© 2000 American Heart Association, Inc.
Thrombosis |
Modification of Protein Moiety of Human Low Density Lipoprotein by Hypochlorite Generates Strong Platelet Agonist
Presented in part at the 17th International Congress of Biochemistry and Molecular Biology, San Francisco, Calif, 1997, and at the XIII Symposium on Platelets, Schloss Seggau, Austria, 1998.
From the Institute of Physiology (I.V., T.M., E.K.), the Institute of Histology and Embryology (E.B.), and the Institute of Biochemistry and Cell Biology (F.K.), University of Vienna, Vienna, Austria.
Correspondence to Dr Elisabeth Koller, Institute of Medical Physiology, University of Vienna, Schwarzspanierstr. 17, A-1090 Vienna, Austria. E-mail elisabeth.koller{at}univie.ac.at
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Abstract |
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Abstract—Conflicting reports exist about the effects of mildly or extensively oxidized low density lipoproteins (LDLs) on the reactivity of human platelets. This platelet response is mainly caused by modification of the protein and lipid moiety, giving rise to very differently modified species with hardly predictable properties. The aim of this study was to prepare oxidized LDL with modifications essentially restricted to the protein moiety and to determine the eventual platelet responses. We treated LDL at 0°C for 10 minutes with a 60- to 1000-fold molar excess of sodium hypochlorite in borate buffer in the presence of the radical scavenger butylated hydroxytoluene. Under these conditions, neither fragmentation of apolipoprotein B-100 nor formation of LDL aggregates was observed, and lipid oxidation products did not exceed the amount present in untreated LDLs. The degree of modification and the respective effects on platelet function were highly reproducible. Hypochlorite-modified LDLs act as strong platelet agonists, inducing morphological changes, dense granule release, and irreversible platelet aggregation. The evoked platelet effects are completely suppressed by inhibitors of the phosphoinositide cycle but not by EDTA or acetylsalicylic acid. Most likely, these effects are transmitted via high-affinity binding to a single class of sites, which does not recognize native or acetylated LDL. Obviously, modified lysines, and the secondary lipid modifications derived from them, are not essential for this interaction. We conclude that bioactive oxidized lipids are not directly involved in the stimulation of platelets by hypochlorite-modified LDLs.
Key Words: atherosclerosis • oxidized LDL • apoB-100 • human platelets • platelet aggregation
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Introduction |
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Platelet–vessel wall interaction (adhesion) and platelet-platelet interaction (aggregation) play a central role in vascular occlusion but are also involved in the earlier stages of development of atherosclerotic plaques.1 Therefore, conditions leading to altered platelet function are accompanied with an enhanced risk of atherosclerosis and thrombosis.2 Among others, severe disorders of plasma lipids lead to enhanced platelet reactivity. In particular, it has been known for >25 years that platelets from hyperlipidemic patients are hyperreactive.3 Furthermore, LDLs from patients with homozygous familial hypercholesterolemia show enhanced susceptibility to oxidative modification,4 thus generating a form that is substantially more atherogenic than unmodified LDLs.5 Native LDLs (nLDLs) reportedly stimulate human platelets,6 7 but minimally modified LDLs rather than nLDLs may be responsible for these effects.8 9 LDLs oxidized in vitro by various agents (oxLDLs) showed even more pronounced platelet activation.10 Because activated platelets may themselves contribute to oxidative modification of LDLs,11 the platelet-stimulatory effect of plasma lipoproteins is potentially a key event in atherogenesis.
Free radicals as well as nonradical oxidants are involved in the oxidative modification of LDL in vivo, so oxLDL certainly does not represent a well-defined species. Accordingly, it proves difficult to assess the atherogenicity contributed by individual modifications. Eventually, all oxidants will cause modification of the lipid moiety (lipid peroxidation, oxidation and loss of cholesterol esters, hydrolysis of phospholipids, and consumption of LDL-bound antioxidants) and the apoB moiety (oxidation and derivatization of amino acids, fragmentation, and cross-linking). The extent and order of these reactions strongly depend on the nature of the oxidizing agent and on the presence of antioxidants within the lipoprotein particle and in its environment. The stimulation of platelets by oxLDLs may be due to the uptake of bioactive oxidized lipids and/or to changes in the lipid domain of platelet membranes. Additionally, the interaction of oxidized areas of apoB with the platelet surface may give rise to signal transduction. Which of these 2 principal possibilities contributes to the effects of oxLDL on platelet function and how much is contributed remain controversial.12 13
We describe the effects of LDLs modified with hypochlorite on washed platelets. Hypochlorite-modified proteins were found in atherosclerotic plaques,14 so the oxidation of LDLs by this agent is likely to occur in vivo. Unlike free radical oxidants, hypochlorite is known to preferentially modify the apoprotein moiety of LDLs,15 and we applied reaction conditions under which only minimal amounts of lipid peroxides were detectable. The reactivity of hypochlorite-modified lipoproteins is highly reproducible, whereas the effects of LDLs oxidized by free radicals are much less predictable.13 The aim of the present study was to assess the specific contribution of the modified apoprotein moiety to the effects of oxLDLs on platelet function.
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Methods |
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NaOCl was from Aldrich; its concentration was determined spectrophotometrically before use (
290=350
L · mol-1 ·
cm-1).
Isolation and Modification of LDLs
Lipoprotein concentrations are always given as microgram protein
per milliliter. All manipulations were performed at 4°C. LDLs
(density 1.019 to 1.063 g/mL) were isolated from fresh normal human
acid-citrate-dextrose plasma by sequential flotation, filtered
(0.45 µm), and immediately used for further modification. Only
LDL preparations with thiobarbituric acid–reactive substances (TBArS)
<0.7 nmol/mg protein were used. KBr was removed by Sephadex G-25
chromatography, and butylated hydroxytoluene (BHT) was
added to a final concentration of 15 µmol/L.
Hypochlorite modification of LDL was based on the method of Arnhold et al16 with the following essential modifications: nLDLs were transferred to 0.1 mol/L sodium borate buffer (pH 7.3) and 0.1 mmol/L EDTA, and 15 µmol/L BHT was added. At 0°C, 10-µL portions of 0.15 mol/L NaOCl were added to give the final concentrations and ratios of NaOCl/LDL as indicated. After 10 minutes, unreacted NaOCl was removed by Sephadex G-25 (0.1 mol/L borate buffer, 0.1 mmol/L EDTA, and 50 mmol/L NaCl, pH 7.3). Hypochlorite-modified LDLs (hypoxLDLs) were always kept on ice and used within 1 week. They were purified by gel filtration on Sepharose-CL 4B immediately before use to remove any aggregates of modified LDL.
Reductive methylation was carried out according to the method of Shepherd and Packard.17 Under these conditions, 60% to 65% of the free amino groups were methylated. Methylated LDLs (metLDLs) were used as such or further modified by hypochlorite (hypmetLDLs), as described above. Acetylation was performed by the method of Basu et al.18 Sixty-five percent to 75% of the free amino groups were modified under these conditions. Acetylated LDLs (acLDLs) were used as such or further modified by hypochlorite (hypacLDLs), as described above.
The electrophoretic mobility of native and modified LDL under nondenaturing conditions was assessed by agarose electrophoresis. SDS-PAGE (3% acrylamide) of nLDL and modified LDL was performed to detect any degradation or cross-linking of the apoB moiety.
Amino groups were estimated with 2,4,6-trinitrobenzenesulfonic acid.19 TBArS were determined according to Mihara and Uchiyama.20 Formation of malondialdehyde and 4-hydroxynonenal was followed by fluorescence measurement at excitation/emission of 400/470 nm and 360/410 to 430 nm, respectively.21
Iodination of Proteins
Radioiodination was performed by the Iodo-Beads method as
described in detail in the online publication (which can be accessed at
http://atvb.ahajournals.org).
Platelet Procedures
Platelets were isolated from freshly drawn blood, as
described previously,22 out of a pool of 34 healthy
donors. In binding studies, adenosine (2 mmol/L) and
theophylline (1 mmol/L) were added to prevent platelet
aggregation. Nonspecific binding was determined as the amount of
platelet-associated radioactivity in the presence of 500 µg/mL of
the unlabeled LDL species. Competition for binding was determined by
the addition of a 7- to 30-fold excess of the unlabeled competitor
species to incubations with either 
5 or 20 µg/mL of the
radiolabeled ligand.
Further details are published online at http://atvb.ahajournals.org.
Electron Microscopy
Aggregation was performed in a final volume of 1 mL with
different concentrations of agonists or hypoxLDL, chosen to yield 50%
or 100% light transmission, respectively. When the desired extent of
aggregation was reached, the reaction was stopped by the addition of an
equal volume of 0.2% aqueous glutardialdehyde (GDA). Samples
were further treated as described elsewhere in
detail.23
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Results |
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Modification of LDLs with hypochlorite was performed in borate buffer at different final concentrations of NaOCl, including the range that may occur in vivo in the course of a phagocytic burst.24 The concentration of nLDL was varied at any given concentration of oxidant, leading to final molar ratios of NaOCl/apoB ranging between
60 and 1000 (based on
Mr 514 000 for apoB-100). Data from
representative experiments are summarized in
Table 1
. The relative
electrophoretic mobility (REM) gives an estimate of the obtained degree
of modification. Electronegativity increased with increasing final
concentration of oxidant and with increasing NaOCl/LDL ratios as well.
Unlike the unpredictable pattern of products obtained when the
oxidation of LDL is catalyzed by transition metal ions, the
modification of LDL by hypochlorite turned out to be highly
reproducible. The observed REM of identically treated lipoproteins from
individual donors generally ranged within a margin of 15%, most likely
reflecting differences in the antioxidant load of the lipoproteins.
Formation of TBArS, indicating lipid peroxidation, was hardly
detectable even in the most highly modified samples. Significant
accumulation of TBArS was, however, observed when BHT was omitted, when
preparations of nLDL with TBArS >1.0 were applied, or when the
modification reaction was performed in phosphate buffer. Additionally,
no specific increase in the relative fluorescence intensity was
detected between 400 and 500 nm after hypochlorite treatment, so
neither malondialdehyde nor 4-hydroxynonenal was formed to any
significant degree. Reportedly, hypoxLDLs show an increased tendency of
self-aggregation, which can be prevented by reductive methylation
before treatment with hypochlorite (hypmetLDLs).25 In
our hands, SDS-PAGE never revealed the formation of the covalently
cross-linked high molecular weight species reported by
others,26 and the gel filtration profiles were virtually
identical to unmodified controls.
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Tracings a to d in Figure 1 show
the effect of hypoxLDL (REM 1.79) on washed human platelets in
suspension, including a control aggregation obtained with ADP. HypoxLDL
induced platelet aggregation in a concentration-dependent manner in
the absence of any further agonist and without the addition of
fibrinogen or Ca2+. The aggregation response was
not enhanced by the addition of either fibrinogen or
Ca2+ (not shown). Even with low doses of
hypoxLDL, the aggregation response was irreversible. Equimolar doses of
nLDL, known to enhance platelet aggregation induced by various
agonists, were without effect on hypoxLDL-induced aggregation (data not
shown).
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The platelet response clearly depended on the degree of LDL
modification (tracings e to k). Of the samples included in Table
1, even very large amounts (up to 830 µg/mL) of slightly
modified hypoxLDL (REM 1.21) failed to induce platelet aggregation.
On the other hand, highly modified LDLs (REM 2.53) induced complete
aggregation (down to 10 µg/mL), and very low doses (1.0 to 2.0
µg/mL) still evoked partial aggregation (30% increase of light
transmission, data not shown). To exclude the possible involvement of
aggregates of hypoxLDL, control studies were performed with hypmetLDLs.
The sample shown in tracing g induced virtually the same platelet
response as the unmethylated hypoxLDLs of comparable electronegativity
(tracing f). The effects of modification of the lysines of apoB are
summarized in Table I (published online at
http://atvb.ahajournals.org). Even high concentrations of metLDL or
acLDL were unable to induce platelet aggregation. However, both
species of modified LDL acquired platelet aggregation potential
after further treatment with NaOCl (hypmetLDL and hypacLDL), and the
behavior of these samples closely matched that of hypoxLDL prepared
with comparable doses of NaOCl.
In platelet-rich plasma, much higher concentrations of hypoxLDL
(between 200 and 300 µg/mL) were necessary to evoke complete
aggregation (Figure IA and IB, published online at
http://atvb.ahajournals.org). Presumably, some plasma component(s)
competes with the platelet surface for interaction with hypoxLDL.
This interaction may include the inactivation (neutralization) of
hypoxLDL by plasma antioxidants, which is currently under
investigation.
Figure 2A demonstrates the correlation
between the aggregation power and the relative mobility in native
electrophoresis, which is proportional to the extent of LDL
modification by hypochlorite. Therefore, different samples of hypoxLDL
are indicated by their respective relative Rf
values (REM) in this paper.
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, 0.25 µmol/L; 
, 0.5 µmol/L; 
,
1 µmol/L; 
, 2 µmol/L; and 
, 3 µmol/L.
The relationship between the conditions of the modification reaction
and the resulting properties of hypoxLDL is documented in more detail
in Figure 2B. For any given constant concentration of
lipoprotein, the platelet aggregation potential increased with
increasing concentrations of NaOCl. However, when prepared at identical
concentrations of NaOCl, the platelet aggregation potential of the
resultant modified LDL decreased with increasing concentrations of
nLDL. Thus, in vitro the reaction of rather low concentrations of
oxidant with low amounts of LDL gives rise to a modified LDL species
that is able to induce platelet aggregation.
The hypoxLDL-induced changes of light transmittance resemble those induced by known platelet agonists, reflecting platelet activation followed by aggregation. Nevertheless, similar traces could be produced by agglutination or cell lysis without activation of the platelets. To assess the nature of the evoked platelet effects, we tested for typical indicators of platelet activation, including morphological changes, release from cellular compartments, and the expression of fibrinogen binding sites.
The morphological effects of hypoxLDL and of different agonists were
compared by electron microscopy. Figure 3
shows a representative series of electron micrographs,
including a control of unstimulated platelets (Figure 3A).
Only low doses of the respective agent were added to the platelets,
which led to a 50% increase in light transmission. In each case, the
formation of mostly small aggregates is evident. ADP-induced aggregates
(Figure 3B) appear rather loose and contain many round cells
with few pseudopodia; hardly any degranulation occurred. The cells in
the aggregates evoked by thrombin (Figure 3C) and collagen
(Figure 3D) are distinctly interdigitating, and the formation of
pseudopodia is more pronounced. With both agonists, degranulation is
evident in individual platelets. HypoxLDLs (REM 2.05, Figure
3E) induce the formation of the tightest aggregates in this
series. Many cells reflect heavy degranulation, and the center of the
aggregates is tightly packed. Generally, the respective activation
state induced by the 4 agonists increased in the following order:
ADP<thrombin
collagen<hypoxLDL. The same conclusion was drawn from
experiments with higher amounts of the examined agonists, ie,
conditions leading to changes in light transmission of 100% (not
shown). Taken together, hypoxLDLs appear to induce true aggregation,
and they show more aggressive power than thrombin and collagen.
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According to these results, hypoxLDLs should evoke release from amine
storage granules. The release of
[14C]5-hydroxytryptamine from
prelabeled platelets was correlated with the aggregation response
(percent changes of light transmission) evoked by different doses of
ADP, thrombin, collagen, and hypoxLDL. Detailed results are shown in
Figure II (published online at http://atvb.ahajournals.org). Under
conditions leading to full aggregation (100% increase in light
transmission), the hypoxLDL-induced release was more pronounced than
that evoked by thrombin and collagen.
We tested the effects of different inhibitors on
platelet aggregation induced by hypoxLDL to find out which known
intracellular second messengers might be involved. The results are
summarized in Table II (published online at
http://atvb.ahajournals.org). Pretreatment of platelets with
10 mmol/L EDTA for 30 minutes did not completely prevent their
aggregation after the addition of hypoxLDL but virtually suppressed any
aggregation response to ADP or thrombin. We conclude that an intact
platelet integrin
(IIb)ß3 (platelet
glycoprotein IIb/IIIa complex) is not required for
activation induced by hypoxLDL. Because
acetylsalicylic acid had no effect, pathways
involving cyclooxygenase(s) are not essential in
hypoxLDL-induced platelet activation. Taken together, the effects
of inhibitors reflect a complex pattern of activation
pathways common with other agonists: elevation of cyclic
nucleotides as well as inhibition of phospholipase
A2 and phospholipase C completely suppressed
platelet aggregation in a dose-dependent manner, whereas inhibition
of protein kinase C caused partial inhibition of platelet
responses.
In the absence of any further agonist, fibrinogen-receptor sites were
expressed on the addition of hypoxLDLs (REM 1.87), which could induce
aggregation, whereas nonaggregating hypoxLDLs (REM 1.34) as well as
nLDLs did not show this effect. Data are given in Table III (available
online at http://atvb.ahajournals.org).
The modification of LDLs by hypochlorite is largely confined to the
protein moiety of the particle; therefore, specific binding of the
modified apoB to the platelet surface is likely to be the basis for
the activation evoked by hypoxLDLs. Because nLDLs and
Cu2+-oxidized LDLs show saturable binding to
sites on the platelet surface (although
nonidentical),27 we also tested whether hypoxLDLs bind to
either of these binding sites. To restrict oxidation of the lipid
moiety to the minimum level, iodination was performed in the presence
of BHT. This procedure slightly reduced the specific radioactivity of
the labeled lipoprotein but kept TBArS in the final product <1.5
nmol/mg protein. Binding isotherms were performed with 3 preparations
of 125I-hypoxLDL modified to different extents
(REM 1.29 to 1.94), and the results are summarized in Table 2. In each case, saturable binding was
observed with only a minor (<15% of the total binding) contribution
of nonspecific binding (ie, binding that could not be displaced by an
excess of the respective unlabeled hypoxLDLs). The apparent binding
strength significantly increases with the increasing extent of
modification. The species that could not induce platelet
aggregation (REM 1.29) apparently bound to a single class of sites,
roughly comparable to the respective behavior of nLDLs. nLDLs
effectively suppressed the binding of this hypoxLDL species, and we
conclude that both lipoprotein species bind to the same receptor sites.
The 2 more extensively modified preparations could induce platelet
aggregation. The species inducing strong platelet aggregation (REM
1.94) bound to a single class of high-affinity sites. This binding
affinity is in perfect accordance with the concentration range, which
is effective in platelet aggregation (Figure III, published online
at http://atvb.ahajournals.org). Binding of this sample was not
affected even by a large excess of unlabeled nLDL (500 µg/mL), so
both species apparently bind to independent sites. Table 2 also
includes the competition of this binding by reductively methylated LDL.
Binding of 125I-hypoxLDL could be displaced by an
excess of hypmetLDL nearly as efficiently as by unlabeled hypoxLDL,
whereas methylated LDL, which was not further modified with
hypochlorite (metLDL), proved as ineffective as nLDL. Maleylated
human serum albumin (malHSA) strongly suppressed the binding of
hypoxLDL, whereas acLDL had no effect at all (data not shown).
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The less extensively modified sample (REM 1.76) revealed intermediate behavior with respect to binding strength and competition for binding by nLDL. We conclude that the regions confining interaction with the nLDL-specific binding sites are partially retained, together with modified stretches responsible for binding to a surface protein specifically recognizing hypoxLDL.
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Discussion |
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Unlike radical-induced oxidation of LDL, controlled modification with hypochlorite gives rise to a lipoprotein species with highly reproducible effects on blood platelets. HypoxLDLs with markedly increased electronegativity (REM
1.5) can induce irreversible
aggregation of resting platelets in suspension as well as in
platelet-rich plasma. HypoxLDLs induce functional morphological
changes and extensive release from dense granules. Key enzymes of the
arachidonate cycle obviously are not required for
platelet activation by hypoxLDLs, whereas the
phosphoinositide cycle appears to be essential for
signal transduction from the platelet surface into the cell. Formation of LDL aggregates on oxidation can be virtually excluded, so platelet aggregation is obviously induced by interaction with monodispersed hypoxLDLs. We report high-affinity binding of hypochlorite-modified LDLs. Dose/response diagrams of the specific binding and the induced platelet aggregation almost perfectly parallel each other, so we conclude that this binding is the limiting step in platelet activation induced by hypoxLDLs. Most likely, this interaction gives rise to transmembrane activation signals. However, we cannot completely rule out the possibility that some modified and thus reactive site of platelet-bound hypoxLDLs might oxidize some component of the platelet plasma membrane on contact. This presumptive oxidation, if taking place at all, apparently is not transmitted by free radicals or free reactive oxygen species, because the addition of oxidant scavengers like catalase or BHT to the platelet-lipoprotein reaction mixtures had no effect (data not shown).
We conclude that 2 independent classes of binding sites exist on the platelet plasma membrane. nLDLs and slightly modified LDLs compete for one type of membrane protein, presumably identified as the platelet integrin (platelet glycoprotein IIb/IIIa complex).28 29 More extensively modified LDLs are no longer recognized by this receptor but tightly bind to a different protein on the platelet surface. Intermediate species can interact with both binding sites, leading to a total binding capacity larger than in the 2 former cases. Experiments to identify the sites to which LDLs bind when sufficiently modified with hypochlorite are under way. Interaction with these sites may be due to a major rearrangement of apoB on the treatment of LDLs with hypochlorite, which exposes parts of the protein otherwise inaccessible,30 as has been reported for several globular proteins.31 Alternatively, some protein in the platelet membrane could recognize (clusters of) modified side chains.32 This possibility is strengthened by the observation that some monoclonal antibodies, which do not cross-react with other types of modified LDLs, also recognize epitopes on other hypochlorite-modified proteins.14 33 34 Furthermore, treatment with hypochlorite can also transform other proteins into platelet-aggregating agents. This includes delipidated HSA and fibrinogen, oxidized with NaOCl/protein ratios between 500 and 1000, whereas several other proteins are ineffective (authors’ unpublished data, 2000). MalHSA, on the other hand, does not activate platelets but strongly inhibits platelet aggregation induced by either thrombin, hypoxLDL, or hypochlorite-treated HSA. Because neither acLDLs nor hypochlorite-modified proteins in general act as platelet agonists, we further conclude that pronounced electronegativity alone is not sufficient to acquire this capability.
The ligand specificity of the platelet receptor recognizing
hypoxLDL reflects some relatedness with the macrophage
scavenger receptor system. Currently, a broad spectrum of partly
unrelated oxLDL receptors has been identified.35 Most of
them, however, are able to bind both oxLDL and acLDL, whereas human
platelets are unable to bind acLDL.27 So far, only 2
strictly oxLDL-specific receptors have been identified, namely, CD32
(the Fc
receptor RIIB2) and CD36; both are present on human
platelets. There is strong evidence that
Cu2+-oxLDL binding to human platelets is
mediated by CD36,27 which therefore represents a
likely candidate for hypoxLDL binding.
One major question concerns the nature of the modification responsible
for the reported platelet effects. It is generally accepted that
hypochlorite preferentially oxidizes apoB15 36 with little
or no lipid peroxidation,15 34 37 that
-tocopherol is not depleted,15 36 and that
LDL cholesterol is not oxidized.15 38
Extensive lipid peroxidation requires prolonged treatment of LDLs or
liposomes with NaOCl at 37°C.39 Very recently, it was
confirmed by Hazell et al40 that most hypochlorite is
rapidly consumed by nonradical reactions with apoB; the formation of
radicals from chloramines with the subsequent oxidation of lipids is a
secondary effect. Large excess of hypochlorite should favor lipid
peroxidation, because amino acid side chains other than amino groups
would be modified, which would more easily give rise to
radicals.41 The reaction conditions stated in the
present study (0°C, rather short incubations, and the addition of
the radical scavenger BHT) appear sufficient to largely restrict lipid
oxidation. BHT reportedly even completely inhibits TBArS formation in
VLDL and phosphatidylcholine vesicles treated with hypochlorite for up
to 3 hours.42
On the other hand, some oxidative modification of the lipid moiety of LDL still may occur. Treatment of phospholipids with hypochlorite could lead to the formation of products not detectable by the methods applied in the present study, eg, the accumulation of small amounts of lysolecithin, chloramines, and other oxidative products derived from the polar part of phospholipids. Additionally, small amounts of oxidized lipids may be formed only transiently during the oxidation reaction. Secondary reactions of these primary products may themselves initiate modifications of the apoprotein, and covalent adducts of oxidized lipids with suitable sites at the polypeptide chain may occur.43 Finally, even trace amounts of lipid peroxides, isoprostanes, and platelet-activating factor–like substances may be important. However, some potentially formed oxidized lipids reportedly inhibit platelet function (eg, oxysterols,44 lysolecithin, and 4-hydroxynonenal45 ); no change in platelet plasma membrane fluidity is induced by Cu2+-oxLDL,46 and hypochlorite-treated phosphatidylcholine vesicles could never induce platelet aggregation (authors’ unpublished data, 2000). Furthermore, platelet shape change is observed immediately after the addition of hypoxLDL, indicating that the transfer of oxidized lipids or lipophilic low molecular weight components of LDL is not essential for hypoxLDL-induced platelet aggregation. Therefore, the reported platelet effects most likely are mediated by the modified apoB of hypoxLDLs. There is increasing evidence that the same may be also true for the interaction of copper-oxidized LDLs with macrophages,43 so the importance of the apoprotein moiety in the tissue effects of oxLDLs may have been generally underestimated. Very recently, it was demonstrated that hypoxLDL induced a macrophage respiratory burst and that this response was not affected by delipidation.47 The results reported in the present study add further evidence that the atherogenic and thrombogenic effects of oxLDL are mediated not only by various bioactive lipids but also by the protein moiety.
The number of intact primary amino groups declines with increasing
electrophoretic mobility of the modified LDLs. However, although this
increase may serve as a suitable parameter to predict the
interaction of hypoxLDLs with platelets, oxidized lysines
apparently are not directly involved. Modification of the majority of
solvent-exposed lysine residues of LDLs (by reductive methylation or by
acetylation) before hypochlorite modification hardly had
any effect on their platelet aggregation power (Figure I and Table
I, published online at http://atvb.ahajournals.org), and hypmetLDL was
indistinguishable from hypoxLDL in binding experiments (Table
2). Obviously, neither intact nor modified lysine residues are
essential in hypoxLDL-platelet interaction, whereas clusters of
charge-neutralized (acetylated but not dimethylated) lysine
residues are responsible for the binding of oxLDL to macrophage
scavenger receptors.19 32 48 Consequently, any secondary
reactions involving chloramines are not likely to be essential for the
conversion of hypoxLDL into a platelet agonist.
Thus, hypoxLDL could serve as an in vitro model of protein-specific atherogenic lipoprotein transformations. HypoxLDL leads to complete platelet aggregation at doses far below those necessary with Cu2+-oxidized LDL to achieve comparable effects. Furthermore, it can induce platelet aggregation even in platelet-rich plasma, although higher doses are required. This demonstrates that plasma components, including other lipoproteins and antioxidants, cannot completely counteract the specific action of hypoxLDL and that platelet stimulation by this lipoprotein species may also occur in vivo.
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Acknowledgments |
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Part of this work was supported by grant No. 6021 of the Jubiläumsfonds der Österreichischen Nationalbank.
Received September 3, 1999; accepted February 3, 2000.
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