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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:867.)
© 2005 American Heart Association, Inc.

Thrombosis

Effect of Oxidation on the Platelet-Activating Properties of Low-Density Lipoprotein

Suzanne J.A. Korporaal; Gertie Gorter; Herman J.M. van Rijn; Jan-Willem N. Akkerman

From the Thrombosis and Haemostasis Laboratory (S.J.A.K., G.G., J.-W.N.A.), Department of Haematology, and Department of Clinical Chemistry (H.J.M.R.), University Medical Center Utrecht, and the Institute for Biomembranes (S.J.A.K., G.G., J.-W.N.A.), University of Utrecht, the Netherlands.

Correspondence to Dr J. W. N. Akkerman, Thrombosis and Haemostasis Laboratory, G.03.647, Department of Haematology, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, the Netherlands. E-mail j.w.n.akkerman{at}azu.nl

	Abstract

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Objective— Because of the large variation in oxidizing procedures and susceptibility to oxidation of low-density lipoprotein (LDL) and the lack in quantification of LDL oxidation, the role of oxidation in LDL–platelet contact has remained elusive. This study aims to compare platelet activation by native LDL (nLDL) and oxidized LDL (oxLDL).

Methods and Results— After isolation, nLDL was dialyzed against FeSO₄ to obtain LDL oxidized to well-defined extents varying between 0% and >60%. The oxLDL preparations were characterized with respect to their platelet-activating properties. An increase in LDL oxidation enhances platelet activation via 2 independent pathways, 1 signaling via p38^MAPK phosphorylation and 1 via Ca²⁺ mobilization. Between 0% and 15% oxidation, the p38^MAPK route enhances fibrinogen binding induced by thrombin receptor (PAR-1)-activating peptide (TRAP), and signaling via Ca²⁺ is absent. At >30% oxidation, p38^MAPK signaling increases further and is accompanied by Ca²⁺ mobilization and platelet aggregation in the absence of a second agonist. Despite the increase in p38^MAPK signaling, synergism with TRAP disappears and oxLDL becomes an inhibitor of fibrinogen binding. Inhibition is accompanied by binding of oxLDL to the scavenger receptor CD36, which is associated with the fibrinogen receptor, _IIbß₃.

Conclusion— At >30% oxidation, LDL interferes with ligand binding to integrin _IIbß₃, thereby attenuating platelet functions.

OxLDL modulates platelet function via distinct mechanisms. At <15% oxidation, agonist-induced platelet functions are enhanced through activation of a p38^MAPK-mediated pathway. At >30% oxidation, oxLDL induces LPA-dependent Ca²⁺ mobilization, leading to immediate aggregation. At >15% oxidation, p38^MAPK-induced sensitization disappears and oxLDL inhibits agonist-induced platelet functions by binding to CD36, thereby interfering with _IIbß₃-mediated outside-in signaling.

Key Words: lipoproteins • platelets • oxidized LDL • scavenger receptor • lysophosphatidic acid

	Introduction

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Patients with familial hypercholesterolemia (FH) show an increased incidence of premature coronary artery disease. These patients lack or have a defective receptor for low-density lipoprotein (LDL), the apolipoprotein (apo) B/E-receptor,¹ which results in an impaired uptake of LDL from the circulation. LDL accumulates and becomes oxidized in the vessel wall at sites of injured endothelium. Uptake of oxidized LDL (oxLDL) transforms macrophages into foam cells, which are characteristic for the fatty streak, the early atherosclerotic lesion.² Plasma levels of oxLDL are higher in coronary artery disease patients (31.1±11.9 mg/L) compared with normal subjects (13.0±8.8 mg/L).³ OxLDL accumulates in atherosclerotic lesions and there is 6-fold more oxLDL in atherosclerotic plaques than in normal intima.⁴ Platelets are key elements in the development of arterial thrombosis and atherosclerosis. They adhere to injured endothelium, to exposed collagen, and to macrophages. On activation, platelets secrete cytokines and growth factors that contribute to migration and proliferation of smooth muscle cells and monocytes. Platelets of FH patients are hyper-reactive and show hyperaggregability in vitro and enhanced activity in vivo as illustrated by increased plasma levels of the -granule product ß-thromboglobulin and an increased prostaglandin (PG) and thromboxane (TxA₂) metabolism.⁵ Moreover, activated platelets have been found in the circulation of FH patients⁶ and high concentrations of oxLDL stimulate platelet adhesion and aggregation via suppression of endothelial production of nitric oxide and stimulation of the synthesis of PG precursors and prostaglandins.⁷ These observations suggest that LDL enhances platelet responsiveness.

Native LDL (nLDL) is a mild activator of platelets via TxA₂-dependent and TxA₂-independent pathways.^8–11 Activation is mediated via a specific LDL receptor, which differs from the classical apoB/E receptor because a similar response is observed after LDL stimulation of platelets from healthy subjects, platelets from FH patients, and platelets that were treated with apoB/E receptor–blocking antibodies.¹² We recently identified apolipoprotein E receptor 2' (apoER2') as a possible candidate for LDL binding to platelets.¹³ apoER2' is a splice variant of apoER2 that has been identified in platelets and megakaryocytic cell lines.¹⁴ At physiological concentrations (0.6 to 0.9 g/L), nLDL increases the sensitivity of platelets for -thrombin, collagen, and ADP but fails to independently induce platelet functions.^8,10,11,15 At higher concentrations (3 g/L), nLDL becomes an independent initiator of platelet activation triggering aggregation and secretion.¹⁶ nLDL-induced platelet sensitization is mediated via the activation of p38 mitogen-activated protein kinase (p38^MAPK), which triggers cytosolic phospholipase A₂ (cPLA₂)-mediated arachidonic acid release and TxA₂ formation.¹⁷ TxA₂ further activates platelets via stimulation of the TxA₂ receptor, causing activation of integrin _IIbß₃ and ligand-induced outside-in signaling through _IIbß₃.¹⁸

The ability of LDL to function as a platelet activator increases on oxidation. CuSO₄-oxidized LDL independently induces platelet functions and acts synergistically with other agonists, leading to faster responses at lower concentrations.^10,19–21 The platelet-activating properties of oxLDL have been attributed to lysophosphatidic acid (LPA), generated during oxidation.²² LPA is present in plasma at a concentration of 0.5 to 1.0 µmol/L and accumulates in atherosclerotic plaques at 10 to 49 pmol/mg, compared with 1.2 to 2.8 pmol/mg in normal arterial tissue.²³ It potently activates platelets and endothelial cells²² via G protein–coupled LPA receptors, which are members of the endothelial differentiation gene receptor family.²⁴ Platelets express LPA₁, LPA₂, and LPA₃.²⁵ Selective antagonists of LPA₁ and LPA₃ block LPA-induced platelet activation, indicating that these receptors respond to LPA.²⁶ At low concentrations (EC₅₀18 nmol/L), LPA activates Rho and Rho-kinase via G_12/13, causing phosphorylation of myosin light chain and changes of the actin cytoskeleton that underlie platelet shape change.^22,27–29 At higher concentrations (EC₅₀1.6 µmol/L), LPA increases intracellular Ca²⁺ levels ([Ca²⁺]_i) and induces platelet aggregation.²⁷

The large variation in oxidizing procedures, the interindividual variation in susceptibility to oxidation of LDL and the lack in quantification of the degree of LDL oxidation have concealed the insight in the role of oxidation in LDL–platelet interactions. The present study was initiated to compare platelet activation by nLDL and by oxLDL. To this end, we prepared LDL preparations oxidized to well-defined extents varying between 0% and >60% oxidation and characterized their platelet-activating properties.

	Methods

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For a detailed Methods section, please see http://atvb.ahajournals.org.

	Results

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Lipoprotein Modification
Table I (available online at http://atvb.ahajournals.org) summarizes the amount of conjugated dienes and relative electrophoretic mobility (REM) of the different oxLDL preparations, defining the extent of lipid and protein modification of the LDL preparations used in this study.

OxLDL-Induced Signaling Via p38^MAPK Activation and Via Ca²⁺ Mobilization
To understand how oxidation changes the p38^MAPK-activating properties of nLDL, platelets were treated with LDL oxidized to different extents. Incubation with nLDL induced p38^MAPK phosphorylation, confirming earlier observations.^17,30 There was little change between 0% and 15% oxidation, but at >15% p38^MAPK phosphorylation increased to an almost 6-fold increase at >60% oxidation (Figure 1A). A similar p38^MAPK phosphorylation was found in the presence of L-NASPA (Figure 1C), which blocks binding of LPA to its receptor, thereby antagonizing platelet functions induced by LPA, such as shape change²² and aggregation.³¹ These results indicate that LPA formed during LDL oxidation did not contribute to p38^MAPK phosphorylation. As expected, oxLDL-induced p38^MAPK phosphorylation was inhibited in the presence of the p38^MAPK inhibitor SB203580 (Figure 1C).

View larger version (19K): [in this window] [in a new window]   Figure 1. OxLDL-induced p38MAPK phosphorylation and Ca2+ mobilization. A, Platelets were stimulated with LDL (1.0 g/L, 5 minutes, 37°C) and fixed with 1% formaldehyde. After centrifugation (30 seconds, 9000g, 20°C), pellets were taken up in Laemmli sample buffer and analyzed by SDS-PAGE to identify dual-phosphorylated and total p38MAPK phosphorylation using a phospho-specific anti-p38MAPK polyclonal antibody (upper panel) or an antibody against p38MAPK as a control for equal lane loading (lower panel). The bars show the semiquantification of dual-phosphorylated p38MAPK. B, Ca2+ mobilization was measured in Ca2+-free buffer on addition of LDL (0.2 g/L, 37°C) to Fura-2/AM-loaded platelets. Inset, Representative traces for Ca2+ mobilization by >60% oxLDL after pre-incubation with vehicle or L-NASPA (10 µmol/L, 5 minutes, 37°C). C, Platelets were incubated with vehicle, L-NASPA, or SB203580 (10 µmol/L, 10 minutes, 37°C), and p38MAPK phosphorylation or Ca2+ mobilization induced by oxLDL (31% to 60%) were measured as described. Data are expressed as fold increase compared with untreated suspensions (ctrl) (means±SEM, n=3, *P<0.05 vs control).

To investigate the contribution of LPA in oxLDL-induced platelet activation, the mobilization of intracellular Ca²⁺ was measured because this is a sensitive marker for LPA-induced signaling.²⁷ Between 0% and 30% oxidation, there was no significant change in [Ca²⁺]_i, but further oxidation strongly increased Ca²⁺ mobilization to a 2-fold increase at >60% oxidation (Figure 1B). The increase in [Ca²⁺]_i was completely blocked by L-NASPA (Figure 1C), indicating that LPA caused the Ca²⁺ mobilization by oxLDL. OxLDL-induced Ca²⁺ mobilization was not inhibited by SB203580 (Figure 1C). Thus, both p38^MAPK phosphorylation and Ca²⁺ mobilization increased at increasing oxidation of LDL with a threshold of 15% to 30% oxidation, below which there was little difference with nLDL. In addition, the findings with L-NASPA and SB203580 suggest that oxLDL activates 2 independent pathways.

OxLDL-Dependent Regulation of cAMP
Platelet agonists initiate aggregation and secretion via G_q-mediated pathways while concurrently suppressing cAMP formation via ADP release and P2Y₁₂-mediated activation of the inhibitory G-protein, G_i.³² Because p38^MAPK activation and Ca²⁺ mobilization sense changes in cAMP, we investigated whether the higher activation observed at more oxidation resulted from suppression of cAMP. Platelets had a basal cAMP concentration of 3.87±1.41 ng/10¹¹ cells, which was not disturbed by nLDL or oxLDL (data not shown). Prostacyclin (PGI₂) induced a 2.6-fold increase in cAMP, which was not changed by LDL preparations oxidized up to 30%. At >30% oxidation, oxLDL reduced the increase in cAMP by 30% to 40% (Table II, available online at see http://atvb.ahajournals.org). Similar results were observed in the presence of L-NASPA, indicating that the reduction was independent of LPA. As expected, -thrombin reduced the PGI₂-induced cAMP accumulation amounting to a decline of 70%, an effect that was independent of LPA. Assuming that the inhibition of PGI₂-induced cAMP accumulation by oxLDL reflects a similar effect on the basal level of cAMP, which is difficult to measure, the platelet-activating properties of oxLDL were enhanced by suppression of cAMP in an LPA-independent manner.

The Effects of oxLDL on Aggregation
nLDL-induced p38^MAPK phosphorylation is an early and rapid step in a slow process that after 5 minutes or more synergistically increases agonist-induced fibrinogen binding, aggregation, and secretion.^8,9,18 In contrast, LPA independently raises [Ca²⁺]_i, inducing shape change, aggregation, and secretion within seconds.^22,27 Because oxLDL was a more potent activator of p38^MAPK phosphorylation than nLDL, and LPA-mediated Ca²⁺ signaling is especially evident at high stages of oxidation, we investigated the functional responses initiated by the 2 pathways. After 5 minutes of pre-incubation, nLDL enhanced thrombin receptor (PAR-1)-activating peptide (TRAP)-induced aggregation (Figure 2A). Surprisingly, this property disappeared on oxidation and at high oxidation oxLDL became an inhibitor of TRAP-induced platelet aggregation (Figure 2A). The inhibition was independent of LPA, because similar results were observed in the presence of L-NASPA (data not shown). In contrast, oxLDL induced aggregation within seconds (Figure 2B). This finding was in agreement with the rapid LPA-dependent mobilization of Ca²⁺ by oxLDL (Figure 1). Aggregation was inhibited in the presence of L-NASPA, indicating that LPA was responsible (data not shown).

View larger version (30K): [in this window] [in a new window]   Figure 2. Loss of LDL-enhanced aggregation and fibrinogen binding. A, Platelets were incubated with vehicle or LDL (0 revelations per minute, 5 minutes, 37°C) and aggregation was stimulated with a suboptimal concentration TRAP (2 µmol/L, 900 revelations per minute, 37°C) in the presence of fibrinogen (1 µmol/L). The tracings are representative for 3 similar experiments. B, Platelets were stimulated with nLDL or oxLDL (900 revelations per minute, 37°C) in the presence of fibrinogen and aggregation was measured. The tracings are representative for three similar experiments. C, Unstirred platelets were pre-incubated with LDL, stimulated with TRAP (15 µmol/L, 5 minutes, 22°C) in the presence of fibrinogen, and fixed after incubation with fluoresceinisothiocyanate (FITC)-conjugated anti-human fibrinogen. Fibrinogen binding was determined by flow cytometry and expressed as percentage of nLDL/TRAP-induced fibrinogen binding (100%). D, Platelets were incubated with nLDL () or oxLDL (31% to 60%; ) at the indicated concentrations, and TRAP-induced fibrinogen binding was determined as described. Data were expressed as percentage of TRAP-induced fibrinogen binding. (means±SEM, n=3, *P<0.05 vs control).

We further investigated the inhibition of aggregation by oxLDL via the p38^MAPK pathway and measured TRAP-induced fibrinogen binding to integrin _IIbß₃ in the presence of oxLDL. In unstirred platelet suspensions, LDL alone failed to induce fibrinogen binding at any oxidation stage (data not shown). In contrast, after 5 minutes of pre-incubation, nLDL enhanced TRAP-induced fibrinogen binding (Figure 2C).⁹ Oxidation to <15% preserved the synergistic properties of nLDL, but further oxidation reduced this property and, at high oxidation, oxLDL inhibited TRAP-induced fibrinogen binding (Figure 2C). Inhibition was already observed at a concentrations of 750 mg/L oxLDL (P=0.0329) and increased at higher concentrations of oxLDL (1.0 g/L: P=0.0220) (Figure 2D). nLDL enhances TRAP-induced fibrinogen binding at this concentration, which indicates that the inhibition by oxLDL was not caused by changes in lipid composition of the medium. The inhibition of TRAP-induced fibrinogen binding was independent of LPA (data not shown).

Inhibition of Platelet Functions by oxLDL
CD36 is a scavenger receptor that is present on platelets and binds oxLDL with high affinity.^33,34 To determine whether binding of oxLDL to CD36 is involved in oxLDL-induced inhibition of platelet aggregation, platelets were treated with the antibody FA6.152 to block binding of oxLDL to CD36,³⁵ before addition of oxLDL and TRAP. Inhibition of oxLDL binding to CD36 by FA6.152 abolished the reduction of TRAP-induced fibrinogen binding by oxLDL in a dose-dependent manner (Figure 3A).

View larger version (36K): [in this window] [in a new window]   Figure 3. OxLDL inhibits ligand binding to IIbß3. A, Platelets were pretreated with FA6.152 (30 minutes, 37°C) at the indicated concentrations before incubation with oxLDL (31% to 60%) and aggregation was stimulated with a suboptimal concentration TRAP (2 µmol/L, 900 revelations per minute, 37°C) in the presence of fibrinogen (1 µmol/L). The tracings are representative for 3 similar experiments. B, Platelets were stimulated with nLDL or oxLDL (31% to 60%) and lysed at the indicated time points. CD36 was immunoprecipitated from platelet lysates with FA6.152 and association with CD61 was analyzed by SDS-PAGE with antibody SZ21 (upper panel). The antibody 131.2 against CD36 was used as a control for equal lane loading (lower panel). A CD11b antibody was used as a nonspecific control compared with CD36. C, Platelets were stimulated with nLDL or oxLDL (31% to 60%) and lysed at the indicated time points. apoB100 was immunoprecipitated from platelet lysates with 1D2, and coprecipitation of CD36 was analyzed by SDS-PAGE using antibody 131.2. The graphs show the semiquantification of the association of apoB100 with CD36. Data are expressed as percentage of the association of apoB100 with CD36 after 3 minutes incubation with oxLDL (100%).

CD36 is known to associate with _IIbß₃ on the plasma membrane of resting platelets.³⁶ Binding of oxLDL to CD36 might therefore inhibit ligand binding to _IIbß₃ or inhibit _IIbß₃ activation. We investigated the association of CD36 with CD61 (the ß₃ subunit of _IIbß₃) in the presence of LDL. CD36 associated with CD61 on resting platelets confirming earlier observations.³⁶ No change in the association was observed on incubation up to 30 minutes with either nLDL or oxLDL (Figure 3B). Immunoprecipitation experiments with a nonspecific antibody failed to immunoprecipitate both CD61 and CD36 (Figure 3B).

To determine whether binding of oxLDL to CD36 might block ligand-binding to or activation of _IIbß₃, platelets were treated with nLDL or oxLDL, apoB100 was immunoprecipitated, and the coimmunoprecipitation with CD36 was determined. Immunoprecipitation of the lipoproteins was associated with the precipitation of the 88-kDa scavenger receptor, CD36 (Figure 3C). Binding of nLDL to CD36 was transient, leading to dissociation after 5 minutes. In contrast, in platelet lysates stimulated with oxLDL, the coassociation between apoB100 and CD36 was persistent, indicating that oxLDL was still bound to CD36 after 5 minutes of oxLDL–platelet interaction. Collectively, these results indicate that the persistent binding of oxLDL to CD36 but not of nLDL is sufficient to block ligand binding to or activation of _IIbß₃ induced by TRAP and thereby to block TRAP-induced fibrinogen binding and aggregation (Figure 2).

nLDL sensitizes platelets to stimulation by collagen and TRAP via ligand-induced outside-in signaling through _IIbß₃.¹⁸ Inhibition of ligand-binding to _IIbß₃ caused by binding of oxLDL to CD36 might impede outside-in signaling through _IIbß₃ and thus inhibit platelet function. To investigate whether outside-in signaling through _IIbß₃ was inhibited, platelets were incubated with LDL and TRAP-induced P-selectin expression was determined as a marker for -granule secretion. Pre-incubation with nLDL for 5 minutes did not influence TRAP-induced P-selectin expression. In contrast, oxLDL inhibited TRAP-induced -granule secretion (Figure 4). This observation indicates that on oxidation, LDL becomes an inhibitor of platelet functions by inhibition of ligand-induced outside-in signaling through _IIbß₃.

View larger version (12K): [in this window] [in a new window]   Figure 4. OxLDL inhibits platelet functions by hindrance of IIbß3-induced outside-in signaling. Platelets were treated with LDL before stimulation with TRAP (15 µmol/L, 5 minutes, 22°C) and fixed after incubation with RPE-conjugated anti-human P-selectin. P-selectin expression was determined by flow cytometry. (means±SEM, n=3, *P<0.05 vs 100%).

	Discussion

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nLDL increases the responsiveness of platelets to activating agents, resulting in faster aggregation and secretion after stimulation with thrombin, ADP, and collagen. This sensitization process is slow,^8,18 requiring 5 minutes or more (37°C), and starts with the rapid activation of p38^MAPK, which via cPLA₂-mediated arachidonic acid release¹⁷ triggers the formation of TxA₂.^9,17 TxA₂ further activates platelets by stimulating the TxA₂ receptor, leading to activation of integrin _IIbß₃ and _IIbß₃-mediated ligand-induced outside-in signaling.¹⁸ Blockade of TxA₂ formation by indomethacin sharply reduces secretion.¹⁵

Oxidation increases the platelet-activating properties of nLDL via 2 mechanisms, which depend on the degree of oxidation and the duration of platelet–LDL contact. The first mechanism involves p38^MAPK activation. In this mechanism, a further increase in nLDL-induced p38^MAPK activation on LDL oxidation leads to a 6-fold increase at >60% oxidation. Despite this increase in the activating pathway, concurrent fibrinogen binding remains constant and even decreases at 16% oxidation or more. The second mechanism is LPA-mediated platelet activation, which is insignificant at low oxidation but becomes a potent activation pathway at >30% oxidation, inducing Ca²⁺ mobilization and aggregation. The observations that LPA is unable to activate p38^MAPK (Figure 1C) ¹⁷ and that p38^MAPK-mediated functions are insensitive to the LPA receptor antagonist L-NASPA illustrate that both pathways are mutually exclusive.

Unexpectedly, at >15% oxidation, synergism through the p38^MAPK pathway does not result in faster platelet functions. Above this threshold, further oxidation decreases the sensitization of TRAP-induced fibrinogen binding and aggregation and inhibits ligand-induced outside-in signaling through _IIbß₃. A similar inhibition is seen with and without L-NASPA, indicating that LPA is incapable of inducing fibrinogen binding after 5 minutes of pre-incubation with oxLDL. Importantly, the inhibition is absent when LDL oxidized at >30% makes immediate contact with stirred platelet suspensions. Apparently, the induction of platelet inhibition by oxLDL is a slow process because it does not interfere with the rapid induction of Ca²⁺ mobilization and platelet aggregation mediated via the LPA pathway. Possibly, the p38^MAPK pathway becomes important at later stages of platelet–LDL contact when LPA receptors become desensitized.^22,27

The cause of the inhibition of platelet aggregation by oxLDL has remained elusive.³⁷ Mechanisms known to attenuate platelet functions are an increase in cAMP, which inhibits platelet activation via protein kinase A, the activation of platelet endothelial cell adhesion molecule-1 (PECAM-1), which generates inhibitory signals, and inhibition of ligand binding to integrin _IIbß₃, which interferes with outside-in signaling through _IIbß₃ thereby attenuating agonist-induced platelet responses.

The basal level of cAMP was unchanged during incubation with oxLDL, but oxLDL reduced the PGI₂-induced accumulation of cAMP. L-NASPA did not abolish this effect, illustrating that oxLDL suppressed the formation of cAMP independent of LPA. Hence, cAMP may be responsible for the inhibition of platelet function through the p38^MAPK pathway by oxLDL.

PECAM-1 is a receptor that on phosphorylation of its immunoreceptor tyrosine-based inhibitory motifs generates inhibitory signals, thereby suppressing platelet activation³⁸ induced by collagen³⁹ and von Willebrand factor.⁴⁰ Interestingly, nLDL activates both p38^MAPK and PECAM-1, albeit with different time courses, thereby regulating initiation and termination of signal generation.⁴¹ Oxidation increases the capacity of LDL to activate PECAM-1, making it a candidate inhibitor of platelet function at extensive oxidation stages (data not shown). However, when PECAM-1 activation was mimicked by treatment with the antibody PECAM-1.3 and cross-linking with F(ab')₂-fragments, there was little interference with TRAP-induced fibrinogen binding, suggesting that PECAM-1 does not mediate platelet inhibition by oxLDL (data not shown).

Treatment of platelets with nLDL or oxLDL and immunoprecipitation with an antibody directed against the apoB100-moiety of LDL showed a clear association of an 88-kDa protein that was identified as CD36, a scavenger receptor that binds nLDL and oxLDL.³⁴ The association of CD36 with nLDL was transient and disappeared after 5 minutes, but with oxLDL a persistent association between apoB100 and CD36 was found. CD36 associates with _IIbß₃ on intact platelets and plasma membrane preparations^36,42 and colocalizes with fibrinogen and _IIbß₃ on immuno-electron micrographs.⁴³ We demonstrated a strong association of CD36 with CD61 on resting platelets, which did not change in the presence of nLDL and oxLDL. Inhibition of oxLDL binding to CD36 abolished the inhibition of TRAP-induced aggregation by oxLDL. Hence, binding of oxLDL to CD36 blocks ligand-binding to _IIbß₃, thereby interfering with outside-in signaling and further platelet activation in a similar way as antibodies directed against _IIbß₃.¹⁸ A similar inhibition is observed with peptides that block ligand binding to _IIbß₃ and thereby block the stimulation of secretion by nLDL.¹⁸ The inhibition of TRAP-induced -granule secretion by oxLDL supports this conclusion. Whether the inhibition of fibrinogen binding, aggregation, and _IIbß₃-mediated outside-in signaling was caused by steric hindrance by oxLDL or inhibited activation of _IIbß₃ remains to be clarified. Collectively, these data suggest that oxLDL directly interferes with fibrinogen binding to _IIbß₃, thereby abolishing outside-in signaling and the stimulation of aggregation and secretion observed with nLDL and LDL preparations oxidized at <15%.

In conclusion, the present study describes distinct mechanisms by which LDL modulates platelets. Between 0% and 15% oxidation, LDL sensitizes platelets to TRAP-induced fibrinogen binding and aggregation through activation of a slow sensitization process mediated by p38^MAPK. At >30% oxidation, the LPA-mediated pathway induces the rapid activation of Ca²⁺ mobilization, leading to immediate aggregation. At >15% oxidation, platelet sensitization via p38^MAPK is abolished and oxLDL inhibits TRAP-induced aggregation and secretion. This mechanism is independent of activation of PECAM-1 but is caused by binding to CD36 and interference with _IIbß₃-mediated ligand-induced outside-in signaling.

	Acknowledgments

 
We thank Dr H.A.M. Voorbij (Department of Clinical Chemistry, University Medical Center Utrecht, Utrecht, the Netherlands) for his assistance. This study was supported in part by a grant from the Netherlands Heart Foundation (1999B061). J.W.N.A is supported by the Netherlands Thrombosis Foundation.

Received June 29, 2004; accepted January 21, 2005.

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