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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@azu.nl
Abstract
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 p38MAPK phosphorylation and 1 via Ca2+ mobilization. Between 0% and 15% oxidation, the p38MAPK route enhances fibrinogen binding induced by thrombin receptor (PAR-1)-activating peptide (TRAP), and signaling via Ca2+ is absent. At >30% oxidation, p38MAPK signaling increases further and is accompanied by Ca2+ mobilization and platelet aggregation in the absence of a second agonist. Despite the increase in p38MAPK 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, IIb?3.
Conclusion— At >30% oxidation, LDL interferes with ligand binding to integrin IIb?3, thereby attenuating platelet functions.
OxLDL modulates platelet function via distinct mechanisms. At <15% oxidation, agonist-induced platelet functions are enhanced through activation of a p38MAPK-mediated pathway. At >30% oxidation, oxLDL induces LPA-dependent Ca2+ mobilization, leading to immediate aggregation. At >15% oxidation, p38MAPK-induced sensitization disappears and oxLDL inhibits agonist-induced platelet functions by binding to CD36, thereby interfering with IIb?3-mediated outside-in signaling.
Key Words: lipoproteins ? platelets ? oxidized LDL ? scavenger receptor ? lysophosphatidic acid
Introduction
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 ?-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 (p38MAPK), 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 IIb?3 and ligand-induced outside-in signaling through IIb?3.18
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 G12/13, 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
For a detailed Methods section, please see http://atvb.ahajournals.org.
Results
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 p38MAPK Activation and Via Ca2+ Mobilization
To understand how oxidation changes the p38MAPK-activating properties of nLDL, platelets were treated with LDL oxidized to different extents. Incubation with nLDL induced p38MAPK phosphorylation, confirming earlier observations.17,30 There was little change between 0% and 15% oxidation, but at >15% p38MAPK phosphorylation increased to an almost 6-fold increase at >60% oxidation (Figure 1A). A similar p38MAPK 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 p38MAPK phosphorylation. As expected, oxLDL-induced p38MAPK phosphorylation was inhibited in the presence of the p38MAPK inhibitor SB203580 (Figure 1C).
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 p38MAPK 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 Gq-mediated pathways while concurrently suppressing cAMP formation via ADP release and P2Y12-mediated activation of the inhibitory G-protein, Gi.32 Because p38MAPK 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 p38MAPK 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 p38MAPK 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).
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 p38MAPK pathway and measured TRAP-induced fibrinogen binding to integrin IIb?3 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).
Figure 3. OxLDL inhibits ligand binding to IIb?3. 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 IIb?3 on the plasma membrane of resting platelets.36 Binding of oxLDL to CD36 might therefore inhibit ligand binding to IIb?3 or inhibit IIb?3 activation. We investigated the association of CD36 with CD61 (the ?3 subunit of IIb?3) 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 IIb?3, 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 IIb?3 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 IIb?3.18 Inhibition of ligand-binding to IIb?3 caused by binding of oxLDL to CD36 might impede outside-in signaling through IIb?3 and thus inhibit platelet function. To investigate whether outside-in signaling through IIb?3 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 IIb?3.
Figure 4. OxLDL inhibits platelet functions by hindrance of IIb?3-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
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 p38MAPK, 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 IIb?3 and IIb?3-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 p38MAPK activation. In this mechanism, a further increase in nLDL-induced p38MAPK 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 p38MAPK (Figure 1C) 17 and that p38MAPK-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 p38MAPK 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 IIb?3. 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 p38MAPK 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 IIb?3, which interferes with outside-in signaling through IIb?3 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 p38MAPK 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 p38MAPK 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 IIb?3 on intact platelets and plasma membrane preparations36,42 and colocalizes with fibrinogen and IIb?3 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 IIb?3, thereby interfering with outside-in signaling and further platelet activation in a similar way as antibodies directed against IIb?3.18 A similar inhibition is observed with peptides that block ligand binding to IIb?3 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 IIb?3-mediated outside-in signaling was caused by steric hindrance by oxLDL or inhibited activation of IIb?3 remains to be clarified. Collectively, these data suggest that oxLDL directly interferes with fibrinogen binding to IIb?3, 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 p38MAPK. At >30% oxidation, the LPA-mediated pathway induces the rapid activation of Ca2+ mobilization, leading to immediate aggregation. At >15% oxidation, platelet sensitization via p38MAPK 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 IIb?3-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.
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