoting
procoagulant activity. Finally, our results demonstrate that COAT-FV
formation is enriched in reticulated platelets, suggesting that young
platelets are more likely than aged ones to undergo this
transformation. Previous studies from our laboratory have demonstrated
that aging platelets lose reactivity toward thrombin23 and
collagen/convulxin (manuscript submitted). It is therefore conceivable
that these age-related changes in reactivity toward single agonists are
especially critical for an activation endpoint (COAT-FV formation),
which relies on both of these agonists.
Two different mechanisms appear to control surface binding of FV
released from -granules. Low-level FV expression can be induced by
all agonists examined and is independent from the exposure of
negatively charged membrane phospholipids, confirming the existence of
a FV binding site other than aminophospholipids.28 One
candidate for a phospholipid-independent FV binding site on activated
platelets is GPIa*/multimerin,29 a large disulfide-linked
multimeric protein stored in -granules30,31 which
colocalizes with FV32 and remains associated with the
platelet surface on activation.30,31 On the other side,
COAT-FV expression is only induced by the combined stimulus of 2 agonists, requires the presence of extracellular calcium, results in
the entire FV molecule being present on the cell surface, and parallels
the exposure of aminophospholipids, although the latter is not
sufficient for its generation. Moreover, only platelets expressing
COAT-FV are able to maximally bind exogenous FXa. This is reminiscent
of the model recently proposed by Bouchard et al33 for
EPR-1 mediated binding of FXa. An FV-specific receptor could be
expressed after maximal platelet stimulation, resulting in the
generation of a highly functional procoagulant surface.
COAT-FV, coinciding with aminophospholipid exposure and the highest
ability to bind FXa, theoretically represents the most efficient
substrate for prothrombinase complex assembly. When 2 stimuli inducing
similar amounts of negatively charged phospholipids are compared, the
stimulus able to induce COAT-FV is more efficient in promoting thrombin
generation. For instance, 500 ng/mL convulxin and the combined effect
of 5 nmol/L thrombin plus 5 ng/mL convulxin both induce
aminophospholipid exposure in about 5% of platelets (Figure 6),
however, only the latter stimulus promotes COAT-FV (Figure 3) and this
correlates with higher initial rates of prothrombin activation (Figure
8B). A similar comparison can be drawn between thrombin alone and the
combination 5 nmol/L thrombin plus 0.5 ng/mL convulxin. These
observations demonstrate that COAT-FV, even though it is present in
just a minority of platelets, is functionally more relevant than
low-level FV. Moreover, ionophore A23187, inducing expression of
negatively charged membrane phospholipids in more than 90% of the
platelets (Figure 6) but no COAT-FV (Figure 3), results in
prothrombinase activity approximating that of the combined stimulus of
5 nmol/L thrombin and 500 ng/mL convulxin (Figure 8B), a combination
that promotes aminophospholipid exposure and COAT-FV in only 30% of
the platelets (Figures 2 and 5). Ionophore is an even weaker promoter
of procoagulant activity than the latter combination shortly after
platelet activation (Figure 10A). Finally, we show that, under our
conditions of dual stimulation with thrombin plus collagen,
platelet-derived MP appear to contribute less than 20% of the
prothrombinase activity in the absence of exogenous Va (Figure 10B).
The difference between ours and previous studies12 in the
relative contribution of platelets and platelet microparticles to
prothrombinase activity may reflect that the latter study was performed
in the presence of exogenous factor Va. Despite the fact that MP
generated in vivo can stimulate coagulation34 and that
MP-related procoagulant activity has been implicated in pathologic prothrombotic states,35 our results agree with previous
observations4,36,37 and are consistent with the concept
that under physiologic conditions an adequate hemostatic response must
be rapid and localized to the site of vascular injury. This concept is
supported by the observation that platelet FV appears to be uniquely
important to hemostasis even in patients with near normal levels of
plasma FV.
The current work presents a new model for vascular hemostasis in which
the simultaneous engagement of the collagen receptor, GP VI, and
thrombin activation generates a unique site on platelets that appears
especially capable of supporting prothrombinase. This would provide for
focal thrombin generation restricted to the site of vascular
compromise. Furthermore, the age-dependence of COAT-FV generation may
well explain the clinically observed hyperfunctionality of young
platelets38 as well as the documented decrease in
hemostatic competence of stored platelets.39
| |
Footnotes |
Submitted April 19, 1999; accepted October 28, 1999.
Supported in part by grants HL53 585 (G.L.D.) and P50 HL54 502
(C.T.E.) from the National Institutes of Health, the W. K. Warren
Medical Research Institute, and the Swiss National Science Foundation
(SSMBS grant, LA; 31-52 396.97, K.J.C.). C.T.E. is an investigator of
the Howard Hughes Medical Institute.
Reprints: George L. Dale, PhD, Department Medicine, BSEB-302,
Oklahoma University Health Sciences Center, PO Box 26901, Oklahoma
City, OK 73190; e-mail: george-dale{at}ouhsc.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Ruggeri ZM.
Mechanisms initiating platelet thrombus formation.
Thromb Haemost.
1997;78:611[Medline]
[Order article via Infotrieve].
2.
Rosing J, van Rijn JL, Bevers EM, van Dieijen G, Comfurius P, Zwaal RFA.
The role of activated platelets in prothrombin and factor X activation.
Blood.
1985;65:319[Abstract/Free Full Text].
3.
Jesty J, Nemerson Y.
The pathways of blood coagulation. In:
Beutler E,Lichtman MA,Coller BS,Kipps TJ, eds.
Williams Hematology. New York, NY: McGraw-Hill; 1995:1227.
4.
Tans G, Rosing J, Thomassen MC, Heeb MJ, Zwaal RFA, Griffin JH.
Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles.
Blood.
1991;77:2641[Abstract/Free Full Text].
5.
Tracy PB, Eide LL, Bowie EJ, Mann KG.
Radioimmunoassay of factor V in human plasma and platelets.
Blood.
1982;60:59[Abstract/Free Full Text].
6.
Chesney CM, Pifer D, Colman RW.
Subcellular localization and secretion of factor V from human platelets.
Proc Natl Acad Sci U S A.
1981;78:5180[Abstract/Free Full Text].
7.
Tracy PB, Giles AR, Mann KG, Eide LL, Hoogendoorn H, Rivard GE.
Factor V (Quebec): A bleeding diathesis associated with a qualitative platelet factor V deficiency.
J Clin Invest.
1984;74:1221.
8.
Grigg AP, Dauer R, Thurlow PJ.
Bleeding due to an acquired inhibitor of platelet associated factor V.
Aust N Z J Med.
1989;19:310[Medline]
[Order article via Infotrieve].
9.
Nesheim ME, Nichols WL, Cole TL, et al.
Isolation and study of an acquired inhibitor of human coagulation factor V.
J Clin Invest.
1986;77:405.
10.
Camire RM, Kalafatis M, Simioni P, Girolami A, Tracy PB.
Platelet derived factor Va/VaLeiden cofactor activities are sustained on the surface of activated platelets despite the presence of activated protein C.
Blood.
1998;91:2818[Abstract/Free Full Text].
11.
Bevers EM, Comfurius P, Zwaal RFA.
Platelet procoagulant activity: physiological significance and mechanisms of exposure.
Blood Rev.
1991;5:146[Medline]
[Order article via Infotrieve].
12.
Sims PJ, Wiedmer T, Esmon CT, Weiss HJ, Shattil SJ.
Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: an isolated defect in platelet procoagulant activity.
J Biol Chem.
1989;264:17,049[Abstract/Free Full Text].
13.
Dachary-Prigent J, Freyssinet J-M, Pasquet J-M, Carron J-C, Nurden AT.
Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups.
Blood.
1993;81:2554[Abstract/Free Full Text].
14.
Polgar J, Clemetson JM, Kehrel BE, et al.
Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor.
J Biol Chem.
1997;272:13,576[Abstract/Free Full Text].
15.
Esmon CT, Esmon NL, Le Bonniec BF, Johnson AE.
Protein C activation.
Meth Enzymol.
1993;222:359[Medline]
[Order article via Infotrieve].
16.
Le Bonniec BF, Esmon CT.
Glu-192 [to] Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin.
Proc Natl Acad Sci U S A.
1991;88:7371[Abstract/Free Full Text].
17.
Le Bonniec BF, Guinto ER, Esmon CT.
The role of calcium ions in factor X activation by thrombin E192Q.
J Biol Chem.
1992;267:6970[Abstract/Free Full Text].
18.
Smirnov MD, Safa O, Regan L, et al.
A chimeric protein C containing the prothrombin Gla domain exhibits increased anticoagulant activity and altered phospholipid specificity.
J Biol Chem.
1998;273:9031[Abstract/Free Full Text].
19.
Esmon CT, Jackson CM.
The factor V activating enzyme of Russell's viper venom.
Thromb Res.
1973;2:509
20.
McEver RP, Bennett EM, Martin MN.
Identification of two structurally and functionally distinct sites on human platelet membrane glycoprotein IIb-IIIa using monoclonal antibodies.
J Biol Chem.
1983;258:5269[Abstract/Free Full Text]
21.
Alberio L, Dale GL.
Flow cytometric analysis of platelet activation by different collagen types present in the vessel wall.
Br J Haematol.
1998;102:1212[Medline]
[Order article via Infotrieve].
22.
Safa O, Morrissey JH, Esmon CT, Esmon NL.
Factor VIIa/tissue factor generates a form of factor V with unchanged specific activity, resistance to activation by thrombin, and increased sensitivity to activated protein C.
Biochem.
1999;38:1829[Medline]
[Order article via Infotrieve].
23.
Peng J, Friese P, Heilmann E, George JN, Burstein SA, Dale GL.
Aged platelets have an impaired response to thrombin as quantitated by P-selectin expression.
Blood.
1994;83:161[Abstract/Free Full Text].
24.
Viskup RW, Tracy PB, Mann KG.
Isolation of human platelet factor V.
Blood.
1987;69:1188[Abstract/Free Full Text].
25.
Dale GL, Friese P, Hynes LA, Burstein SA.
Demonstration that thiazole orange-positive platelets in the dog are less than twenty-four hours old.
Blood.
1995;85:1822[Abstract/Free Full Text].
26.
Ault KA, Knowles C.
In vivo biotinylation demonstrates that reticulated platelets are the youngest platelets in circulation.
Exp Hematol.
1995;23:996[Medline]
[Order article via Infotrieve].
27.
Rinder HM, Tracey JB, Recht M, et al.
Differences in platelet -granule release between normals and immune thrombocytopenic patients and between young and old platelets.
Thromb Haemost.
1998;80:457[Medline]
[Order article via Infotrieve].
28.
Nesheim ME, Furmaniak-Kazmierczak E, Henin C, Cote G.
On the existence of platelet receptors for factor V(a) and factor VIII(a).
Thromb Haemost.
1993;70:80[Medline]
[Order article via Infotrieve].
29.
Polgar J, Magnenat E, Wells TNC, Clemetson KJ.
Platelet glycoprotein Ia* is the processed form of multimerin isolation and determination of N-terminal sequences of stored and released forms.
Thromb Haemost.
1998;80:645[Medline]
[Order article via Infotrieve].
30.
Bienz D, Clemetson KJ.
Human platelet glycoprotein Ia*: One component is only expressed on the surface of activated platelets and may be a granule constituent.
J Biol Chem.
1989;264:507[Abstract/Free Full Text].
31.
Hayward CPM, Smith JW, Horsewood P, Warkentin TE, Kelton JG.
p-155, a multimeric platelet protein that is expressed on activated platelets.
J Biol Chem.
1991;266:7114[Abstract/Free Full Text].
32.
Hayward CPM, Furmaniak-Kazmierczak E, Cieutat AM, et al.
Factor V is complexed with multimerin in resting platelet lysates and colocalizes with multimerin in platelet alpha-granules.
J Biol Chem.
1995;270:19,217[Abstract/Free Full Text].
33.
Bouchard BA, Catcher CS, Thrash BR, Adida C, Tracy PB.
Effector cell protease receptor-1, a platelet activation-dependent membrane protein, regulates prothrombinase catalyzed thrombin generation.
J Biol Chem.
1997;272:9244[Abstract/Free Full Text].
34.
Nieuwland R, Berckmans RJ, Rotteveel-Eijkman RC, et al.
Cell derived microparticles generated in patients during cardiopulmonary bypass are highly procoagulant.
Circulation.
1997;96:3534[Abstract/Free Full Text].
35.
Geiser T, Sturzenegger M, Genewein U, Haeberli A, Beer JH.
Mechanisms of cerebrovascular events as assessed by procoagulant activity, cerebral microemboli and platelet microparticles in patients with prosthetic heart valves.
Stroke.
1998;29:1770[Abstract/Free Full Text].
36.
Zwaal RFA, Comfurius P, Bevers EM.
Platelet procoagulant activity and microvesicle formation: its putative role in hemostasis and thrombosis.
Biochim Biophys Acta.
1992;1180:1[Medline]
[Order article via Infotrieve].
37.
Swords NA, Tracy PB, Mann KG.
Intact platelet membranes, not platelet-released microvesicles, support the procoagulant activity of adherent platelets.
Arteriosclerosis Thromb.
1993;13:1613[Abstract/Free Full Text].
38.
Harker LA, Slichter SJ.
The bleeding time as a screening test for evaluation of platelet function.
N Engl J Med.
1972;287:155
39.
Seghatchian J, Krailadsiri P.
The platelet storage lesion.
Transfusion Med Rev.
1997;11:130[Medline]
[Order article via Infotrieve].
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L. Leo, J. Di Paola, B. A. Judd, G. A. Koretzky, and S. R. Lentz
Role of the adapter protein SLP-76 in GPVI-dependent platelet procoagulant responses to collagen
Blood,
September 26, 2002;
100(8):
2839 - 2844.
[Abstract]
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D. M. Monroe, M. Hoffman, and H. R. Roberts
Platelets and Thrombin Generation
Arterioscler Thromb Vasc Biol,
September 1, 2002;
22(9):
1381 - 1389.
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K. Furihata, K. J. Clemetson, H. Deguchi, and T. J. Kunicki
Variation in Human Platelet Glycoprotein VI Content Modulates Glycoprotein VI-Specific Prothrombinase Activity
Arterioscler Thromb Vasc Biol,
November 1, 2001;
21(11):
1857 - 1863.
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S. Kamath, A.D. Blann, and G.Y.H. Lip
Platelet activation: assessment and quantification
Eur. Heart J.,
September 1, 2001;
22(17):
1561 - 1571.
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J. J. Briede, G. Tans, G. M. Willems, H. C. Hemker, and T. Lindhout
Regulation of Platelet Factor Va-dependent Thrombin Generation by Activated Protein C at the Surface of Collagen-adherent Platelets
J. Biol. Chem.,
March 2, 2001;
276(10):
7164 - 7168.
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Top
Abstract
Introduction