Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:377-384

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Malle, E.
Right arrow Articles by Sattler, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Malle, E.
Right arrow Articles by Sattler, W.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:377-384.)
© 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

Ernst Malle; Anton Ibovnik; Hans J. Leis; Gerhard M. Kostner; Peter F. J. Verhallen; Wolfgang Sattler

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.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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{alpha}. In contrast to native lipoproteins, HNE- or MDA-modified LDL and Lp(a) ({approx}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) ({approx}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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {alpha}-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 {alpha}-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 {varepsilon}-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 {varepsilon}-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 {varepsilon}-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).


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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{alpha} (D4-PGF2{alpha}) 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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Treatment of lipoproteins with different concentrations of HNE or MDA resulted in modification of free {varepsilon}-amino groups and changes in net charge and relative electrophoretic mobility (REM) as a further consequence (Table 1Down). 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%.


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of Aldehyde Modification on REM and {epsilon}-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 2Down) 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 2Down). 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{alpha} 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


View this table:
[in this window]
[in a new window]
  		Table 2. Platelet Aggregability of Native and Modified LDL and Lp(a)

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 1ADown) or Lp(a) (Fig 1BDown) 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.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 1. Graphs showing influence of native and 4-hydroxy-2,3-transnonenal (HNE)–modified LDL (A) and lipoprotein(a) [Lp(a); B] on 5-hydroxytryptamine release of gel-filtered platelets (GFPs) after thrombin-induced platelet aggregation. HNE modification of lipoproteins was performed as described in "Methods." Symbols represent control lipoproteins (closed circles) and lipoproteins modified in the presence of HNE 0.1 mmol/L (closed squares), 1 mmol/L (closed diamonds), 5 mmol/L (closed triangles), and 10 mmol/L (hatched squares). The amount of free amino groups modified is shown in Table 1Up. Aggregation was induced by adding 0.1 to 0.2 U thrombin and continued for 6 minutes at 37°C at a constant stirrer speed of 1000 rpm. [14C]Serotonin was measured in the supernatant. 5-Hydroxytryptamine release is expressed as percentage of 14C activity released in the absence of lipoproteins. Results shown represent means of four independent experiments using different lipoprotein preparations in duplicate determinations (SD is shown for native lipoproteins only). Total amount of serotonin released from stimulated platelets incubated in the absence of lipoproteins (30 minutes) is given as 100%. SD is lower than 9%.

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 2ADown). 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 2BDown). A similar pattern of serotonin release reaction was found when collagen was used to activate platelets.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 2. Graphs showing influence of native and malondialdehyde (MDA)-modified LDL (A) or lipoprotein(a) [Lp(a); B] on 5-hydroxytryptamine release of gel-filtered platelets (GFPs) after thrombin-induced platelet aggregation. MDA modification of lipoproteins was performed as described in "Methods." Symbols represent control lipoproteins (closed circles) and lipoproteins modified in the presence of MDA 1 mmol/L (closed squares), 5 mmol/L (closed diamonds), 10 mmol/L (closed triangles), 25 mmol/L (hatched squares), and 50 mmol/L (open diamonds). The amount of free amino groups modified is shown in Table 1Up. Incubation of GFPs with lipoproteins, aggregation of GFPs, and secretion of 5-hydroxytryptamine were performed as described in Fig 1Up legend. Results shown represent means of four independent experiments using different lipoprotein preparations in duplicate determinations. SD is lower than 7%.

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 3Down). 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.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 3. Graph showing influence of native and acetylated (ac) LDL or lipoprotein(a) [Lp(a)] on 5-hydroxytryptamine release of gel-filtered platelets (GFPs) after thrombin-induced platelet aggregation. Acetylation of lipoproteins was performed as described in "Methods." Symbols represent native LDL (closed circles), native Lp(a) (closed diamonds), acLDL (open circles), and acLp(a) (open diamonds). The amount of free amino groups is 31±10% and 37±8% for acLDL and acLp(a), respectively. Incubation of GFPs with lipoproteins, aggregation of GFPs, and measurements of 5-hydroxytryptamine were performed as described in Fig 1Up legend. Results shown represent means of four independent experiments in duplicate determinations using different lipoprotein preparations.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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 {alpha} dense granules (Figs 1Up and 3Up) 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 {varepsilon}-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 2Up 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.


*    Acknowledgments
 
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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Stamler J, Wentworth D, Neaton JD. Is relationship between serum cholesterol and risk of premature death from coronary heart disease continuous and graded? Findings in 356,222 primary screenees of Multiple Risk Factor Intervention Trial (MRFIT). JAMA. 1986;251:2823-2828.
  2. Goldstein JL, Brown MS. The low density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem. 1977;46:897-930. [Medline] [Order article via Infotrieve]
  3. Weisgraber KH, Innerarity TL, Mahley RW. Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts. J Biol Chem. 1978;253:9053-9059. [Abstract/Free Full Text]
  4. Haberland ME, Fogelman AM, Edwards PA. Specificity of receptor-mediated recognition of malondialdehyde-modified low density lipoproteins. Proc Natl Acad Sci U S A. 1982;79:1712-1716. [Abstract/Free Full Text]
  5. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modification of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915-924. [Medline] [Order article via Infotrieve]
  6. Haberland ME, Steinbrecher U. Modified low-density lipoproteins: diversity and biological relevance to atherogenesis. Monogr Hum Genet. 1992;14:35-61.
  7. Haberland ME, Fong D, Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science. 1988;241:215-218. [Abstract/Free Full Text]
  8. Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoproteins in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086-1095.
  9. Jürgens G, Chen Q, Esterbauer H, Mair S, Ledinski G, Dinges HP. Immunostaining of human autopsy aortas with antibodies to modified apolipoprotein B and apoprotein(a). Arterioscler Thromb. 1993;13:1689-1699. [Abstract/Free Full Text]
  10. Palinski W, Ord VA, Plump AS, Breslow JL, Steinberg D, Witztum JL. ApoE-deficient mice are a model of lipoprotein oxidation in atherosclerosis: demonstration of oxidation-specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum. Arterioscler Thromb. 1994;14:605-616. [Abstract/Free Full Text]
  11. Marcus AJ, Safier LB. Thromboregulation: multicellular modulation of platelet reactivity in hemostasis and thrombosis. FASEB J. 1993;7:516-522. [Abstract]
  12. Fuhrmann B, Brook JG, Aviram M. Activated platelets secrete a protein-like factor that stimulates oxidized-LDL receptor activity in macrophages. J Lipid Res. 1991;32:1113-1123. [Abstract]
  13. Malle E, Sattler W. Platelets and the lipoproteins: native, modified and platelet modified lipoproteins. Platelets. 1994;5:70-83.
  14. Pedreño J, de Castellarnau C, Cullaré C, Sánchez J, Gómez-Gerique J, Ordónez-Llanos J, Gonzáles-Sastre F. LDL binding sites on platelets differ from the "classical" receptor of nucleated cells. Arterioscler Thromb. 1992;12:1353-1362. [Abstract/Free Full Text]
  15. Koller E, Koller F, Binder BR. Purification and identification of the lipoprotein-binding proteins from human blood platelet membrane. J Biol Chem. 1989;264:12412-12418. [Abstract/Free Full Text]
  16. Malle E, Ibovnik A, Steinmetz A, Kostner GM, Sattler W. Identification of glycoprotein IIb as the lipoprotein(a)-binding protein on platelets: lipoprotein(a) binding is independent of an arginyl-glycyl-aspartate tripeptide located in apolipoprotein(a). Arterioscler Thromb. 1994;14:345-352. [Abstract/Free Full Text]
  17. Shmulewitz A, Brook JG, Aviram M. Native and modified low-density-lipoprotein interaction with human platelets in normal and homozygous familial-hypercholesterolaemic subjects. Biochem J. 1984;224:13-20. [Medline] [Order article via Infotrieve]
  18. Aviram M, Brook JG, Lees AM, Lees RS. Low density lipoprotein binding to human platelets: role of charge and of specific amino acids. Biochem Biophys Res Commun. 1981;99:308-318. [Medline] [Order article via Infotrieve]
  19. Utermann G. The mysteries of lipoprotein(a). Science. 1989;246: 904-910.
  20. Selley ML, McGuinnes JA, Jenkin LA, Bartlett MR, Ardlie NG. Effect of 4-hydroxy-2,3-trans-nonenal on platelet function. Thromb Haemost. 1988;59:143-146. [Medline] [Order article via Infotrieve]
  21. Hurst JS, Slater TF, Lang J, Juergens G, Zollner H, Esterbauer H. Effects of the lipid peroxidation product 4-hydroxynonenal on the aggregation of human platelets. Chem Biol Interact. 1987;61:109-124. [Medline] [Order article via Infotrieve]
  22. Sattler W, Kostner GM, Waeg G, Esterbauer H. Oxidation of lipoprotein Lp(a): a comparison with low-density lipoproteins. Biochim Biophys Acta. 1991;1081:65-74. [Medline] [Order article via Infotrieve]
  23. Malle E, Sattler W, Prenner E, Leis HJ, Hermetter A, Gries A, Kostner GM. Effects of dietary fish oil supplementation on platelet aggregability and platelet membrane fluidity in normolipemic subjects with and without high plasma Lp(a) concentrations. Atherosclerosis. 1991;88:193-201. [Medline] [Order article via Infotrieve]
  24. Lowry OH, Rosebrough NJ, Farr AL, Randall RL. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275. [Free Full Text]
  25. Jürgens G, Lang H, Esterbauer H. Modification of human low-density lipoprotein by the lipid peroxidation product 4-hydroxynonenal. Biochim Biophys Acta. 1986;875:103-114. [Medline] [Order article via Infotrieve]
  26. Haberland ME, Fless GM, Scanu AM, Fogelman AM. Malondialdehyde modification of lipoprotein(a) produces avid uptake by human monocyte-macrophages. J Biol Chem. 1992;267:4143-4151. [Abstract/Free Full Text]
  27. Basu SK, Goldstein JL, Anderson RGW, Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976;73:3179-3182.
  28. Steinbrecher UP. Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products. J Biol Chem. 1987;262: 3603-3608.
  29. Malle E, Gleispach H, Kostner GM, Leis HJ. Isotope dilution gas chromatography-mass spectrometry for the study of eicosanoid metabolism in human blood platelets. J Chromatogr Biomed Appl. 1989;488:283-293.
  30. Malle E, Nimpf J, Leis HJ, Wurm H, Gleispach H, Kostner GM. Cyclooxygenase and lipoxygenase metabolites during platelet aggregation: quantitative measurement by negative ion chemical ionization-gas chromatography/mass spectrometry. Prostaglandins Leukot Med. 1987;27:53-70. [Medline] [Order article via Infotrieve]
  31. Holmsen H, Dangelmaier CA. Measurement of secretion of serotonin. Methods Enzymol. 1989;169:205-210. [Medline] [Order article via Infotrieve]
  32. Hoff HF, O'Neil J, Chisholm GM III, Cole TB, Quehenberger O, Esterbauer H, Jürgens G. Modification of low-density lipoprotein with 4-hydroxynonenal induces uptake by macrophages. Arteriosclerosis. 1989;9:538-549. [Abstract/Free Full Text]
  33. Hassall DG, Owen JS, Bruckdorfer KR. The aggregation of isolated human platelets in the presence of lipoproteins and prostacyclin. Biochem J. 1983;216:43-49. [Medline] [Order article via Infotrieve]
  34. Aviram M, Brook JG. Platelet interaction with high and low density lipoproteins. Atherosclerosis. 1983;46:259-268. [Medline] [Order article via Infotrieve]
  35. Aviram M, Brook JG. Characterization of the effects of plasma lipoproteins on platelet function in vitro. Haemostasis. 1983; 13:344-350.
  36. Aviram M, Sirtori CR, Colli S, Maderna P, Morazzoni G, Tremoli E. Plasma lipoproteins affect platelet malondialdehyde and thromboxane B2 production. Biochem Med. 1985;34:29-36. [Medline] [Order article via Infotrieve]
  37. Beitz J, Block HU, Beitz A, Müller G, Winkler L, Dargel R, Mest HJ. Endogenous lipoproteins modify the thromboxane formation capacity of platelets. Atherosclerosis. 1986;60:95-99. [Medline] [Order article via Infotrieve]
  38. Beitz J, Mest HJ. Thromboxane A2 (TXA2) formation by washed human platelets under the influence of low and high density lipoproteins from healthy donors. Prostaglandins Leukot Med. 1986;23:303-309. [Medline] [Order article via Infotrieve]
  39. Colli S, Maderna P, Tremoli E, Baraldi A, Rovati GE, Gianfranceschi G, Nicosia S. Prostacyclin-lipoprotein interaction: studies on human platelet aggregation and adenylate cyclase. Biochem Pharmacol. 1985;34:2451-2457. [Medline] [Order article via Infotrieve]
  40. Surya II, Gorter G, Mommersteeg M, Akkerman JWN. Enhancement of platelet function by low density lipoproteins. Biochim Biophys Acta. 1992;1165:19-26. [Medline] [Order article via Infotrieve]
  41. Meraji S, Moore CE, Skinner VO, Bruckdorfer KR. The importance of oxidation or glycosylation of low-density lipoproteins in relation to platelet activation. Platelets. 1992;3:155-162.
  42. Nimpf J, Malle E, Wurm H, Kostner GM. Is there any correlation between platelet activation and plasma lipoprotein? Agents Actions. 1987;22:366-368.
  43. Dunn RC, Schachter M, Miles CMM, Feher MD, Tranter PR, Bruckdorfer KR, Sever PS. Low-density lipoprotein increase intracellular calcium in aequorin-loaded platelets. FEBS Lett. 1988;238: 357-360.
  44. Block LH, Knorr M, Vogt E, Locher R, Vetter W, Groscurth P, Qiao BY, Pometta D, James R, Regenass M, Pletscher A. Low density lipoprotein causes general cellular activation with increased phosphoinositol turnover and lipoprotein catabolism. Proc Natl Acad Sci U S A. 1988;85:885-889. [Abstract/Free Full Text]
  45. Andrews HE, Aitken JW, Hassall DG, Skinner VO, Bruckdorfer KR. Intracellular mechanisms in the activation of human platelets by low-density lipoproteins. Biochem J. 1987;242:559-564. [Medline] [Order article via Infotrieve]
  46. Van Willigen G, Gorter G, Akkerman JWN. LDLs increase the exposure of fibrinogen binding sites on platelets and secretion of dense granules. Arterioscler Thromb. 1994;14:41-46. [Abstract/Free Full Text]
  47. Ardlie NG, Selley ML, Simons LA. Platelet activation by oxidatively modified low density lipoproteins. Atherosclerosis. 1989; 76:117-124.
  48. Aviram M. Modified forms of low density lipoprotein affect platelet aggregation in vitro. Thromb Res. 1989;53:561-567. [Medline] [Order article via Infotrieve]
  49. Pedreño J, de Castellarnau C, Cullaré C, Ortín R, Sánchez JL, Llopart R, Gonzáles-Sastre F. Platelet LDL receptor recognizes with the same apparent affinity both oxidized and native LDL: evidence that the receptor-ligand complexes are not internalized. Arterioscler Thromb. 1994;14:401-408. [Abstract/Free Full Text]
  50. Endemann G, Stanton LW, Madden KS, Bryant CM, White RT, Protter AA. CD36 is a receptor for oxidized low density lipoprotein. J Biol Chem. 1993;268:11811-11816. [Abstract/Free Full Text]
  51. Vanderyse L, Devreese AM, Baert J, Vanloo B, Lins L, Ruysschaert JM, Rosseneu M. Structural and functional properties of apolipoprotein B in chemically modified low density lipoproteins. Atherosclerosis. 1992;97:187-199. [Medline] [Order article via Infotrieve]
  52. Koller E. Lipoprotein-binding proteins in the human platelet plasma membrane. FEBS Lett. 1986;200:97-102. [Medline] [Order article via Infotrieve]
  53. Surya II, Gorter G, Akkerman JWN. Arachidonate transfer between platelets and lipoproteins. Thromb Haemost. 1992;68: 719-726.
  54. Tertov VV, Orekhov AN, Sobenin IA, Morrisett JD, Gotto AM Jr, Guevara JG Jr. Carbohydrate composition of protein and lipid components in sialic acid-rich and -poor low density lipoproteins from subjects with and without coronary artery disease. J Lipid Res. 1993;34:365-375. [Abstract]
  55. Watanabe J, Wohltmann HJ, Klein RL, Colwell JA, Lopes-Virella MF. Enhancement of platelet aggregation by low-density lipoproteins from IDDM patients. Diabetes. 1988;37:1652-1657. [Abstract]
  56. Koslovsky N, Livne AA. Protein kinase C of human platelets: resolution of Ca2+-dependent and independent forms by measuring endogenous phosphorylation. Platelets. 1993;4:332-337.
  57. Malle E, Leis HJ, Karádi I, Kostner GM. Lipoxygenases and hydroperoxy/hydroxyeicosatetraenoic acid formation. Int J Biochem. 1987;19:1013-1021. [Medline] [Order article via Infotrieve]
  58. Galle J, Bauersachs J, Busse R, Bassenge E. Inhibition of cyclic AMP- and cyclic GMP-mediated dilations in isolated arteries by oxidized low-density lipoproteins. Arterioscler Thromb. 1992;12: 180-186.
  59. Weisser B, Locher R, Mengden T, Vetter W. Oxidation of low-density lipoprotein enhances its potential to increase intracellular free calcium concentration in vascular smooth muscle cells. Arterioscler Thromb. 1992;12:231-236.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
G. Marsche, P. G. Furtmuller, C. Obinger, W. Sattler, and E. Malle
Hypochlorite-modified high-density lipoprotein acts as a sink for myeloperoxidase in vitro
Cardiovasc Res, July 1, 2008; 79(1): 187 - 194.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
S. Bacot, N. Bernoud-Hubac, B. Chantegrel, C. Deshayes, A. Doutheau, G. Ponsin, M. Lagarde, and M. Guichardant
Evidence for in situ ethanolamine phospholipid adducts with hydroxy-alkenals
J. Lipid Res., April 1, 2007; 48(4): 816 - 825.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
L. D Tsironis, J. V Mitsios, H. J Milionis, M. Elisaf, and A. D Tselepis
Effect of lipoprotein (a) on platelet activation induced by platelet-activating factor: role of apolipoprotein (a) and endogenous PAF-acetylhydrolase
Cardiovasc Res, July 1, 2004; 63(1): 130 - 138.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Marsche, R. Zimmermann, S. Horiuchi, N. N. Tandon, W. Sattler, and E. Malle
Class B Scavenger Receptors CD36 and SR-BI Are Receptors for Hypochlorite-modified Low Density Lipoprotein
J. Biol. Chem., November 28, 2003; 278(48): 47562 - 47570.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
S. Bacot, N. Bernoud-Hubac, N. Baddas, B. Chantegrel, C. Deshayes, A. Doutheau, M. Lagarde, and M. Guichardant
Covalent binding of hydroxy-alkenals 4-HDDE, 4-HHE, and 4-HNE to ethanolamine phospholipid subclasses
J. Lipid Res., May 1, 2003; 44(5): 917 - 926.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
C. M. Hackeng, M. Huigsloot, M. W. Pladet, H. K. Nieuwenhuis, H. J. M. van Rijn, and J.-W. N. Akkerman
Low-Density Lipoprotein Enhances Platelet Secretion Via Integrin-{alpha}IIbß3–Mediated Signaling
Arterioscler. Thromb. Vasc. Biol., February 1, 1999; 19(2): 239 - 247.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
M. R. McCall, J. Y. Tang, J. K. Bielicki, and T. M. Forte
Inhibition of Lecithin-Cholesterol Acyltransferase and Modification of HDL Apolipoproteins by Aldehydes
Arterioscler. Thromb. Vasc. Biol., October 1, 1995; 15(10): 1599 - 1606.
[Abstract] [Full Text]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
E. Malle, L. Hazell, R. Stocker, W. Sattler, H. Esterbauer, and G. Waeg
Immunologic Detection and Measurement of Hypochlorite-Modified LDL With Specific Monoclonal Antibodies
Arterioscler. Thromb. Vasc. Biol., July 1, 1995; 15(7): 982 - 989.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Malle, E.
Right arrow Articles by Sattler, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Malle, E.
Right arrow Articles by Sattler, W.