© 1995 American Heart Association, Inc.
Articles |
Lysine Modification of LDL or Lipoprotein(a) by 4-Hydroxynonenal or Malondialdehyde Decreases Platelet Serotonin Secretion Without Affecting Platelet Aggregability and Eicosanoid Formation
From the Karl-Franzens University, Institute of Medical Biochemistry (E.M., A.I., G.M.K., W.S.), and the Institute of Paediatrics (H.J.L.), Department of Mass Spectrometry, Graz, Austria, and the Research Laboratories of Schering AG (P.F.J.V.), Berlin, FRG.
Correspondence to Ernst Malle, Karl-Franzens University, Institute of Medical Biochemistry, Harrachgasse 21, A-8010 Graz, Austria.
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Abstract |
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Abstract The effects of lysine-modified atherogenic plasma lipoproteins, known to be constituents of human atherosclerotic plaques, were studied on platelet function in vitro. LDL and lipoprotein(a) [Lp(a)] modified with secondary breakdown products of lipid peroxidation (4-hydroxy-2,3-trans-nonenal [HNE] 0.1 to 10 mmol/L or malondialdehyde [MDA] 1 to 50 mmol/L) induced neither spontaneous platelet aggregation nor secretion of 5-hydroxytryptamine (5-HT) from platelet amine-storage granules. Incubation of platelets with HNE- or MDA-modified LDL or Lp(a) (up to 1200 µg protein/mL) prior to thrombin (0.2 U/mL)– or collagen (2 µg/mL)–induced aggregation did not enhance platelet aggregability or formation of eicosanoids, ie, thromboxane A2 or prostaglandins E2 and F2
. In contrast to native
lipoproteins, HNE- or MDA-modified LDL and Lp(a) (
20% to 30% of
total apolipoprotein lysine residues modified) exerted a pronounced
dose-dependent inhibition of 5-HT release from activated platelets in
the following order: HNE LDL (50%)>HNE Lp(a) (40%)>MDA LDL
(20%)>MDA Lp(a) (5%). Preincubation of human blood platelets with
acetylated LDL or Lp(a) (60% to 70% of total lysine residues
modified) prior to aggregation impaired serotonin secretion by 50%
compared with native LDL or Lp(a). These findings suggest that the
interaction of platelets with aldehyde-modified atherogenic plasma
lipoproteins should not necessarily be considered as proatherogenic
with respect to the effects observed in our in vitro studies.
Key Words: platelet-lipoprotein interaction • acetylation • dense-granule secretion • eicosanoids • gas chromatography–mass spectrometry
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Epidemiological studies have shown that high levels of atherogenic plasma lipoproteins are considered a prerequisite for most forms of atherosclerosis.1 Acquisition of cholesterol from the circulation is performed by receptor-mediated endocytosis of LDL.2 For the interaction of LDL with its receptor, intact lysine residues of apolipoprotein (apo) B-100 are required. However, when the extent of modified lysines exceeds about 15%, these particles are no longer recognized by the LDL receptor.3 The uptake and degradation of modified lipoproteins by monocyte-derived macrophages is considered the leading event in the formation of cholesterol-enriched foam cells, which are the hallmark of fatty streaks and the earliest recognizable lesion of atherosclerosis.4 5 6 Some of these lysine modifications, eg, malondialdehyde (MDA)- and 4-hydroxynonenal (HNE)–modified LDL, are of pathophysiological relevance in the development of atherosclerosis. MDA- and HNE-modified atherogenic lipoproteins have been found entrapped in arterial walls of both humans and animals.7 8 9 10
Besides macrophages, blood platelets play a central role during
thrombogenesis and atherosclerosis.11 Exposure of
subendothelium due to vascular damage results in immediate platelet
adherence to subfibrillar collagen. The release of ADP and
5-hydroxytryptamine (5-HT; serotonin) from dense granules,
platelet-derived proteins from the -granules, and thromboxane
A2 (TXA2) formed from arachidonic acid (AA) are
early events in platelet activation and consolidation of thrombi. In
addition, platelets can display atherogenic properties by upregulation
of macrophage scavenger receptor activity, mainly by -granule
release of platelet-derived growth factors in vitro.12 It
is assumed that atherogenic plasma lipoproteins may enhance platelet
function in vivo. Different authors have demonstrated in vitro that LDL
may alter platelet aggregability and release of 5-HT and
TXA2 (for review, see Reference 1313 ), two compounds known as
potent biological vasoactive compounds and thus relevant for vascular
occlusion.11 In contrast to LDL, antiatherogenic HDL are
assumed to exert beneficial effects on platelet function and release
reaction.13 Platelets, like a variety of other cells,
express high-affinity binding sites for atherogenic plasma lipoproteins
that are, however, immunologically different from the apoB/E receptor,
the "classic" receptor of nucleated cells14 ;
platelet membrane glycoproteins, ie, glycoproteins IIb (GPIIb) and
GPIIIa, are responsible for the interaction of platelets with native
atherogenic and antiatherogenic plasma lipoproteins.15 16
Since cyclohexanedione (CHD)-modified LDL loses its ability to bind to
intact platelets,17 it seems reasonable to assume that
free 
-amino groups are involved in lipoprotein-platelet
interactions.18
Therefore, the present study was performed to investigate the
interaction of platelets with lysine-modified atherogenic LDL and
lipoprotein(a) [Lp(a)]. Lp(a) contains all constituents of LDL and an
additional glycoprotein, designated apo(a), with striking homology
to human plasminogen (for review, see Reference 1919 ). Thus, the role of
Lp(a) seems to extend from atherogenesis to thrombogenesis, and Lp(a)
has been considered a "missing link" between atherosclerosis and
thrombosis. To achieve blockage of lysine -amino groups of
lipoproteins, LDL and Lp(a) particles were exposed to increasing
concentrations of HNE and MDA. MDA is formed in vivo, both
nonenzymatically as a product of lipid peroxidation and enzymatically
as a product of the cyclooxygenase pathway during platelet aggregation
at concentrations equimolar to TXA2. In addition, MDA, like
HNE, another secondary breakdown product of lipid peroxidation from AA,
is also known to react with -amino groups of lysine residues by
Schiff-base adduct formation. HNE potentiates aggregation and increases
TXA2 formation in washed platelets challenged with ADP,
thrombin, or ionophore A23187; platelet responses to collagen,
epinephrine, and AA, however, are not affected by HNE (10 to 100
µmol/L).20 HNE concentrations higher than 100 µmol/L
appear to inhibit platelet activation and platelet release reaction in
general,20 21 possibly by modulation of functionally
important SH groups of phospholipase A2.20
Hurst et al21 report an inhibitory effect of HNE on
platelet aggregations of washed platelets in response to collagen,
thrombin, and AA even at low concentrations of HNE (12 to 84
µmol/L).
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Methods |
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Isolation and Purification of Lipoproteins
Protease inhibitors, aprotinin, 0.01 mmol/L phenylmethylsulfonic acid fluoride (Sigma Chemical Co, 0.001 mmol/L D- phenylalanyl-L-propyl-L-arginine chloromethylketone (Calbiochem), 0.01% Na2-EDTA, and 1 mmol/L sodium azide were added to donor plasma immediately after separation. LDL was isolated from apparently Lp(a)-negative plasma by sequential ultracentrifugation at density intervals of 1.020 to 1.050 g/mL and further purified by density gradient centrifugation in an SW-41 rotor (Beckman).22 Lp(a) was isolated by ultracentrifugation (d=1.060 to 1.125 g/mL) from donors with plasma Lp(a) levels >30 mg/dL followed by size-exclusion chromatography on Bio-Gel A-5M. The purity of the Lp(a) fractions was assessed by double-decker rocket immunoelectrophoresis22 by using polyclonal anti-human apoB and anti-human apo(a) antisera (Behring). Separation of apo(a) isoforms was performed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 3.75% slab gels) using a discontinuous buffer and gel system. Phenotyping of apo(a) was performed by means of different apo(a) isoform standards (Immuno) using monoclonal and polyclonal anti-human apo(a) antibodies followed by peroxidase-conjugated immunoglobulins using 4-chloro-1-naphthol as a substrate.23 The different apo(a) isoforms were B type, B/S1 type, S1 type, S2 type, and S2 low type (migrating slightly faster in 3.75% SDS-PAGE than S2).
Modification of Lipoproteins
Prior to modification, lipoproteins were desalted and purified
from preservatives by dialysis or size-exclusion chromatography on
Sephadex PD-10 columns (Pharmacia). The chemical composition of the
lipoprotein fractions (triglycerides, phospholipids, total cholesterol,
and cholesterol esters) was estimated by using commercially available
enzymatic test kits and found to be in a similar range as
described.22 The protein content of the samples was
estimated according to the method of Lowry et al24 using
bovine serum albumin as a standard. To avoid inadvertent oxidation of
lipoproteins during the modification procedures, all buffers were
stored over Chelex 100 (BioRad) to remove contaminating,
redox-available transition metals. All buffers were extensively purged
with nitrogen before dialysis.
HNE Modification
HNE modification of lipoproteins (1 to 1.5 mg protein/mL)
was performed with an aqueous HNE solution for 4 hours at 37°C under
nitrogen after acidic saponification of
HNE-diethylacetal.25 Final HNE concentrations were 0.1, 1,
5, and 10 mmol/L, respectively. HNE LDL and HNE Lp(a) were extensively
dialyzed against Ca2+-free Tyrode's solution (in
mmol/L): 137 NaCl, 2.68 KCl, 0.42
NaH2PO4, and 1.7
MgCl2, pH 7.35.23
MDA Modification
MDA modification of lipoproteins was performed as described by
Haberland et al.26 Briefly, LDL and Lp(a) (1 mg protein/mL
phosphate-buffered saline, pH 7.4) were incubated in the presence of
increasing volumes of a freshly prepared MDA solution (0.2 mol/L in 0.1
mol/L sodium phosphate buffer, pH 6.4) for 3 hours at 37°C under
nitrogen to obtain an increasing degree of lysine modification. The
final MDA concentrations in the reaction mixture were 1, 5, 10, 25, and
50 mmol/L, respectively. The reaction was stopped by dialysis against
Ca2+-free Tyrode's solution at 4°C.
Acetylation
Acetylation of lipoproteins was performed by the method of Basu
et al.27 Briefly, 1 mL 0.15 mol/L NaCl containing 15 mg
LDL or Lp(a) protein was added to 1 mL of saturated solution of sodium
acetate under continuous stirring under nitrogen at 0°C.
Subsequently, multiple 2-µL aliquots of acetic anhydride were added
to the stirred solution (final ratio of protein/acetic anhydride,
1:1.5, wt/wt). After stirring for an additional 30 minutes at 0°C,
the reaction solution was extensively dialyzed against
Ca2+-free Tyrode's solution at 4°C.
Electrophoretic mobility of native, HNE- and MDA-treated, and acetylated lipoproteins was assessed by agarose electrophoresis using the lipidophor system (Immuno).
Estimation of Reactive Amino Groups
Reactive apoprotein amino groups were estimated with
trinitrobenzenesulfonic acid.28 Protein (50 µg) from
native or modified LDL or Lp(a) was mixed with 1 mL NaHCO3
(4%, wt/vol; pH 8.4) and 50 µL trinitrobenzenesulfonic acid in
H2O (0.1%, vol/vol). After incubation for 1 hour at
37°C, 100 µL HCl (1N) and 100 µL SDS (10%) were added.
Absorbance was measured at 340 nm. The standard curve (using valine as
a standard) was linear in the range 5 to 50 nmol NH2.
Platelet Aggregation Studies and Eicosanoid Analysis
Human gel-filtered platelets (GFPs) were isolated from
platelet-rich plasma (PRP) by column chromatography on a Sepharose 2B
column that had been equilibrated with freshly prepared
Ca2+-free Tyrode's solution (pH 7.35; 0.2% human
serum albumin).23 29 Platelet counts were adjusted to
200 000 GFP/µL by means of a Thrombocounter-C system. GFPs were
incubated with different concentrations of native or modified LDL or
Lp(a) (up to 1200 µg protein/mL) at 37°C in tightly closed
Eppendorf cups for up to 30 minutes. Platelet aggregations induced with
collagen (0.2 or 2 µg/mL) or thrombin (0.2 or 0.5 U/mL) under
continuous stirring (1000 rpm for 6 minutes at 37°C) were monitored
by the change in light transmission at 640 nm.29 After
acidification to pH 3.2 and the addition of
18 O2-labeled TXB2 and
D4-prostaglandin F2
(D4-PGF2) and
D4-PGE2 (20 ng per 50 µL methanol) as
internal standard, eicosanoids were extracted with diethylether,
purified by silicic acid column chromatography, and estimated by
negative-ion chemical ionization–gas chromatography–mass
spectrometry.29 Preparation of the trimethylsilyl
derivatives of eicosanoids was performed as
described.30
Measurement of 5-HT Release
For serotonin release measurements, PRP was prelabeled with
[14C]5-HT (0.868 mmol/L; Sigma; specific activity, 2.13
GBq/mmol; final concentration, 1.5 µmol/L) for 15 minutes at 25°C
in tightly closed Eppendorf cups. After gel filtration of
serotonin-labeled PRP, the resulting GFP suspension was incubated in
the presence of native or modified lipoproteins at 37°C for 30
minutes. Prior to thrombin- or collagen-induced stimulation of GFPs,
platelet-lipoprotein suspensions (500 µL) were incubated with
imipramine (2 µmol/L; Sigma) to prevent uptake of released
serotonin.31 Thrombin- or collagen-induced 5-HT release
was stopped by adding 80 µL formaldehyde (final concentration, 6.33
mmol/L) and EDTA (final concentration, 6.5 mmol/L).
Platelet-lipoprotein suspensions were centrifuged at 10 000 rpm for 3
minutes, and aliquots (100 µL) of supernatants were counted on a beta
counter (LKB).
Measurement of Intracellular Cyclic Nucleotides
cAMP and cGMP were measured by an enzyme-immunoassay system
according to the manufacturer's suggestions (Amersham). After
incubation of GFPs with lipoproteins, cells were lysed with ice-cold
ethanol; after centrifugation, supernatants were dried under nitrogen
and dissolved in assay buffer prior to analysis.
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Results |
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Treatment of lipoproteins with different concentrations of HNE or MDA resulted in modification of free
-amino groups and changes in
net charge and relative electrophoretic mobility (REM) as a further
consequence (Table 1
). Low concentrations of HNE (0.1
and 1 mmol/L) or MDA (1 and 5 mmol/L) resulted in slightly increased
REM and loss of free LDL and Lp(a) NH2 groups. The higher
the degree of lysine modification (MDA, 10 and 25 mmol/L; HNE, 5 and 10
mmol/L), the higher the REM of the corresponding lipoprotein. The most
pronounced effect on REM was observed for MDA-treated LDL. Treatment of
Lp(a) with HNE resulted in a reduced trinitrobenzoic acid reactivity by
30% and 33%; the corresponding values for LDL were 10% and 17%.
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-Amino
Group Content of LDL and Lp(a)We first examined whether lysine-modified lipoproteins may directly activate platelets. Incubation (5 to 30 minutes) of platelets (2x107 to 2x108 GFP/mL) with increasing concentrations of HNE- or MDA-modified LDL/Lp(a) (10 to 1200 µg protein/mL) led to neither spontaneous aggregation nor secretion of serotonin from platelet amino-storage granules. Under these conditions cAMP and cGMP concentrations remained at the basal values of 5.28±0.86 and 2.55±0.34 pmol/108 GFPs, respectively.
We then studied aggregations from activated platelets that were
preincubated with lipoproteins (up to 1200 µg protein/mL). Neither
native LDL or Lp(a) nor the HNE- or MDA-modified lipoproteins
influenced platelet aggregation (including shape change, slope values,
and maximal aggregation wave expressed as percent change in light
transmission) in response to thrombin (Table 2) or
collagen (data not shown). To confirm that the percentage of lysine
modification was not a determining factor for altered platelet
aggregation, the same experiments were performed in the presence of
acetylated lipoproteins (about 70% of total reactive amino groups are
lost). Neither acetylated LDL (acLDL; REM, 2.9±0.31; 31±10% free
amino groups, n=6) nor acLp(a) (REM, 2.18±0.35; 37.2±8% free amino
groups, n=6) significantly altered platelet aggregability (Table 2).
Parallel measurements of platelet-derived cyclooxygenase metabolites by
gas chromatography–mass spectrometry analysis revealed similar
concentrations of eicosanoids secreted upon platelet incubation in the
presence of native or lysine-modified lipoproteins. The concentrations
of TXB2, the stable hydrolysis product of
TXA2, ranged between 13.1 and 21.5 ng
TXB2/1x108 GFPs. Levels of
PGE2 and PGF2 were between 1.5 and 2.6 and
0.82 to 1.2 ng/108 GFPs, respectively. Collagen-induced
platelet aggregation (final concentration, 2 µg/mL platelet
suspension) led to similar levels of TXB2 (21.4±3.5
ng/1x108 GFPs) when platelets were preincubated with
either native or HNE- or MDA-modified LDL or Lp(a). These data agree
with reports of GFP response after stimulation with thrombin or
collagen without the addition of lipoproteins.29 30
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As 5-HT response might be considered a more sensitive parameter than
aggregability, we studied serotonin secretion of thrombin-activated
platelets after preincubation with increasing concentrations of
lipoproteins. Under our experimental conditions neither native LDL nor
Lp(a) (up to 750 µg protein/mL) induced changes in
[14C]serotonin secretion from the amine-storage granules
of labeled GFPs. A dose-dependent [14C]serotonin
secretion was found when platelets were preincubated with LDL (Fig 1A) or Lp(a) (Fig 1B) modified with different HNE
concentrations. The maximum inhibitory effect of HNE LDL (10 mmol/L
HNE, 84% free amino groups) was observed at a protein concentration of
200 µg HNE LDL protein. HNE Lp(a) (10 mmol/L HNE, 65% free amino
groups) resulted in 40% inhibition of platelet-derived 5-HT at low
protein concentrations [40 µg HNE Lp(a)/0.4x108
GFP/mL]. At protein concentrations of 200 to 400 µg/mL and high HNE
concentrations (10 mmol/L, 20% of total amino groups blocked), LDL
inhibited [14C]serotonin secretion slightly more than the
corresponding Lp(a) sample (about 35% of total amino groups blocked).
The maximum inhibition in platelet-derived 5-HT release reaction was
52% for LDL (10 mmol/L HNE) and 40% for Lp(a) (5 and 10 mmol/L HNE),
respectively. HNE Lp(a) (5 and 10 mmol/L HNE) displayed its maximum
inhibitory effect on serotonin release reaction in the presence of 50
µg Lp(a) protein. The same was true for HNE LDL at a threshold
protein concentration of 200 µg HNE LDL/mL. Lipoproteins modified
with HNE concentrations higher than 10 mmol/L are not suitable for
studying their effects on platelet function in vitro due to aggregation
of lipoproteins.32 An increase from 400 to 1200 µg
HNE-modified lipoproteins/mL did not significantly alter serotonin
release reaction of activated platelets.
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In our experiments MDA LDL (40 to 400 µg protein/mL, 25 mmol/L MDA,
69±7% free amino groups) inhibited serotonin secretion by only 20%
(Fig 2A). MDA LDL preparations with 12% to 24% of
lysine residues modified (1 to 10 mmol/L MDA) failed to significantly
inhibit 5-HT secretion. Preincubation of platelets with MDA Lp(a) (up
to 400 µg Lp(a) protein/mL) only marginally influenced serotonin
secretion from prelabeled platelets even with a high degree of MDA
modification (25 and 50 mmol/L, 
30% of free amino groups blocked)
(Fig 2B). A similar pattern of serotonin release reaction was found
when collagen was used to activate platelets.
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To further study the effects of lysine modification of lipoproteins,
platelets were preincubated with acLDL or acLp(a) (comparable
modification rates with respect to free amino groups) prior to thrombin
stimulation. At protein concentrations lower than 40 µg/mL, acLp(a)
(37% free amino groups) slightly increased serotonin release reaction
compared with acLDL (31% free amino groups) (Fig 3).
Higher concentrations of acLp(a) and acLDL (100 to 400 µg
protein/mL), however, decreased the release of serotonin in a
dose-dependent manner up to 50% compared with platelets preincubated
with native LDL or Lp(a). At acLDL concentrations of 400 and 750 µg,
serotonin secretion was inhibited by 48±7% (n=6) and 51±9%,
respectively (n=3); inhibition of serotonin release in the presence of
acLp(a) was in a similar range compared with acLDL and was independent
of the various apo(a) isoforms present in the different Lp(a)
preparations.
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Discussion |
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We investigated the functional properties of chemically modified LDL and Lp(a) on platelet function with special emphasis on the potential of lysine-modified lipoproteins to influence platelet aggregability and release reaction. Acetylated or HNE- or MDA-modified LDL or Lp(a) neither induced spontaneous platelet aggregation nor enhanced platelet aggregability and concomitant release of cyclooxygenase metabolites compared with native LDL or Lp(a). A dose-dependent inhibition of 5-HT secretion by HNE and either acLDL or Lp(a) indicated specific effects of the degree of lysine modification on release reaction from
dense granules (Figs 1
and 3) rather than
aggregability.
The concept of platelet activation and recruitment leading to
progressive occlusion lesions in the coronary or cerebral circulation
are assumed to be related to high plasma concentrations of native
and/or modified atherogenic plasma lipoproteins. LDL and high
concentrations of HDL (2000 µg protein/mL) can induce spontaneous
platelet aggregation.33 At protein concentrations between
25 and 2000 µg/mL, LDL sensitizes platelets to a variety of
stimulating agents, including ADP, calcium ionophore A23187, thrombin,
epinephrine, collagen, or AA, resulting in increased aggregability or
release of serotonin, MDA, or TXA2 from activated
platelets.17 33 34 35 36 37 38 39 40 This phenomenon, however, could be the
result of inadvertent lipid peroxidation processes during LDL
isolation. Meraji and coworkers41 have clearly
demonstrated that a low degree of oxidation (37 to 56 µmol lipid
hydroperoxides/g LDL protein) significantly enhances aggregation of
washed platelets, whereas LDL preparations free of lipid hydroperoxides
have no proaggregatory effect on platelet sensitivity. The present
findings, ie, platelet aggregation of GFPs in the presence of native
LDL and Lp(a) (1200 µg lipoprotein/mL), are in line with these
observations.41 Moreover, native Lp(a) does not alter the
aggregation pattern or the TXB2 or
12-hydroxyeicosatetraenoic acid formation in PRP stimulated with
collagen, thrombin, or ADP.42
Although platelet GPIIb is the main binding protein for native LDL and Lp(a) on human thrombocytes,15 16 the precise regulatory mechanisms of LDL/Lp(a)–platelet interactions have not been revealed in detail. Increased concentrations of platelet-derived TXA2 in the presence of LDL via a phospholipase A2– and Ca2+-mediated mechanism have been discussed.43 LDL may cause increased hydrolysis of polyphosphoinositides and consequently phosphoinositol triphosphate release.44 Stimulation of platelet phospholipase C by LDL may promote phosphorylation of a 47-kD protein considered a sensitive indicator of protein kinase C activity,45 findings that are not consistent with other data.40 LDL may enhance platelet secretion by TXA2-dependent and TXA2-independent mechanisms.40 Although ligand blotting experiments reveal that LDL and HDL may bind to GPIIb and GPIIIa, respectively,15 only LDL seems to increase exposure of fibrinogen binding sites on ADP-stimulated GFPs.46 Van Willigen et al46 have shown that exposure of the GPIIb-IIIa complex, the so-called fibrinogen receptor, is not mediated by formation of proaggregatory eicosanoids, ie, PGG2/H2 and TXA2. Thus, considering all the different effects of LDL on platelets, it is not clear which mechanism predominates at different stages during platelet activation.
It was generally assumed that the higher the degree of oxidation, the
more intensive the proaggregatory effect of LDL. However, the presence
of lipid hydroperoxides significantly in excess of 100 µmol/g LDL
protein does not result in direct aggregation of
platelets.41 Findings that Cu2+-induced
oxidation of LDL may increase47 or decrease48
platelet aggregability might be a result of the different LDL
subspecies, antioxidant content, and the amount of lipid hydroperoxides
formed during Cu2+-mediated oxidation. The presence of
heterogenous lipid and protein degradation products in different
Cu2+-oxidized LDL and oxidatively modified Lp(a)
preparations (Reference 2222 , Fig 6) might additionally contribute to
these controversial findings. Pedreño et al49 report
that Cu2+-oxidized LDL is able to bind to washed platelets
with the same apparent affinity as native LDL. In contrast, Endemann et
al50 provide evidence that platelet GPIV must be
considered the receptor for Cu2+-oxidized
125I-LDL. The inability of native and acLDL to compete with
Cu2+-oxidized LDL binding on washed platelets clearly shows
different binding properties for oxidatively modified and acetylated
lipoproteins to platelets. This is not surprising since aldehyde- and
copper-oxidized modified lipoproteins differ in their physical and
chemical properties.51 Modification of free -amino
groups by HNE or MDA to an extent observed with other lysine
modifications17 may abolish specific binding of these
lysine-modified lipoproteins to resting and/or activated platelets.
However, in ligand blotting experiments no differences in binding
properties of native and CHD LDL to platelet membrane protein fractions
have been observed.52 Surya et al40 report
that native LDL has the same effect on thrombin-induced 5-HT secretion
as does lysine-modified LDL. This suggests that the specific binding
sites on platelets that bind intact LDL but not lysine-modified LDL are
not involved. After carefully studying the influence of prolonged
incubation (4 hours) with lipoproteins (2 mg/mL) on platelet function,
Surya and coworkers53 report a considerable 5-HT secretion
and TXB2 production of LDL-treated platelet suspensions
even in the absence of agonists. Lysine-modified (carbamylated) LDL
induced these responses to a lesser extent than that observed for
unmodified LDL (Reference 5353 , Table 2 and Fig 7). Shmulewitz et
al17 also report a dose-dependent decrease of
thrombin-induced platelet aggregation (25% to 47%) and serotonin
secretion (52% to 59%) by increasing concentrations of CHD LDL (25 to
200 µg protein/mL). Hassall and coworkers33 have further
shown that ADP- and epinephrine-induced platelet aggregations are not
influenced by CHD LDL even at concentrations of 3000 µg protein/mL;
our data on platelet aggregability and TXB2 formation of
thrombin- and collagen-stimulated GFPs preincubated with acetylated or
HNE- or MDA-treated LDL or Lp(a) (up to 1200 µg protein/mL) agree
with these findings.33
The carbohydrate composition of apoB-100 may significantly affect the interaction of LDL with its receptor on different cells.54 Watanabe et al55 report that the incubation of platelets with LDL glycated in vitro enhanced the reactivity of washed platelets to thrombin, collagen, and ADP and the products of TXB2 to a greater extent than incubation with control LDL. However, both the reactivity of platelets to the aggregating agents and the production of TXB2 were similar for the various LDL preparations, although their degree of glycosylation (another form of lysine modification) varied according to the concentration of glucose (10 to 150 mmol/L) in the incubation media. In contrast to these results,55 Meraji et al41 have shown that glycosylation of LDL does not increase the sensitivity of platelets under conditions in which all precautions were taken to avoid parallel oxidation of LDL.
The mechanism of reduced serotonin secretion mediated by lysine-modified lipoproteins as observed in the present study suggests effects independent of aggregability, eicosanoid secretion, and cyclic nucleotide levels. Whether lysine-modified LDL or Lp(a) may activate platelet protein kinase C via Ca2+-dependent or Ca2+-independent mechanisms56 is currently under investigation. One may speculate that lysine-modified lipoproteins may influence platelet thiol content, resulting in a disturbed pattern of endogenous lipoxygenase metabolites.20 Impaired concentrations of glutathione and reduced glutathione peroxidase activity may lead to increased concentrations of 12-hydroperoxyeicosatetraenoic acid, subsequently reducing platelet serotonin secretion.57
The vascular response to the activation of platelets is a balance of vasodilator activity mediated primarily by adenine nucleotides and vasoconstrictor activity mediated by serotonin and thromboxane. The reduced serotonin release in vitro may imply reduced serotonergic amplification of platelet reaction, observations that might be relevant for physiological or pathophysiological processes during platelet–vessel wall interaction, which so far have only been investigated using oxidatively modified LDL.58 59 Our results indicate that the effects of modified lipoproteins with respect to their atherogenic properties might be quite versatile in different experimental systems. The degree of lysine modification is apparently not the solely responsible factor for this phenomenon, as MDA-modified LDL and Lp(a) have a similar percentage of free NH2 groups as found for HNE-modified LDL. One could speculate that attachment of modified LDL and Lp(a) to platelets provides an important prerequisite to induce subsequent changes in platelet function which, however, may also occur independent of specific binding sites, as discussed for native LDL.40 From our findings, it is apparent that further studies are required to elucidate the role of MDA- and HNE-modified lipoproteins, their interaction with platelets, and subsequent regulatory effects on macrophages and endothelial cells to understand their role in atherogenesis.
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Acknowledgments |
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We acknowledge financial support from the Austrian FWF to E.M. (P 8433 and P 12354 MED), H.J.L. (P 11043), and W.S. (P 10145 MED) and to the Franz Lanyar Foundation (KFU-Graz). The authors are indebted to Professor Esterbauer (KFU-Graz) for providing HNE diethylacetal as well as helpful discussions. We would like to thank H. Dieplinger (Innsbruck, Austria) for providing monoclonal anti–human apo(a) antibodies.
Received August 26, 1994; accepted December 29, 1994.
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References |
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