© 1999 American Heart Association, Inc.
Original Contributions |
Low-Density Lipoprotein Enhances Platelet Secretion Via Integrin-
IIbß3–Mediated Signaling
From the Departments of Clinical Chemistry (C.M.H., M.H., M.W.P., H.J.M.v.R.) and Haematology (C.M.H., M.H., H.K.N., J.-W.N.A.), University Hospital Utrecht, and Institute for Biomembranes, Utrecht University, Utrecht, The Netherlands.
Correspondence to Prof dr Jan-Willem N. Akkerman, Department of Haematology, University Hospital Utrecht, PO Box 85500, 3508 GA Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail J.W.N.Akkerman{at}laboratory.azu.nl
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Abstract |
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Abstract—LDL is known to increase the sensitivity of human platelets for agonists and to induce aggregation and secretion independently at high concentrations, but its mechanism of action is largely obscure. To clarify how LDL increases platelet sensitivity, cells were incubated in lipoprotein-poor plasma and treated with collagen at a concentration that induced
20% secretion of
14C-serotonin. Preincubation with LDL (30
minutes at 37°C) enhanced secretion in a dose-dependent manner to
60±14% at a concentration of 2 g LDL protein/L. Similar
stimulation by LDL was seen when secretion was induced by the thrombin
receptor–activating peptide. This enhancement was strongly reduced (1)
in the presence of monoclonal antibody PAC1 against activated
IIbß3, a polyclonal antibody
against IIb, and in the presence of the fibrinogen
peptides GRGDS and HHLGGAKQAGDV; (2) in
IIbß3-deficient platelets; and (3)
after dissociation of IIbß3. In contrast,
binding of 125I-LDL to normal platelets in the presence
of PAC1, anti-IIb, GRGDS, and HHLGGAKQAGDV, and to
IIbß3-deficient platelets was normal.
LDL increased the surface expression of fibrinogen in lipoprotein-poor
plasma and fibrinogen-free medium, suggesting that extracellular and
granular fibrinogen bind to IIbß3 after
platelet-LDL interaction. Platelets deficient in fibrinogen
(<0.5% of normal) or von Willebrand Factor (<1% of normal)
but containing normal amounts of other ligands for
IIbß3 preserved responsiveness to LDL,
indicating that occupancy of IIbß3 was not
restricted to fibrinogen. Inhibition of protein kinase C
(bisindolylmaleimide) diminished fibrinogen binding and sensitization
by LDL; inhibition of tyrosine kinases (herbimycin A) left fibrinogen
binding unchanged but diminished sensitization by LDL. We conclude that
an increased concentration of LDL, such as observed in homozygous
familial hypercholesterolemia, sensitizes
platelets to stimulation by collagen and thrombin
receptor–activating peptide via ligand-induced outside-in signaling
through integrin-IIbß3.
Key Words: lipoproteins • LDL • platelet activation • integrin IIbß3 • protein kinases • signal transduction
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Platelets possess specific high-affinity binding sites for LDL, ranging from 1000 to 8000 copies per platelet with a dissociation constant (KD) value between 40 and 100 nmol/L.1 2 3 4 5 In subjects deficient in apolipoprotein (apo) B or E receptor, LDL binding to platelets is preserved, indicating that another binding site must be involved.3 Platelet aggregation is mediated via fibrinogen binding to sites on the integrin-
IIbß3 complex
that become exposed once the cell is activated. The same
complex has been implicated in the binding of LDL because the separate
subunits bound 125I-LDL on Western blots. Results
of studies in which platelets remained intact have been
contradictory, describing both inhibition2 and no
effect6 on 125I-LDL binding
by antibodies against ß3. Modification of
lysine and arginine residues in apoB100 abolished LDL
binding,7 indicating that the protein moiety of
LDL served as the binding locus.
Several reports have described the synergistic enhancement of
platelet responses by LDL particles. LDL enhanced fibrinogen
binding by ADP8 ; aggregation,
thromboxane A2 formation, and
serotonin secretion by thrombin9 10 ;
and platelet responsiveness to epinephrine and
Ca2+-ionophore.11 Acting as
an independent agonist, very low concentrations of LDL (10 mg
protein/L) induced a rise in cytosolic Ca2+ and
inositol phosphate turnover,12 13
physiological concentrations induced changes in
shape (0.25 to 0.5 g/L) and aggregation (
0.75
g/L),14 and, at concentrations >3 g/L, LDL
triggered phosphorylation of pleckstrin, the 47-kDa
substrate of protein kinase C (PKC).15
Apart from receptor-mediated signaling, LDL may affect platelets by lipid exchange. LDL is a donor of lecithins16 and an acceptor of arachidonic acid.17 LDL-induced sensitization is accompanied by decreased angular movement in the platelet membrane, possibly caused by cholesterol transfer,11 and platelets enriched with cholesterol show increased arachidonic acid release and thromboxane B2 formation.18 Platelets from hypercholesterolemic patients are hyperresponsive (reviewed by Betteridge et al19 ), whereas platelets from patients with abetalipoproteinemia, lacking apoB, respond poorly to different agonists20 or are affected by the abnormal HDLs in these patients.21
In the current study, we investigated the synergism between LDL and a
platelet agonist in more detail. Because platelets are
extremely sensitive to variations in the surrounding medium, cells were
suspended in lipoprotein-poor (LP) plasma to optimally preserve their
responsiveness.9 The results, which were
significant at LDL concentrations equivalent to those typical of
patients with homozygous familial
hypercholesterolemia (10 g LDL mass/L or
more), suggest that the synergistic effects of LDL depend on the active
involvement of
integrin-IIbß3
(glycoprotein IIb/IIIa).
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Methods |
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Materials
LP plasma was obtained from Perimmune Inc, and BSA (demineralized) was obtained from Organon Teknika. Sepharose 2B and protein A-Sepharose were from Pharmacia Biotech, and hirudin was from Behring. 5-Hydroxy [side-chain-2-14C] tryptamine creatinine sulfate ([14C]serotonin; specific activity, 1.85 GBq/mmol) and horseradish peroxidase–linked protein A were purchased from Amersham, and Horm collagen was from Nycomed. 32P-Phosphoric acid and enhanced chemiluminescence reagent were from NEN-Dupont. Thrombin receptor–activating peptide SFLLRN (TRAP) was synthesized with a semiautomatic peptide synthesizer (Labortec AG SP650) according to the method of van Scharrenburg et al.22 Malonaldehyde bis-(dimethyl acetal) was from Brunschwig. Herbimycin A was from Biomol. The bisindolylmaleimide GF 109203X, okadaic acid, and N-octylglucoside were from Boehringer Mannheim. Trypsin inhibitor was from Sigma Chemical Co. Reinforced nitrocellulose sheets were from Schleicher and Schuell. Fluorescein isothiocyanate (FITC)–conjugated monoclonal antibody (mAb) P2 against the intact
IIbß3
complex23 was from Immunotech, and
FITC-conjugated anti-human fibrinogen and the negative control
X949-FITC were from Dako.
Antiphosphotyrosine mAb 4G10 was from Upstate Biotechnology, and
anti-phosphotyrosine mAb PY20 was from Santa Cruz Biotechnology. The
mAb PAC1, against the activated
IIbß3 complex
(immunoglobulin M), was a gift of Dr S.J. Shattil (Scripps Research
Institute, La Jolla, Calif). The fibrinogen-derived peptides GRGDS and
HHLGGAKQAGDV (
400–411) were provided by Dr
H.M. Verheij (Department of Biochemistry, Utrecht University,
Utrecht, Netherlands). The mAb against coagulation factor XI
(XI-3) was a gift of Dr P.A.K. von dem Borne (Department of Hematology,
University Hospital Utrecht). The polyclonal antibody
(pAb) against IIb (IIb), mAb against GPIb
(6F6),24 and mAb RUUSPI.18 (against
P-selectin)25 were raised in our laboratory. All
other chemicals used were of analytical grade.
LDL Isolation
Fresh, nonfrozen plasma from 4 donors, each containing <200 mg
lipoprotein(a) [Lp(a)] per liter, was pooled, and LDL (density, 1.019
to 1.063 kg/L) was isolated by sequential flotation in a Beckman L-70
ultracentrifuge.26 To prevent lipid
modification and bacterial contamination, 0.25 mmol/L PMSF,
0.2 mmol/L thimerosal, 2 mmol/L NaN3,
and 4 mmol/L EDTA (final concentrations) were present during
the first run (20 hours, 175 000g, 10°C). Subsequent runs
(20 hours, 175 000g, 10°C) were performed in the absence
of additives except for NaN3 and EDTA. LDL was
filtered through a 0.45-µm filter (Millipore) and subsequently
dialyzed against 103 vol of 150 mmol/L NaCl
containing 1.5 mmol/L NaN3 and 1 mmol/L
EDTA. LDL was stored at 4°C under nitrogen for no longer than 14 days
and, before each experiment, dialyzed overnight against
104 vol of 150 mmol/L NaCl.
Analysis of LDL Preparations
The purity of LDL preparations was assessed by agarose gel
electrophoresis followed by Fat Red staining (Titan Lipoprotein Gel,
Helena Laboratories). Levels of apoB100 and apoA-1 were measured using
the Behring Nephelometer 100. Possible oxidative modification was
measured by (1) the thiobarbituric acid-reactive substances (TBARS)
method using malonaldehyde bis-(dimethyl acetal) as
standard27 and (2) determination of lipid
peroxides with H2O2 as
standard.28 On agarose gel, LDL was present
as a single band (not shown). Lp(a) (Apotech, Organon Technika) was
<14±7 mg/L (n=6); TBARS values were 0.20±0.07 nmol/mg B100 (n=7),
and lipid peroxides were 6.7±1.9 nmol/mg (n=8). These data are in the
range of recently reported values of 0 to 0.15,11
0.7,28 1.5,29 and
1.6530 nmol/mg apoB100 for TBARS and
5.4±0.328 and 2231 nmol/mg
apoB100 for lipid peroxides. Six LDL preparations were analyzed
for possible contamination by fibrinogen (Laurell technique),
fibronectin (enzyme-linked immunosorbent assay [ELISA]), or von
Willebrand Factor (ELISA); all concentrations were below
detection limits, which were <50 µg fibrinogen, <50 ng fibronectin,
and <5 ng von Willebrand factor/g B100 protein. All LDL
concentrations are expressed as grams of apoB100 per liter, which is
equivalent to total LDL mass per liter after multiplication by a factor
of 5.
Analysis of LP Plasma
Plasma apoproteins were measured with the Behring Nephelometer
100. Fibrinogen concentration was determined according to the methods
of Laurell32 (IEF agarose was from Pharmacia, and
rabbit anti-human fibrinogen, from Behring). Osmolality was determined
by using the Advanced Microosmometer 3 MO Plus (Advanced Instruments).
All other electrolytes and proteins were determined by using the Kodak
Ektachem 750 XRC analyzer (Rochester, NY) according to standard
laboratory techniques. The composition of LP plasma was as follows
(reference values are in parentheses): Na+,
162 mmol/L (136 to 146); K+, 4 mmol/L
(3.8 to 5.0); Ca2+, <0.12 mmol/L (2.2 to
2.6); glucose <0.6 mmol/L but supplemented to 5.0 (3.6 to 5.6);
albumin, 25 g/L (35 to 50); fibrinogen, 0.03 g/L (2.0 to 4.0);
fibronectin, 0.16 g/L (0.2 to 0.4); von Willebrand factor,
0.003 g/L (0.006 to 0.015); apoB100, <0.012 g/L (0.6 to 0.9); and
apoA-1, 0.05 g/L (1.2 to 1.6). The osmolality of LP plasma was 296
mmol/kg (275 to 300 mmol/kg). Because the LP plasma had been
defibrinated by thrombin by the manufacturer, hirudin (final
concentration, 20 U/mL) was added to prevent thrombin-mediated
platelet activation.
Platelet Isolation
Freshly drawn venous blood from healthy volunteers and patients
with different types of bleeding disorders was collected into 0.1 vol
of 130 mmol/L trisodium citrate. Donors claimed not to have taken any
medication 2 weeks before blood collection. Platelet-rich plasma
(PRP) was prepared by centrifugation (10 minutes,
200g, 22°C). Gel-filtered platelets (GFPs) were
isolated by gel filtration through Sepharose 2B equilibrated in
Ca2+-free Tyrode's solution (137 mmol/L
NaCl, 2.68 mmol/L KCl, 0.42 mmol/L
NaH2PO4, 1.7 mmol/L
MgCl2, and 11.9 mmol/L
NaHCO3, pH 7.25) containing 0.2% BSA and 5
mmol/L glucose. GFPs were adjusted to a final count of
2x1011 platelets/L.
Measurement of Dense-Granule Secretion
PRP was incubated with 1 µmol/L
[14C]serotonin for 30 minutes at
37°C, followed by gel filtration as described above. GFPs were
adjusted to pH 6.5 with acid-citrate-dextrose solution for 10
minutes at 400g. The pellet was resuspended in LP or normal
plasma (10 minutes, 500g, 22°C) to a final count of
2x1011 platelets/L. Imipramine (2
µmol/L) was added to prevent reuptake of
[14C]serotonin. Platelets were
incubated with antibodies (2 mg/L) or peptides (100 µmol/L)
before LDL incubation (15 minutes, 37°C) as indicated in the Results
section. Subsequently, platelet suspensions were incubated with
2 g of LDL protein per liter for 30 minutes at 37°C or an equal
volume of 150-mmol/L NaCl unless stated otherwise. After preincubation,
samples were stimulated by collagen, stirred (8 minutes, 900 rpm,
37°C), and collected in 0.15 vol of 1.035-mol/L formaldehyde (0°C,
freshly prepared). After centrifugation (1 minute,
10 000g, room temperature), the supernatant was
analyzed for
[14C]serotonin. Data were expressed
as a percentage of maximal secretion, defined as the secretion induced
by 5 mg/L collagen under the same conditions, unless stated otherwise.
Maximal secretion was 68±12% of total
[14C]serotonin taken up by the
platelets (n=26).
In a few experiments, platelets were treated with the PKC inhibitor bisindolyl maleimide (GF 109203X)33 (5 µmol/L, 1 minute, 22°C) or with the tyrosine kinase inhibitor herbimycin A34 (25 µmol/L, 5 minutes, 22°C) before incubation with LDL.
Dissociation of the IIbß3
Complex
[14C]Serotonin-labeled GFPs
were incubated with EGTA (2 mmol/L, 45 minutes, 37°C) to
dissociate the IIbß3
complex.35 36 37 38 Dissociation was stopped by an
equimolar amount of CaCl2, and platelets were
resuspended in LP plasma as described. Concurrently, the degree of
dissociation was determined by incubating 5x106
GFPs with 10 µL of P2-FITC (50 mg/L, 30 minutes, 37°C), a mAb
specific for the intact
IIbß3
complex.23 Subsequently, 500 µL of Tyrode's
solution containing 10 µg/L of prostaglandin
I2 (pH 6.5) was added and platelets were
washed once (10 minutes, 400g) and taken up in BSA-free
Tyrode's solution supplemented with 1% formaldehyde.
Fluorescence was measured on a Becton-Dickinson FACScan. The
degree of dissociation was measured as the mean fluorescence of
the treated platelets and compared with that of GFPs incubated with
X949-FITC as a negative control. X949-FITC fluorescence was
<0.5% of the fluorescence of P2-FITC.
Binding of 125I-LDL to Human Platelets
LDL was labeled with 125I according to the
method by McFarlane,39 modified by Bilheimer et
al.40 GFPs in Tyrode's solution or LP plasma
were incubated with 125I-LDL (45 minutes,
37°C), and 100-µL aliquots were layered on top of 100 µL of 25%
(wt/vol) sucrose in Tyrode's solution in microsedimentation tubes
(Sarstedt). Cells were separated from medium by
centrifugation (2 minutes, 12 000g,
22°C), and both fractions were counted in a Cobra gamma-counter
(Packard). Aspecific binding (10% of total binding) was determined
by addition of a 30-fold excess of unlabeled LDL and subtracted from
total binding to obtain specific-binding data.
Measurement of Platelet-Bound Fibrinogen
GFPs in Tyrode's solution or LP plasma were treated as
indicated in Results. Samples of 5 µL were added to 5 µL of
anti-fibrinogen-FITC in 40 µL HEPES-Tyrode buffer (145 mmol/L
NaCl, 5 mmol/L KCl, 1 mmol/L MgSO4, and
10 mmol/L HEPES supplemented with 1.0% BSA and 0.1% glucose, pH
7.4). After 30 minutes of incubation (22°C), 0.5 mL of
HEPES-Tyrode's solution with 1% formaldehyde (without BSA and
glucose) were added. Fluorescence was measured on the FACScan.
Platelets incubated with LDL (2 g/L, 30 minutes, 37°C) in the
absence of anti-fibrinogen-FITC did not emit fluorescence.
Pleckstrin Phosphorylation
PRP was labeled with 32P-orthophosphoric
acid (60 minutes, 37°C), and GFPs were prepared as described earlier.
Platelets (108 cells) were incubated with the
indicated LDL concentrations (30 minutes, 37°C) or 0.2 U/mL thrombin
(2 minutes, 22°C) in the presence of okadaic acid (1 µmol/L).
The cells were centrifuged through a dibutyl/dinonylphtalate
(60:40 vol/vol) layer (4 minutes, 12 000g) to remove
excess LDL into sample buffer containing ß-mercaptoethanol, SDS, and
glycerol. Quantities of lysate with the same radioactivity were
separated on a 10% SDS-polyacrylamide gel. Proteins were
visualized by using autoradiography.
Immunoprecipitation of Tyrosine-Phosphorylated Proteins
Platelets (108 cells) were treated as
indicated in Results, and incubation was stopped in ice-cold 10x lysis
buffer (1:10 vol/vol) containing 10% Nonidet P-40, 5%
N-octylglucoside, 10 mmol/L
Na3VO4, 20 mmol/L
PMSF, 200 µg/mL trypsin inhibitor, 50 mmol/L
N-ethylmaleimide, and 100 mmol/L benzamidine in Tyrode's
solution. Tyrosine-phosphorylated proteins were
precipitated using 1 µg of PY20 and protein A-Sepharose (100 µL of
a 1% suspension of protein A-Sepharose in lysis buffer) for 5 hours at
4°C. Precipitates were washed 5 times with lysis buffer and taken up
in sample buffer. Proteins were separated by SDS-polyacrylamide
gel electrophoresis (PAGE) using a 7.5% gel and transferred to a
nitrocellulose membrane. Proteins were visualized by incubation with
antiphosphotyrosine mAb 4G10 (0.5 µg/mL, 15 hours, 4°C) and
peroxidase-linked protein A (1:10 000 vol/vol, 1 hour, 4°C) and by
enhanced chemiluminescence.
Patients
Five unrelated patients with Glanzmann's thrombastenia
(CAS, MAV, CPW, NH, and AV), one patient with afibrinogenemia (SS), and
one patient with severe von Willebrand's disease type 3 (AS)
were studied. The diagnosis was based on a markedly prolonged bleeding
time (Simplate, >30 minutes [normal, <8]). The case of patient CAS
has been reported previously.41 On 2-dimensional
(2D) electrophoresis, neither IIb nor
ß3 were detectable after silver staining.
Fluorescence-activated cell sorter (FACS) data revealed
0.2% IIbß3-positive
cells. Patient MAV had thrombocytopenia (72x109
platelets/L), and a faint signal for IIb
and ß3 was detected on 2D gels after silver
staining and radiography of labeled platelet
lysate. FACS analysis revealed 0.3%
IIbß3-positive cells.
The platelet fibrinogen level was normal. Patients CPW, NH, and AV
had 0.4%, 0.7%, and 0.2%
IIbß3-positive
platelets, respectively. In plasma from patient SS, no fibrinogen
clotting activity was detectable and fibrinogen protein content was 2
mg/L42 ; platelet fibrinogen was 10
µg/1011 cells (control, 3
mg/1011 cells).42 Patient
AS had low factor VIII activity (1% of normal), whereas von
Willebrand factor antigen was <1%, and ristocetin-induced
agglutination could not be detected.
Statistics
Data are expressed as the mean±SD with the number of
observations (n) and were analyzed with Student's t
test for unpaired observations. Differences were considered significant
at P<0.05.
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Results |
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LDL Enhances Collagen-Induced Dense-Granule Secretion
Figure 1A
illustrates the
dose-response curve for collagen-induced secretion of
[14C]serotonin for platelets
resuspended in normal and LP plasma. There was a slight decrease in
responsiveness, possibly due to the low concentrations of lipoproteins
in LP plasma. These data indicated that GFPs resuspended in LP plasma
closely resembled PRP in collagen-induced secretion responses.
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) or normal plasma (
) were stimulated with collagen
(0 to 5 mg/L). Dense-granule secretion was measured by extrusion of
[14C]-serotonin and expressed as a percentage
of total [14C]-serotonin. Curves are
representative of 5 experiments. B, GFPs in LP plasma
were incubated with LDL (30 minutes, 37°C) at the following
concentrations: 0 g (
), 1 g (
), and 2 g (
) LDL
protein/L. Dense-granule secretion was measured after stimulation with
collagen (8 minutes, 37°C, 900 rpm). Data are expressed as a
percentage of maximal secretion (5 mg/L collagen, 8 minutes, 37°C,
900 rpm). *P<0.05 (n=4). **P<0.001
(n=26) compared with control values without LDL (n=26).
A 30-minute preincubation (37°C) with 2 g/L LDL failed to induce
secretion. However, this treatment enhanced the responsiveness to 1
mg/L collagen and increased the release of
[14C]serotonin from 20% to 40%
(1 g/L LDL) and 60% (2 g/L LDL) of maximal secretion (Figure 1B
).
Similar results were obtained with stirred suspensions stimulated with
TRAP (3 µmol/L): 2 g/L LDL increased
[14C]serotonin release from
30±15% to 90±13% (n=4, P<0.001). When the suspensions
were not stirred, these data were 47±5% and 75±5% (n=3,
P<0.05), which indicates that the sensitization induced by
LDL was not restricted to stimulation by collagen and was found both
with and without concurrent aggregation.
Role of IIbß3 in LDL-Induced
Sensitization
Because IIbß3 has
been proposed as a binding site for LDL,2 we
investigated whether antibodies directed against
IIbß3 affected the
sensitization induced by LDL. Figure 2A
illustrates the effect of a mAb against the activated
IIbß3 complex (PAC1),
a pAb against IIb (IIb), mAb against GPIb
(6F6), and mAb against coagulation factor XI (XI-3). Furthermore,
peptides derived from the fibrinogen -chain (GRGDS, which binds to
ß3) and the fibrinogen -chain
(400–411, which binds to
IIb)43 were used.
Control experiments showed that these antibodies and peptides did not
change [14C]serotonin secretion
induced by 1 mg/L collagen in the absence of LDL provided that
secretion was 20% (data not shown). A strong decrease in
LDL-induced sensitization was observed with mAb PAC1 and pAb IIb.
Strong inhibition was also seen with GRGDS and
400–411. In contrast, mAb 6F6 and mAb XI-3
(negative controls) had no effect. Thus, interference with ligand
binding to exposed
IIbß3 abolished the
sensitization induced by LDL.
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To investigate whether the inhibitory antibodies and
peptides affected binding of LDL, incubations were repeated in the
presence of 125I-labeled LDL (Figure 2B). Binding
of 125I-LDL was unchanged in the presence of
PAC1, pAb IIb, GRGDS, and 400–411. This
suggests that the interference with LDL-induced sensitization was not
the result of impaired LDL-platelet contact. This conclusion was
confirmed in experiments with platelets from patients with
Glanzmann's thrombastenia, who are deficient in
IIbß3. In the absence
of LDL, Glanzmann's platelets responded normally to collagen,
but the stimulation by LDL was only 3%, compared with 27% in
concurrent incubations with normal platelets (Figure 3A). Separate binding studies of
125I-LDL to platelets from 4 patients
revealed a number of binding sites,
Bmax of 4883±1855 sites per
platelet, and a KD of 123±40 nmol/L
(Figure 3B). These values were similar to those found in control
subjects (Bmax=5222±2025 sites per
platelet, KD=110±57 nmol/L, n=4).
Thus, the lack of sensitization induced by LDL was accompanied by a
99% reduction in
IIbß3 but a normal
number of LDL-binding sites, indicating that the absence of this
integrin caused the sensitization defect.
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To further characterize the role of
IIbß3 in LDL-induced
sensitization, platelets were incubated with EGTA (2 mmol/L,
45 minutes, 37°C). This treatment is known to dissociate the
complex,35 36 37 38 as confirmed by FACS
analysis of P2-FITC binding, a mAb specific for the intact
complex.23 EGTA treatment reduced the number of
intact IIbß3 complexes
by 76±6% (n=5) (Figure 4A).
Collagen-induced [14C]-serotonin
secretion was not affected by EGTA treatment (data not shown). However,
LDL-induced sensitization was reduced from 23% to 10% (Figure 4B),
indicating that the intact
IIbß3 complex is
required for LDL-induced sensitization.
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LDL Initiates Outside-In Signaling Through
IIbß3
To characterize the role of
IIbß3 in LDL-induced
sensitization in more detail, platelets were incubated with LDL and
ligand binding to this integrin was evaluated by FACS analysis
using an FITC-labeled anti-fibrinogen antibody. LDL induced binding of
fibrinogen to IIbß3 in
a dose-dependent manner and a proportional enhancement of
collagen-induced secretion (Figure 5 and
the Table). Because the LP plasma
contained a low (1% of normal) but still significant amount of
fibrinogen, it seemed feasible that the medium was the source of
fibrinogen. However, when incubations were repeated with platelets
suspended in Tyrode's solution, similar data were obtained, indicating
that endogenous fibrinogen could play a role in
IIbß3-mediated
sensitization induced by LDL. This implied that secretion of
-granule contents contributed to the stimulatory effect of LDL. The
secretion was small, however, ranging between 5% and 10% on the basis
of expression of the -granule marker P-selectin (CD62P) and taking
expression induced by 20 µmol/L TRAP (5 minutes) as the maximum.
Restoration of a normal fibrinogen concentration (2 g/L) increased
collagen-induced secretion from 34±9% to 50±8% in the absence of
LDL and from 58±6% to 76±7% (n=4) in LDL-treated suspensions,
illustrating that the stimulation was left intact.
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To investigate ligand specificity of
IIbß3, the studies
were repeated in LP plasma with platelets deficient in fibrinogen
(0.3% of normal) and platelets deficient in von Willebrand
factor (<1% of normal). The LDL-induced increase in secretion by
these platelets was 53±4% and 20±3%, respectively, indicating
that stimulation by LDL was preserved. Thus, other ligands for
IIbß3 present in
the LP plasma and the platelet -granules probably replaced the
deficient proteins.
Protein phosphorylation reactions have been implicated
in both exposure of binding sites on
IIbß3 (inside-out
signaling) and activation pathways induced by ligand-occupied
IIbß3 (outside-in
signaling).44 The involvement of serine/threonine
kinases and tyrosine kinases was investigated using the PKC
inhibitor bisindolylmaleimide and the protein tyrosine
kinase inhibitor herbimycin A.33 34
Expression of surface-bound fibrinogen by LDL was totally inhibited by
pretreatment with bisindolylmaleimide, suggesting that PKC mediated the
LDL-induced exposure and ligand binding to
IIbß3. As expected,
this treatment partly inhibited the secretion induced by 1 mg/L
collagen alone (to 45±17%) since secretion depends on PKC activation.
The enhancement in secretion by LDL was reduced to 15%. In contrast,
herbimycin A failed to affect LDL-induced ligand binding to
IIbß3 but strongly
interfered with the effect of LDL on collagen-induced secretion
(Table).
To confirm that LDL indeed induced protein
phosphorylation, 32P
incorporation in pleckstrin, a major 47-kDa substrate for PKC, and
tyrosine phosphorylation of a number of proteins were
measured. LDL indeed induced phosphorylation of
pleckstrin (Figure 6A and 6B). However,
this phosphorylation was apparent only in the presence
of okadaic acid, an inhibitor of serine/threonine
phosphatases, indicating that LDL is a very weak activator
of PKC.
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LDL induced tyrosine phosphorylation of several
proteins (Figure 6C). This phosphorylation was
diminished in platelets pretreated with bisindolylmaleimide,
suggesting that this step occurred downstream of PKC. Tyrosine
phosphorylation was also reduced in the presence of the
400–411 peptide, indicating that ligand
binding to IIbß3
contributed to outside-in signaling before stimulation with
collagen.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The data presented here indicate that 2 g/L LDL increases the sensitivity of platelets to agonists via outside-in signaling through integrin
IIbß3. This
concentration is above the range of normal subjects (0.6 to 0.9 g/L)
but typical of patients with
hypercholesterolemia. A first step in this
mechanism involves binding of LDL to specific sites on the platelet
surface. The observation that antibodies and fibrinogen-derived
peptides that block ligand binding to
IIbß343
do not disturb the binding of LDL suggests that LDL-binding sites are
not on or near the integrin. Also, the normal binding of LDL to
platelets deficient in
IIbß3 suggests a
distinct LDL-binding site. These findings agree with those recently
reported by Pedreño et al.6 Curtiss and
Plow1 reported that LDL binding to platelets
was unaffected by prolonged incubation with EDTA (3 mmol/L,
37°C, 2 hours), although this treatment dissociates the
IIbß3
complex,36 37 again separating LDL-binding sites
from IIbß3.
The next step is slow induction of fibrinogen binding to surface
IIbß3. This step is
mediated by PKC and independent of tyrosine
phosphorylations. The degree of PKC activation by LDL
is small and can be detected only when
dephosphorylation of pleckstrin is prevented with an
inhibitor of serine/threonine phosphatases. The fact that
LDL activates PKC agrees with results reported
earlier15 and makes the LDL-binding site a true
signaling receptor. The role of PKC in
IIbß3 exposure agrees
with the fibrinogen binding induced by phorbol
ester,45 the sensitivity of thrombin-induced
fibrinogen binding to inhibitors of
PKC,46 47 and the stoichiometric correlation of
ligand binding and phosphorylation of the
ß3 subunit seen in thrombin-stimulated
platelets.46 On the other hand, ADP-induced
ligand binding seems not to depend on PKC.47 48
Normally, signal generation by platelet agonists is extremely
rapid, raising cytosolic Ca2+ and activating PKC
within seconds, which results in immediate exposure of
IIbß3 and induction of
secretion responses. In the current study, a relatively long incubation
with LDL was required before an effect on the platelets could be
observed. Even after 30 minutes of LDL-platelet contact, the
release of 14C-serotonin was the same
as in LDL-free suspensions (<5% of maximal). Also, Weidtmann et
al49 reported that native LDL failed to induce
dense-granule secretion. However, Kaplan et al50
reported that -granule secretion precedes dense-granule secretion at
low concentrations of thrombin, collagen, and
Ca2+-ionophore, resulting in release of minor
amounts of granular fibrinogen. It is possible that a similar
membrane-bound rearrangement of granular fibrinogen mediates the
signaling properties of LDL illustrated in the present report. The
FACS data point to 2 platelet populations, 1 free of fibrinogen and
the other showing maximal binding. The same "all-or-none" response,
defined as "quantal activation" by Frojmovic et
al,51 was reported earlier for platelets
treated with increasing concentrations ADP.
The third step in LDL-induced sensitization involves ligand-induced
outside-in signaling through
IIbß3. This step is
mediated by tyrosine phosphorylations, but concurrent
involvement of PKC cannot be ruled out. Extensive outside-in signaling,
as seen in aggregating platelets, is accompanied by tyrosine
phosphorylation of the
ß3-subunit52 and the
focal adhesion kinase
pp125FAK,53 whereas, under
nonaggregating conditions, occupied
IIbß3 initiates
tyrosine phosphorylation of pp50 to 68 and
pp140.54 Indeed, we found that LDL induces
tyrosine phosphorylation of several proteins. The data
also show that both extracellular and -granular fibrinogen can serve
as ligands for IIbß3.
However, a similar sensitization induced by LDL is seen with
fibrinogen-free platelets and with platelets that are deficient
in von Willebrand factor, a second -granule protein that can
bind to IIbß3. Thus,
in the absence of 1 granule-stored ligand for
IIbß3, a second ligand
can occupy IIbß3 and
preserve the activating properties of LDL. A likely candidate is
fibronectin, which is present in -granules at a relatively high
concentration (3 µg/109
platelets55 ).
Finally, LDL-induced sensitization was evaluated with a secretion test
after stimulation with collagen and TRAP. It is largely uncertain how
collagen induces aggregation and secretion, but
phosphorylation of phospholipase C-2 might be a
central step.56 Similar LDL-induced activation is
seen with TRAP, indicating that platelets also get sensitized for
receptor-coupled G protein signaling. This agrees with results of
earlier studies by Surya et al,10 who reported
that LDL increased the sensitivity of platelets to thrombin. This
sensitization depended in part on cyclooxygenase
activity and was unchanged after lysine modification of apoB100.
Van Willigen et al8 found that ADP-induced
fibrinogen binding was strongly enhanced by LDL, but this property
depended on intact apoB100-lysine residues. These findings favor 2
distinct activating mechanisms of LDL. In contrast, Malle et
al57 found no stimulation by LDL of thrombin- and
collagen-induced dense granule secretion, but their experimental
conditions induced optimal responses, leaving little room for further
stimulation by LDL.
Our results indicate that LDL increases the sensitivity of
platelets to agonists via outside-in signaling through
IIbß3. This
sensitization may have little effect on platelets in the
circulation as long as the LDL concentration is within the normal
range. In contrast, in pathological conditions, such as heterozygous
familial
hypercholesterolemia,58
familial combined
hyperlipidemia,59 nephrotic
syndrome60 (1.6 to 2.0 g/L LDL), and especially
homozygous familial hypercholesterolemia (3.0
to 5.5 g/L LDL),61 62 LDL may induce a state of
hypersensitivity in the platelets that contributes to the
thrombotic tendency observed in these patients.
|
Acknowledgments |
|---|
We are grateful to Drs G. Boonen, I. Hers, and G. van Willigen for fruitful discussions and to Dr E. Harthoorn, Groot Ziekengasthuis, `s Hertogenbosch, for her cooperation. This study was supported by the University Hospital Utrecht.
Received June 13, 1997; accepted June 18, 1998.
|
References |
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