© 1995 American Heart Association, Inc.
Articles |
Mildly Oxidized LDL Induces Platelet Aggregation Through Activation of Phospholipase A2
From the Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, University of Munich, Germany.
Correspondence to Prof Dr W. Siess, Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Universität München, Pettenkoferstr 9, 80336 München, Germany.
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Abstract |
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Abstract Native LDL and LDL oxidized under various conditions were compared in terms of their ability to activate platelets. Native LDL did not induce platelet shape change or aggregation, even at high concentrations (2 mg protein/mL). LDL was mildly oxidized with either CuSO4 (mox-LDL) or 3-(N-morpholino)sydnonimine (SIN-1–LDL). Analysis of mox-LDL and SIN-1–LDL showed a small increase of dienes (E234nm from 0.28±0.04 to 0.55±0.09, mean±SD) and thiobarbituric acid–reactive substance (from 0 to 10.6±1.5 nmol/mg, mean±SEM), no change in apo B electrophoretic mobility, and a minor (12% to 30%) decrease in polyunsaturated fatty acid content. Interestingly, this small oxidative modification of LDL dramatically changed its effect on platelets. Irreversible aggregation and secretion were induced by a threshold concentration of 0.4 mg protein/mL. In contrast, LDL thoroughly oxidized with CuSO4 (ox-LDL) did not aggregate platelets. Although mox-LDL was depleted in antioxidants (
- and
-tocopherol, - and ß-carotene, and other
carotenoids), incubation of mox-LDL with exogenous
-tocopherol did not reverse its ability to induce
platelet aggregation and secretion. Preincubation of platelets
with the cyclooxygenase inhibitor
aspirin or the phospholipase A2 inhibitors
trifluoperazine, quinacrine, 4-bromophenacyl bromide, and
propranolol completely prevented platelet aggregation
and secretion caused by mox-LDL or SIN-1–LDL. These results indicate
that mildly oxidized LDL activates platelets through a
phospholipase A2/cyclooxygenase–dependent pathway.
The complete inhibition of mox-LDL–induced platelet aggregation by
aspirin could contribute to its beneficial effect in
cardiovascular disease.
Key Words: platelet activation • LDL peroxidation • tocopherol • aspirin • SIN-1–LDL • LDL fatty acids
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Introduction |
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The involvement of platelets in arterial thrombus formation, which triggers myocardial infarction and ischemic stroke, has been well established.1 There is also substantial evidence that platelets contribute to the formation of the atherosclerotic plaque.2 3 4 By interacting with macrophages, platelets are able to increase foam cell formation,5 6 7 the hallmark of the early arteriosclerotic lesion. By secreting mitogens such as platelet-derived growth factor from
-granules, platelets stimulate the chemotaxis and proliferation
of smooth muscle cells that lead to intimal
hyperplasia.3 LDL is a major risk factor in cardiovascular disease. Its interaction with platelets may play an important role in the pathogenesis of atherosclerosis. LDL, but not HDL, has been reported to sensitize platelets to aggregation by physiological stimuli.8 9 10 LDL binding sites on platelets, different from the classic LDL receptor, have been described.11 12 13 14 15 However, the evidence regarding the induction of platelet aggregation by LDL is equivocal. In some studies no platelet aggregating effect of native LDL alone has been observed.8 9 16 17 In others, platelet-stimulating effects of either low (10 to 50 mg LDL protein per liter)14 18 19 or high concentrations of LDL (more than 1 to 2 g LDL protein per liter)20 21 have been described. These discrepancies might be related to differences in LDL isolation. Oxidative modification of LDL, which plays an important role in the pathogenesis of atherosclerosis, has been reported to enhance its ability to activate platelets in some16 17 22 but not all studies.15 23
In none of the above studies was an attempt made to chemically characterize the platelet-activating LDL. LDL oxidation may occur in vivo, and it continues during isolation of LDL in vitro. Thus, in the present study we measured several chemical parameters of native and oxidized LDL in relation to their platelet-stimulating effect. The mechanism of platelet activation by oxidatively modified LDL was also studied.
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Methods |
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Materials
Iloprost was purchased from Schering AG. PBS was from GIBCO, and 3-(N-morpholino)sydnonimine (SIN-1) was kindly provided by Cassella.
-Tocopherol, probucol, ADP, bovine serum
albumin, EGTA, staurosporine, and thrombin receptor
activating peptide and the phospholipase A2
inhibitors quinacrine, trifluoperazine, and 4-bromophenacyl
bromide were obtained from Sigma Chemical Co. Propranolol
(Dociton) was from Rhein-Pharma. Platelet activating factor (PAF)
was purchased from Bachem, the PAF antagonist L652,989 was
from Merck, Sharp & Dohme, and aspirin was from Fluka.
4-Hydroxy-2,3-trans-nonenal was a gift from Prof Esterbauer
(University of Graz, Austria). The thromboxane A2
receptor antagonist BM-13.505 was a gift from Prof
Patscheke (Klinikum Karlsruhe, Germany).
Isolation of Human Platelets
Human platelets were isolated from freshly drawn blood (50
mL) as described previously.24 25 Blood was obtained from
healthy volunteers aged 20 to 35 years who had not taken any medication
for at least 10 days before sampling. The blood was anticoagulated with
1:10 volume of 3.8% (wt/vol) trisodium citrate and
centrifuged for 20 minutes at 180g to yield
platelet-rich plasma. After incubation of platelet-rich plasma
with iloprost (100 nmol/L for 10 minutes at 37°C), platelets were
pelleted by centrifugation at 800g for 20
minutes and resuspended in a buffer, prewarmed to 37°C (pH 6.2),
containing HEPES (20 mmol/L), NaCl (138 mmol/L), KCl (2.9 mmol/L), and
MgCl (1 mmol/L). After centrifugation at
800g for 20 minutes, platelets were resuspended in 8 mL
of the same buffer (but at pH 7.4), and glucose (final concentration 5
mmol/L) was added. The platelet suspension was kept at room
temperature for at least 30 minutes before the measurements.
Preparation of Native Lipoproteins
Blood (400 mL) from unmedicated fasting healthy volunteers was
anticoagulated with EDTA (2.7 mmol/L) and centrifuged at
1500g for 30 minutes at 4°C. Plasma was treated with
NaN3 (7.7 mmol/L), gentamicin sulfate (0.11 mmol/L), ethyl
mercurithiosalicylate (thimerosal) (0.25 mmol/L), and
phenylmethylsulfonyl fluoride (1 mmol/L). LDL (d=1.019 to 1.063
g/mL) was isolated by sequential flotation
ultracentrifugation with a Beckman Ti 50.2 rotor
according to the method of Schumaker and Puppione26 in the
presence of EDTA (1 mmol/L), NaN3 (2 mmol/L), thimerosal
(0.25 mmol/L), gentamicin sulfate (0.11 mmol/L), chloramphenicol (0.25
mmol/L), benzamide (1 mmol/L), and glutathione (0.65 mmol/L). LDL was
dialyzed at 4°C for 36 hours against 15 L of an
N2-saturated buffer (pH 7.4) containing NaCl (150 mmol/L),
chloramphenicol (0.15 mmol/L), and EDTA (0.24 mmol/L) and was then
filter-sterilized (0.22 µm) and stored at 4°C. All LDL
concentrations are given in terms of their protein content as
determined by a modified Lowry method27 with bovine serum
albumin used as a standard.
Lipoprotein Modification
To yield EDTA-free LDL, the freshly dialyzed LDL containing 0.24
mmol/L EDTA was loaded onto an Econo-Pac 10 DG column (Bio-Rad) and
recovered in PBS. Lipoproteins were then concentrated to a final
concentration of 20 mg protein/mL by Centricon-100 concentrators
(Amicon). LDL was mildly oxidized by one of two methods: incubation of
EDTA-free LDL (20 mg protein/mL) with CuSO4 (final
concentration, 640 µmol/L) for 20 hours at 37°C (mox-LDL) or with
SIN-1 (final concentration, 10 mmol/L) for 20 hours at 37°C
(SIN-1–LDL). To produce thoroughly oxidized LDL (ox-LDL), EDTA-free
LDL (0.06 mg protein/mL) was incubated with CuSO4 (final
concentration, 1.66 µmol/L) for 14 hours at room temperature as
described by Esterbauer et al28 and concentrated by
Centricon-100 concentrators to give a final concentration of 20 mg
protein/mL. Acetylated LDL (acetyl-LDL) was prepared as
described,29 washed, and concentrated to 20 mg protein/mL.
The degree of LDL oxidation and acetylation was assessed by
agarose gel electrophoresis (Ciba Corning) as described.30
The kinetics of LDL lipid peroxidation were determined by monitoring of
the formation of conjugated dienes at 234 nm with a Uvicon 930
spectrophotometer (Kontron), after dilution of the concentrated LDL
samples to a concentration of 0.05 mg protein/mL with PBS at the given
time intervals. The thiobarbituric acid–reactive substance (TBARS)
content of native and mox-LDL was determined photometrically as
described.31
Determination of Fatty Acids
Aliquots of the different LDL preparations were diluted to a
final concentration of 1 mg protein/mL in PBS containing 1 mmol/L EDTA
as antioxidant. Lipids were extracted with chloroform:methanol (2:1)
containing 0.2% butylated hydroxytoluene. Phospholipids,
cholesterol esters, triglycerides, and free
fatty acids were separated on aminopropyl-bonded phase Bond-Elut
columns (Baker) as described.32 The lipid eluates were
transesterified with methanolic HCl (90°C, 1 hour) in the presence of
C17:0 as internal standard. Fatty acid methyl esters were recovered
in petroleum benzene and quantified with a Hewlett-Packard 5890A gas
chromatograph by use of a 2.5 mmx30 m DB-225 fused silica
capillary column. Carrier gas was helium at a flow rate of 5 mL/min.
Injection and ionization temperatures were 90°C and 200°C,
respectively.
Determination of Antioxidants
Aliquots of the different LDL solutions were diluted to a final
concentration of 0.7 mg protein/mL in PBS containing 1 mmol/L EDTA.
Proteins were precipitated with ethanol and total lipids extracted with
hexane. Antioxidants were separated on a C18 reverse-phase column
(Ultrasphere ODS, 0.5x15 cm, Beckman) with
acetonitrile/dichloromethane/methanol/NH4 acetate
(70:20:10:0.01) as the mobile phase at a flow rate of 1.5
mL/min,33 and they were detected on a programmable
multiwavelength detector (Waters 490 E, Millipore).
Platelet Aggregation and Secretion
Platelet aggregation was measured by use of either a
double-channel aggregometer (Fresenius) or a Lumi-Aggregometer
(Chronolog).24 The Lumi-Aggregometer was calibrated with
0.4 mL platelet resuspension buffer (see above) containing 2%
platelet-poor plasma. ATP secretion was measured in the
Lumi-Aggregometer after addition of 40 µL of a mixture of
luciferin/luciferase (Chronolog). Samples (0.4 mL) of platelet
suspension were transferred into aggregometer cuvettes,
platelet-poor plasma (2%) was added, and platelets were
incubated for 1 minute at 37°C while being stirred at 1100 rpm and
were then exposed to mox-LDL, SIN-1–LDL, or ox-LDL.
Statistical Analysis
Statistical analysis was performed with Student's
t test for paired data. Results are expressed as
mean±SEM.
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Results |
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Effect of LDL Oxidized by Various Methods on Platelets
To study the interaction of LDL with platelets, we used suspensions of washed platelets to circumvent the possible interference of plasma proteins such as HDL or fibrinogen. Native LDL (2 mg protein/mL) isolated under conditions to prevent oxidation did not cause shape change or aggregation (Fig 1
, top).
Platelet aggregation was observed only in the presence of
subthreshold concentrations of other agonists such as ADP, indicating a
platelet-sensitizing effect of native LDL (data not shown).
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It has previously been shown that oxidation renders LDL either
more16 17 22 or less23 reactive to
platelets. We oxidized LDL by three different methods. The tracings
shown in Fig 1
demonstrate that the method of LDL oxidation was crucial
to the effect on platelets.
mox-LDL produced by incubation of LDL (20 mg protein/mL) with 640
µmol/L Cu2+ for 20 hours at 37°C induced shape change,
maximal irreversible platelet aggregation, and dense granule
secretion (see Fig 2). The average threshold
concentration of mox-LDL was 0.4 mg protein/mL, but varied among LDL
prepared from different donors. Within single LDL preparations, the
dose-response curve of mox-LDL on platelet aggregation showed a
very narrow range. A small, reversible aggregation was observed with
0.3 mg mox-LDL protein per milliliter, whereas 0.4 mg mox-LDL protein
per milliliter induced a maximal irreversible aggregation (data not
shown). Incubation of platelets with CuSO4 alone (final
concentration, 64 µmol/L) did not evoke platelet activation.
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ox-LDL, produced as described by Esterbauer et al,28 induced shape change only, and did not evoke platelet aggregation.
SIN-1–LDL caused platelet shape change and aggregation (final
concentration, 2 mg protein/mL), although significantly less than
mox-LDL (Fig 1, top). It has recently been shown that the sydnonimine
SIN-1 can initiate the peroxidation of LDL in vitro.34 35
SIN-1 liberates both superoxide and nitric oxide during its
auto-oxidation, resulting in the formation of hydroxyl radicals. Nitric
oxide liberated from SIN-1 is also known to inhibit platelets and
thus might antagonize the proaggregatory effect of SIN-1–LDL. Indeed,
10 mmol/L SIN-1 incubated in buffer for 20 hours at 37°C completely
inhibited irreversible platelet aggregation induced by 10 µmol/L
thrombin receptor activating peptide (data not shown). The
platelet-aggregating effect of SIN-1–LDL may thus be much more
pronounced, but it is apparently counterbalanced by the
simultaneous production of the
platelet-inhibiting nitric oxide. LDL incubated under the same
conditions with only 100 µmol/L SIN-1 or PBS had no
platelet-activating effect (data not shown).
Acetyl-LDL, a known ligand for the scavenger receptor that in other
cell types binds ox-LDL,36 did not induce platelet
aggregation (Fig 1, top).
Chemical Properties of LDL Oxidized by the Various
Methods
Electrophoretic Mobility of Apo B
Oxidation of LDL can lead to a modification of the apoprotein B100
that increases its electrophoretic mobility on agarose
gels.37 We found that the electrophoretic mobility of both
acetyl-LDL and ox-LDL was increased, as described
previously.37 38 In contrast, mox-LDL and SIN-1–LDL
showed only a minor change in electrophoretic mobility (Fig 1, bottom).
Thus, the apoprotein B100 was only minimally modified by the methods
used to produce the latter two lipoproteins.
Diene Production and TBARS
To determine the degree of lipid peroxidation of LDL oxidized by
the various methods, we measured the increase in absorbance at 234 nm
by conjugated dienes. mox-LDL showed only a small increase of diene
production after 20 hours compared with native LDL. Absorption
at 234 nm increased only twofold, from 0.28±0.04 to 0.55±0.09
(mean±SD; n=10, P<.0009). Similar values were obtained
with SIN-1–LDL (data not shown). In contrast, the diene absorption
increased fourfold to values of 1.2 to 1.3 in ox-LDL produced according
to the method of Esterbauer et al.28 We found that not
only the ratio between LDL and Cu2+ concentration but
also the absolute concentration of the former determined the kinetics
of diene production (data not shown). High LDL concentrations
(20 mg protein/mL) seemed to protect against oxidation although
Cu2+ was present in excess. This might be explained by
a decreased probability of contact of all LDL particles with molecular
oxygen. In support of this explanation, we found that the diene
absorption of mox-LDL increased to a value of 1.3 after the
concentrated mox-LDL sample (20 mg protein/mL) was diluted to 0.05 mg
LDL protein per milliliter and 1.6 µmol/L Cu2+ and
incubated for 1 hour at 20°C (data not shown). Continuous measurement
of diene formation during oxidation of the concentrated LDL (20 mg
protein/mL) was not possible because of its interfering absorption at
234 nm. By measuring dienes in diluted LDL samples at 15-minute
intervals during oxidation, we found that the amount of dienes formed
over time varied among the different LDL preparations (Table 1). Although the absolute diene content of mox-LDL was
not predictive of its platelet-aggregating effect (data not shown),
a threshold value of diene production of 
E
0.08 (absorption
after oxidation minus absorption before oxidation) appeared to be
necessary to obtain platelet aggregation.
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No TBARS were detected in native LDL. mox-LDL contained 10.6±1.5 nmol TBARS per milligram of protein (mean±SEM).
Fatty Acid Composition
Polyunsaturated fatty acids (PUFAs) constitute about 50% of
total fatty acids of native LDL. Linoleic acid (C18:2 n-6), the
predominant PUFA, is present mainly as cholesterol
ester. Arachidonic (C20:4 n-6) and docosahexaenoic
(C22:6 n-3) acids, present in smaller amounts, are most prevalent
in the phospholipids (Table 2). The LDL fatty acid
composition changes during oxidation because of the oxidative
degradation of PUFAs.28 39 As shown in Table 2, the degree
of change in fatty acid composition was dependent on the method of
oxidation used. The fatty acid composition of mox-LDL or SIN-1–LDL
changed only slightly. In both, the content of PUFAs such as
arachidonic and docosahexaenoic acids decreased, by
12% to 29%. Linoleic acid (C18:2 n-6) content did not change in any
lipid fraction. Linoleic acid, because of its primary location in the
LDL core and its lower unsaturation, is less susceptible to oxidation
than the other PUFAs. In ox-LDL produced according to the method of
Esterbauer et al,28 all of the PUFAs were dramatically
diminished.
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Antioxidant Content
LDL contains numerous lipophilic antioxidants that protect it from
free radical attack and oxidation. The consumption of these
antioxidants is known to be an early event of LDL oxidation, because
the lipid peroxidation process only enters into a propagating chain
reaction after the complete depletion of
antioxidants.39
As shown in Table 3, the content and distribution of
antioxidants varied greatly between different native LDL preparations.
This is most probably due to different dietary habits of the donors.
High concentrations of the tocopherols seemed to prevent
the complete consumption of carotenes during LDL oxidation. mox-LDL was
completely depleted of tocopherols. In two of three mox-LDL
preparations, all antioxidants were consumed. Similarly, no
antioxidants were detected when LDL was oxidized with 10 mmol/L SIN-1
or according to the method of Esterbauer et al28 (ox-LDL).
Oxidation of LDL with a lower concentration (100 µmol/L) of SIN-1 did
not significantly reduce the tocopherol content of LDL.
This LDL also did not cause platelet aggregation. The results lead
us to assume that the loss of tocopherols from LDL is a
prerequisite for aggregation of platelets by mildly oxidized
LDL.
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Effect of Antioxidants on Platelet Aggregation Caused by
mox-LDL
We examined the hypothesis that the effect of mildly oxidized LDL
on platelet aggregation is mediated by peroxyl radicals formed
during LDL oxidation. mox-LDL was incubated with
-tocopherol for 10 minutes before the mixture was added
to platelets. -Tocopherol is known to be a potent
peroxyl radical scavenger40 and thus should inhibit the
effect of mox-LDL if peroxyl radicals are involved. Surprisingly,
-tocopherol did not inhibit platelet activation by
mox-LDL, as shown in Fig 3. Similar results were
obtained with probucol, a lipid-lowering drug with antioxidative
properties. The results also indicate that neither vitamin E
depletion per se nor peroxyl radicals are responsible for the
platelet-aggregating effect of mox-LDL.
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Mechanism of Platelet Aggregation and ATP Secretion Induced
by mox-LDL
Platelet activation induced by mox-LDL and SIN-1–LDL could be
due to increased production of prostaglandin (PG)
endoperoxides and thromboxane
A2, attributable to increased mobilization of
arachidonic acid. To examine this hypothesis, we
investigated the effect of aspirin, a
cyclooxygenase inhibitor, on
platelet aggregation caused by mox-LDL. As seen in Fig 4, preincubation of platelets with aspirin (1
mmol/L) completely inhibited the effect of mox-LDL (2 mg protein/mL) on
platelet aggregation and ATP secretion (data not shown). Shape
change was not affected. The same results were obtained with the PG
endoperoxide/thromboxane A2
receptor antagonist BM 13.505 (1 µmol/L; data not shown).
Aspirin also inhibited platelet aggregation but not shape change in
response to SIN-1–LDL. These results indicate that mildly oxidized LDL
(mox-LDL and SIN-1–LDL) aggregates platelets through a mechanism
mediated by the formation and action of PG
endoperoxides and thromboxane
A2.
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Surya et al41 recently reported on
arachidonate transfer between lipoproteins and
platelets. To investigate whether the increased production
of cyclooxygenase metabolites leading to enhanced
platelet response was due to a transfer of
arachidonic acid from LDL to platelets, we studied
the effect of phospholipase A2 inhibitors on
platelet aggregation and ATP secretion induced by mox-LDL. As shown
in Fig 2, platelet aggregation as well as ATP
secretion induced by mox-LDL was completely inhibited by preincubation
of platelets with the phospholipase A2
inhibitors quinacrine,42
trifluoperazine,43
propranolol,44 45 and 4-bromophenacyl
bromide.46 The mobilization of arachidonic
acid from platelet phospholipids seems therefore to be essential
for platelet activation by mox-LDL.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The results of our study demonstrate that mildly oxidized LDL (mox-LDL and SIN-1–LDL) but not ox-LDL or native LDL induces platelet aggregation and secretion. The mild oxidation was achieved by incubating highly concentrated LDL (20 mg protein/mL) with CuSO4 or SIN-1. This LDL concentration, which is 100-fold higher than the concentration used conventionally, obviously protected LDL against strong oxidation. In most of the experiments, LDL needed to be incubated with CuSO4 for at least 8 hours at 37°C for us to observe a significant diene formation. In some of the experiments, an incubation time of more than 24 hours was necessary to produce mox-LDL with elevated diene content and platelet-stimulating properties (eg, experiment 4 in Table 1
). The
TBARS content of mox-LDL was also only moderately raised. Although -
and -tocopherols were completely absent in mox-LDL and
SIN-1–LDL, it is possible that LDL was not completely
homogeneously oxidized because of differences in contact
with molecular oxygen. Thus, mox-LDL and SIN-1–LDL could be mixtures
of more and less mildly oxidized LDL. The rather wide protein bands of
mox-LDL and SIN-1–LDL (Fig 1, bottom) also suggest that these
preparations were somewhat heterogeneous.
Interestingly, the incubation of LDL with SIN-1 also resulted in a
mildly oxidized LDL preparation (Tables 2 and 3). SIN-1 liberates
nitric oxide and superoxide (O2-), molecules
that can also be generated by activated macrophages and
endothelial cells of the arterial wall. The
simultaneous generation of superoxide and nitric oxide
results in the formation of an oxidant with hydroxy radical–like
activity capable of initiating oxidation of LDL.34 35 We
found that SIN-1–LDL also induced platelet aggregation and
secretion. In contrast, ox-LDL did not induce platelet aggregation,
but it did induce platelet shape change. The latter observation is
in agreement with a recent morphological study47
describing shape change and pore formation in the platelet plasma
membrane due to LDL (100 µg/mL) heavily oxidized according to the
method of Esterbauer et al.28 Our results, however, are in
contrast to those of another study, in which ox-LDL was found to elicit
platelet aggregation that was, notably, insensitive to inhibition
by aspirin.17 Cellular uptake of acetyl-LDL and ox-LDL is
mediated by scavenger receptors. We found that acetyl-LDL did not
activate platelets and ox-LDL only induced shape change.
These results support the idea that scavenger receptors, if present
on platelets, do not mediate the effect of mildly oxidized LDL.
What is the active component in mildly oxidized LDL responsible
for platelet aggregation? Treatment of mox-LDL with a high
concentration of -tocopherol did not reverse its
platelet-aggregating effect. Therefore, the loss of
tocopherols itself is unlikely to be responsible for the
change of biologically inactive LDL to platelet-activating LDL. The
addition of H2O2 to platelets has been
reported to sensitize platelets to stimuli such as
arachidonic acid and thrombin.48 49 50 51
H2O2 and superoxide anions apparently increase
the amount of free arachidonate in platelets and
enhance its conversion to thromboxanes.50 51 The
absence of an inhibitory effect of
-tocopherol and probucol on mox-LDL–induced
platelet aggregation, however, argues against the possibility that
accumulation of superoxide anions and other oxygen radicals in mox-LDL
is responsible for its platelet-aggregating effect. We did not
observe the platelet activation elicited by
-tocopherol (1 mmol/L) alone that was reported
previously,52 possibly because of the presence of plasma
in our platelet buffer.
It has been reported that minimally modified LDL produced by mild
oxidation with soybean lipoxygenase or phospholipase
A2 activates endothelial
cells.53 One of the active principles of minimally
modified LDL seems to reside in a class of oxidized phospholipids that
are PAF-like substances.53 We found that the effect of
mox-LDL was not inhibited by L 652 989, an antagonist of
the platelet PAF receptor (data not shown). It is therefore
unlikely that the active component of mox-LDL belongs to this class of
PAF-like substances. F2-isoprostanes are biologically
active lipids that are generated by free radical–catalyzed
peroxidation of arachidonic acid and have been found in
Cu2+-exposed LDL.54 55 One of these compounds,
8-epiprostaglandin F2, has been shown
to induce platelet shape change and a slight, reversible
platelet aggregation, both of which are inhibitable by PG
endoperoxide/thromboxane A2
receptor antagonists but not by blockade of platelet
cyclooxygenase.56 In contrast, we
observed that the inhibition of both cyclooxygenase
and the PG endoperoxide/thromboxane
A2 receptor inhibited platelet aggregation but not
shape change induced by mox-LDL or SIN-1–LDL.
8-Epiprostaglandin F2, although
possibly present in mox-LDL, is therefore unlikely to be a
candidate for mediating platelet shape change and platelet
aggregation induced by mox-LDL or SIN-1–LDL.
Another possible biologically active lipid generated during LDL oxidation is 4-hydroxy-2,3-trans-nonenal. In our study this compound, even at low concentrations, inhibited platelet aggregation, as observed previously,57 and did not stimulate platelet aggregation.58
We observed that the active component responsible for platelet activation resided within the mox-LDL particle, because the aqueous vehicle of mox-LDL had no effects on platelets. Recently, it has been demonstrated that both native LDL and ox-LDL interacted with the same high-affinity binding site on platelets.15 Mildly oxidized LDL might bind to the same site but activate the receptor differently and induce a more efficient signal transduction than native LDL or ox-LDL.
We propose that mildly oxidized LDL induces platelet aggregation through activation of phospholipase A2, the liberation of arachidonate from phospholipids, and arachidonate's subsequent metabolism to biologically active prostaglandin endoperoxides and thromboxane A2. Using four different inhibitors of platelet phospholipase A242 43 44 45 46 and a specific inhibitor of platelet cyclooxygenase, we demonstrated that aggregation and secretion induced by mox-LDL and SIN-1–LDL were completely blocked. In previous studies, an increase of [3H]arachidonate and its metabolites has been demonstrated in short-term (10 seconds to 2 minutes) and long-term (4 hours) incubations of [3H]arachidonate-labeled platelets with native LDL.20 41 In one of these studies, platelet aggregation by native LDL was inhibited by indomethacin or aspirin.20 In contrast, no inhibition of serotonin secretion by indomethacin was found in the other study.41 We observed that the effect of mox-LDL resembled the effect of collagen. Platelet aggregation after a low dose of collagen occurs with a time lag and is completely dependent on the release of arachidonate and the formation of cyclooxygenase metabolites.59 In contrast to our findings with mox-LDL, we observed no platelet-aggregating effect of native LDL, although native LDL did sensitize platelets to low concentrations of ADP (data not shown), a finding consistent with previous studies.8 14 15 19 23 60 61 We suggest that the inconsistency of the platelet-aggregating effects of native LDL alone in previous studies is due to the presence of variable amounts of mildly oxidized LDL in the LDL used.
Evidence is accumulating that oxidatively modified LDL contributes to the initiation and propagation of the atherosclerotic lesion.62 Our finding that mildly oxidized LDL (mox-LDL and SIN-1–LDL) but not ox-LDL aggregates platelets is especially intriguing. Mildly oxidized LDL could be formed by activated endothelial cells and monocytes and hence might be present in the circulation at sites of endothelial injury. In contrast to ox-LDL, mildly oxidized LDL is unlikely to be rapidly cleared from the circulation, because its apo B, only minimally modified, will not be recognized by cellular scavenger receptors.63 Mildly oxidized LDL might bind to and activate specific LDL receptors on the platelet surface.12 15 Receptor activation could, through various mechanisms—stimulation of G proteins, protein kinases, or Ca2+ mobilization—induce phospholipase A2 activation.64 65 In the present study, the release of arachidonate from platelet phospholipids and its subsequent cyclooxygenase-dependent conversion to PG endoperoxide and thromboxane A2 were found to be entirely responsible for platelet aggregation and secretion induced by mildly oxidized LDL. The finding that aspirin completely prevented platelet aggregation by mildly oxidized LDL might indicate a novel antiatherogenic action of aspirin that would contribute to its beneficial effects in cardiovascular diseases.
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Acknowledgments |
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This study was supported by grants from the Deutsche Forschungsgemeinschaft (Si 274; A. We 681) and the August-Lenz-Stiftung. The results are part of the thesis (in preparation) of A. Weidtmann at the University of Munich. The authors thank E. Bretzke for expert secretarial help and F. Haag for the skillful photographic reproduction.
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Footnotes |
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Presented, in part, at the Joint 12th World Congress of Cardiology and 16th Congress of the European Society of Cardiology, Berlin, Germany, September 10-14, 1994.
Received May 31, 1994; accepted April 20, 1995.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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