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Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:2476-2483
Published online before print August 30, 2007, doi: 10.1161/ATVBAHA.107.150698
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:2476.)
© 2007 American Heart Association, Inc.

Thrombosis

Platelet Activation by Oxidized Low Density Lipoprotein Is Mediated by Cd36 and Scavenger Receptor-A

Suzanne J.A. Korporaal; Miranda Van Eck; Jelle Adelmeijer; Martin Ijsseldijk; Ruud Out; Ton Lisman; Peter J. Lenting; Theo J.C. Van Berkel; Jan-Willem N. Akkerman

From the Department of Clinical Chemistry and Haematology (S.J.A.K., J.A., M.I., T.L., P.J.L., J.-W.N.A.), University Medical Center Utrecht, and Institute of Biomembranes, Utrecht University, the Netherlands; and the Division of Biopharmaceutics (S.J.A.K., M.V.E., R.O., T.J.C.V.B.), Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, the Netherlands.

Correspondence to Prof Dr J.W.N. Akkerman, Department of Clinical Chemistry and Haematology (G03.550), University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, the Netherlands. E-mail j.w.n.akkerman{at}umcutrecht.nl


*    Abstract
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 *Abstract
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Objective— The interaction of platelets with low density lipoprotein (LDL) contributes to the development of cardiovascular disease. Platelets are activated by native LDL (nLDL) through apoE Receptor 2' (apoER2')-mediated signaling to p38MAPK and by oxidized LDL (oxLDL) through lysophosphatidic acid (LPA) signaling to Rho A and Ca2+. Here we report a new mechanism for platelet activation by oxLDL.

Methods and Results— Oxidation of nLDL increases p38MAPK activation through a mechanism that is (1) independent of LPA, and (2) unlike nLDL-signaling not desensitized by prolonged platelet-LDL contact or inhibited by receptor-associated protein or chondroitinase ABC. Antibodies against scavenger receptors CD36 and SR-A alone fail to block p38MAPK activation by oxLDL but combined blockade inhibits p38MAPK by >40% and platelet adhesion to fibrinogen under flow by >60%. Mouse platelets deficient in either CD36 or SR-A show normal p38MAPK activation by oxLDL but combined deficiency of CD36 and SR-A disrupts oxLDL-induced activation of p38MAPK by >70%.

Conclusion— These findings reveal a novel platelet-activating pathway stimulated by oxLDL that is initiated by the combined action of CD36 and SR-A.

Platelets are activated by nLDL through binding to apoER2', which signals to p38MAPK. Oxidation induces more p38MAPK activation through loss of apoER2' binding thereby initiating the combined activity of CD36 and SR-A.


Key Words: platelets • LDL • oxidized LDL • CD36 • scavenger receptor-A


*    Introduction
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An elevated level of native low density lipoprotein (nLDL) is a risk factor for arterial thrombosis and atherosclerosis as demonstrated in familial hypercholesterolemia, where defective apoB/E receptors fail to remove nLDL from the circulation. Atherogenesis starts when nLDL accumulates in the vessel wall at sites of injury and is oxidized by products from macrophages, smooth muscle cells, and endothelial cells.1 Oxidized LDL (oxLDL) accumulates in monocytes that have infiltrated the subendothelium and differentiated into macrophages. The resulting foam cells are characteristic for the early atherosclerotic lesion.2 OxLDL further contributes to atherosclerosis because it contains lysophosphatidic acid (LPA), which starts platelet shape change and aggregation.3

In healthy individuals, the concentration of oxLDL is low. The normal intima contains little oxLDL (1.86±0.59 ng/µg apolipoprotein B100 [apoB100]) but levels increase 6-fold in atherosclerotic lesions (11.9±1.7 ng/µg apoB100).4 Blood from atherosclerotic patients contains autoantibodies that react with oxidation-specific epitopes in both the lipid and protein moiety of oxLDL,5,6 indicating that oxLDL is also present in the circulation. Hence, in the circulation, platelets can come into contact with oxLDL and become activated, thereby contributing to thrombotic occlusion.

The oxidation of nLDL in vivo can be mimicked in vitro by treatment of nLDL with FeSO4. These oxLDL preparations resemble in vivo oxidized LDL with respect to electrophoretic mobility, density, LPA content, fragmentation of apoB100, chemotactic activity for monocytes, and susceptibility to degradation by macrophages.3,7–9 LPA makes oxLDL a potent platelet activating agent3 through activation of its LPA1 and LPA3 receptors,10 which are members of the endothelial differentiation gene receptor family. At low concentrations, LPA stimulates Rho, Rho-kinase, and myosin light chain phosphorylation resulting in platelet shape change caused by changes in the actin cytoskeleton.11 At high concentrations, LPA stimulates Ca2+ mobilization and the tyrosine kinases Syk and Src resulting in integrin {alpha}IIbβ3 activation and aggregation.12

Recently, our laboratory identified the signaling receptor through which nLDL changes the behavior of platelets: the apolipoprotein E Receptor 2' (apoER2').13 It is a 130-kDa splice variant of apoER2, a member of the LDL receptor family, also known as LRP8. The ligand-binding domain of full-length apoER2 contains 7 complement type A binding repeats14 and the apoER2' variant lacks binding repeats 4 to 6.15 ApoER2' is activated by contact with the receptor-specific domain within apoB100 of LDL, called the B-site, with amino acid sequence RLTRKRGLKLA.13 Receptor activation starts signaling through p38MAPK and cytosolic phospholipase A2 leading to formation of thromboxane A2, a potent platelet activating agent.16 The result is an increase in responsiveness to thrombin, collagen, and ADP, leading to enhanced aggregation and secretion on contact with nLDL.

To understand how oxidation enhances the platelet-activating properties of nLDL, signaling through p38MAPK has been compared with LPA-dependent platelet activation at different degrees of LDL oxidation.17 Both pathways were mutually independent because oxLDL signaling to p38MAPK was unaffected by L-NASPA, an inhibitor of LPA receptors,17 and LPA was incapable of activating p38MAPK.16 Below 30% oxidation, activation occurred primarily through the p38MAPK pathway resulting in sensitization, whereas at higher oxidation levels also the LPA pathway was initiated resulting in aggregation.17

In an attempt to understand platelet activation by oxLDL in more detail, we compared the activation of the p38MAPK pathway by oxLDL with that induced by nLDL. We found that oxidation led to a strong increase in p38MAPK signaling caused by loss of apoER2' activation and appearance of the combined activity of CD36 and scavenger receptor-A.


*    Methods
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 *Methods
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To elucidate the receptors mediating platelet activation by oxLDL, human and murine platelets from wild-type C57Bl/6 mice and mice deficient in either CD36, SR-A, or both were incubated with 1.0 g/L native or partially oxidized LDL and phosphorylation of apoER2' and/or p38MAPK was determined as a measure of activation. In these experiments, nLDL and oxLDL signaling to p38MAPK was separated by prolonged incubation of platelets with nLDL before treatment with nLDL or partially oxidized LDL. To investigate functional consequences of oxLDL signaling through CD36 and SR-A, human platelets were incubated with partially oxidized LDL (31% to 60% oxidation) and adhesion to immobilized fibrinogen was measured at a shear rate of 300 s–1 in the absence and presence of inhibitors.

A full description of the preparation of native and modified LDL, the isolation of human and murine platelets, measurement of phosphorylation of apoER2' and p38MAPK, and adhesion to immobilized fibrinogen is available in the detailed Methods section at http://atvb.ahajournals.org.


*    Results
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 *Results
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P38MAPK Signaling by oxLDL and nLDL
To understand how oxidation changes the regulation of the p38MAPK pathway, LDL was oxidized for 30% to 60% and compared with nLDL. At saturating concentrations (1.0 g/L), partially oxidized LDL induced about 2-fold more p38MAPK phosphorylation than nLDL without affecting the transient kinetics of this activation. (Figure 1A and 1B). This agrees with earlier observations showing that oxidation between 0 and >60% led to a proportional increase in p38MAPK activation.17 Thus, oxidation increases activation of the p38MAPK pathway. Recently, we described that nLDL activates the p38MAPK pathway through apoER2' thereby increasing the sensitivity of platelets to agonist stimulation.13 To determine whether the enhanced activation of p38MAPK by partially oxidized LDL was the result of better activation of apoER2', platelets were incubated with both types of LDL and tyrosine phosphorylation of apoER2' was measured. Both LDL preparations induced a similar activation of apoER2' reaching a maximum after 30 seconds and returning to prestimulation values after 10 minutes (Figure 1C). Thus, the extra activation of p38MAPK by partially oxidized LDL could not be attributed to stronger apoER2' activation.


Figure 1
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  		Figure 1. P38MAPK signaling by oxLDL and nLDL. Platelets were incubated with nLDL or partially oxidized LDL (31% to 60% oxidation) at 37°C for the indicated time periods (1.0 g/L (A)) or at the indicated concentrations (1 minute) (B) and p38MAPK phosphorylation was determined. Data were expressed as percentage of the density of incubation with nLDL at 1 minute. C, Platelets were incubated with 1.0 g/L nLDL or partially oxidized LDL. At the indicated time points, apoER2' was immunoprecipitated from platelet lysates and tyrosine phosphorylation was detected. Data were expressed as percentage of the density after 30 seconds of incubation with nLDL. Means±SEM, n=3.

Oxidation of nLDL Introduces ApoER2'-Independent Platelet Signaling
To identify the pathway through which oxLDL activates p38MAPK, platelets were first treated with 1.0 g/L nLDL for 1 minute to saturate apoER2' signaling to p38MAPK and thereafter treated with a second dose of either nLDL or partially oxidized LDL. A second addition of nLDL did not further increase p38MAPK phosphorylation, confirming maximal activation by the first dose. In contrast, a second addition of a saturating concentration of partially oxidized LDL increased p38MAPK activity 4-fold to the range found with a single dose of oxLDL (supplemental Figure IA). A second addition of nLDL or oxLDL after a first treatment with oxLDL did not change p38MAPK activation by oxLDL. These data fit to the concept that oxidation of LDL introduces a second property that activates p38MAPK and is independent of the activation induced by nLDL.

Receptor-associated protein (RAP) is a specific blocker of ligand binding to LDL receptor family members. RAP induced a dose-dependent inhibition of p38MAPK phosphorylation induced by nLDL, which is in agreement with involvement of apoER2', an LDL receptor family member (supplemental Figure IB). In contrast, p38MAPK phosphorylation induced by oxLDL was hardly affected by RAP, the minor decrease probably reflecting residual nLDL in this partially oxidized preparation. This conclusion was supported by the observation that apoER2' tyrosine phosphorylation induced by nLDL and oxLDL was equally inhibited by RAP (supplemental Figure IC). Chondroitinase ABC removes chondroitin and dermatan sulfate side chains from proteoglycans, which are important for nLDL binding to its receptor.18 Chondroitinase ABC completely abolished p38MAPK phosphorylation by nLDL, but hardly interfered with oxLDL-induced phosphorylation, the minor decrease again probably resulting from residual nLDL (supplemental Figure ID). Treatment with L-NASPA to block LPA binding to its receptors did not affect p38MAPK activation by oxLDL (data not shown). Thus, the receptor that mediates the extra p38MAPK activation by oxLDL is therefore not a member of the LDL receptor or LPA receptor families.

Desensitization of nLDL-Induced Signaling
A property of many receptors is the ability to become desensitized after prolonged ligand contact. To assess whether receptors mediating nLDL and oxLDL signaling to p38MAPK could be desensitized, platelets were incubated with nLDL (1.0 g/L) for 30 minutes and thereafter stimulated with a second dose of nLDL or with partially oxidized LDL. Indeed, a first treatment with nLDL almost completely abolished p38MAPK activation by a second dose of nLDL, indicating that this treatment arrested nLDL signaling (Figure 2A). In contrast, p38MAPK activation by partially oxidized LDL remained mostly intact after a first treatment with nLDL, the minor decrease reflecting residual nLDL (Figure 2A). Figure 2B illustrates the desensitization of nLDL signaling by preincubation with nLDL and residual nLDL present in 2 partially oxidized LDL preparations. OxLDL signaling was not desensitized. Thus, prolonged contact with nLDL desensitized nLDL signaling but not oxLDL signaling to p38MAPK.


Figure 2
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  		Figure 2. Desensitization of nLDL-induced signaling. A, Platelets were treated with vehicle (left panel) or nLDL (1.0 g/L, 30 minutes, right panel), stimulated with 1.0 g/L nLDL or partially oxidized LDL and p38MAPK phosphorylation was determined. B, Platelets were treated with vehicle, 1.0 g/L nLDL, or 2 preparations of oxidized LDL (31% to 60% and >60% oxidation) for 30 minutes, stimulated with vehicle, 1.0 g/L nLDL, or partially oxidized LDL and p38MAPK phosphorylation was determined. C, Platelets were treated with vehicle or 1.0 g/L nLDL for 30 minutes, then stimulated with 1.0 g/L LDL oxidized to the indicated extents (1 minute) and phosphorylation of p38MAPK was determined. D, Platelets were incubated with vehicle (left panel) or nLDL (1.0 g/L, 30 minutes, right panel), again stimulated with 1.0 g/L nLDL (1 minute), and tyrosine phosphorylation of apoER2' was measured. A and D, Data were expressed as percentage of the density of incubations with 1 minute nLDL before desensitization. C, Data were expressed as percentage of the density of incubations with oxLDL before desensitization (white bars=100%). Means±SEM, n=3.

The possibility to separate nLDL and oxLDL signaling to p38MAPK by prolonged incubation with nLDL was used to quantify the contribution of oxidation to the signaling properties of LDL. nLDL preparations were oxidized to different extents, and p38MAPK was activated before and after the desensitization phase induced by nLDL (Figure 2C). nLDL contained <15% oxidized LDL and was responsible for >90% of total p38MAPK phosphorylation through the nLDL pathway. As more LDL was oxidized, the contribution of nLDL signaling to p38MAPK decreased and was replaced by oxLDL signaling. LDL preparations of >60% oxidation activated the platelets almost exclusively through the oxLDL receptor.

The loss of nLDL signaling after prolonged nLDL-platelet contact appears a logical consequence of desensitization at the level of the receptor. To confirm this concept, apoER2' phosphorylation by nLDL was analyzed before and after 30 minutes incubation with nLDL (Figure 2D). Unexpectedly, a similar receptor activation was observed, illustrating that ligand activation of apoER2' is fully reversible. Apparently, the cause of desensitization of p38MAPK signaling is not at the level of the receptor and must be sought downstream of apoER2'.

CD36 and Scavenger Receptor-A Mediate oxLDL-Induced Activation of p38MAPK in Human and Murine Platelets
Scavenger receptors (SRs) are membrane glycoproteins that bind oxLDL on cells like macrophages, smooth muscle cells, and platelets.19 To investigate whether SRs play a role in p38MAPK activation, platelets were incubated with antibody FA6.152 against CD36,20 which is a class-B SR and also known as glycoprotein IV, and with fucoidan, which inhibits oxLDL binding to class A SRs.21 Subsequent stimulation with nLDL as a control and with partially oxidized LDL (31% to 60% oxidation) showed that each treatment failed to change the p38MAPK activation by both nLDL and oxLDL. In contrast, combined incubation with both inhibitors sharply decreased p38MAPK activation by oxLDL by >40% to the level found with nLDL (Figure 3A). Thus, the combined blockade of CD36 and SR-A leads to complete inhibition of oxLDL signaling to p38MAPK leaving nLDL signaling to p38MAPK undisturbed. The specificity of this inhibition was confirmed by the inability of RAP to interfere with oxLDL-induced signaling and of fucoidan with nLDL-induced signaling (Figure 3B and 3C).


Figure 3
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  		Figure 3. CD36 and SR-A mediate oxLDL-induced activation of p38MAPK in human platelets. A, Platelets were treated with vehicle, FA6.152 (anti-CD36; 4 µg/mL, 30 minutes), and fucoidan (anti–SR-A; 50 µg/mL, 30 minutes), stimulated with nLDL or partially oxidized LDL (1.0 g/L, 1 minute), and p38MAPK phosphorylation was determined. Data were expressed as percentage of the density of incubations with nLDL. Means±SEM, n=3. B and C, Platelets were treated with vehicle, (B) GST-RAP (50 µg/mL, 10 minutes), or (C) fucoidan, stimulated with the indicated concentrations (B) partially oxidized LDL or (C) nLDL (1 minute), and p38MAPK phosphorylation was determined.

To confirm these observations by targeted interference with receptor expression, incubations with oxLDL were repeated with murine platelets deficient in either CD36 (CD36–/–), SR-A (SR-A–/–), or both (CD36xSR-A double knockout [dKO]). Murine wild-type (w.t.) platelets showed the same p38MAPK activation by nLDL and oxLDL as human platelets and again only the combined addition of FA6.152 and fucoidan brought oxLDL activation by >70% back to levels found with nLDL (Figure 4). A similar blockade of oxLDL signaling was induced in CD36–/– platelets incubated with fucoidan and in SR-A–/– platelets incubated with FA6.152. The combined deletion of CD36 and SR-A expression sharply reduced oxLDL signaling even in the absence of additions. These results support the findings in human platelets and show that interference with both CD36 and SR-A is required for full inhibition of oxLDL signaling to p38MAPK.


Figure 4
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  		Figure 4. CD36 and SR-A mediate oxLDL-induced activation of p38MAPK in murine platelets. WT murine platelets, CD36–/–, SR-A–/–, and CD36xSR-A dKO platelets were treated with vehicle, FA6.152, or fucoidan before stimulation with 1.0 g/L nLDL or partially oxidized LDL, and p38MAPK phosphorylation was determined. Data were expressed as percentage of the density of incubations with nLDL Means±SEM, n=3. *P<0.05, **P<0.01.

CD36 and SR-A Mediate oxLDL-Induced Platelet Adhesion to Immobilized Fibrinogen
To investigate functional consequences of oxLDL signaling through CD36 and SR-A, human platelets were incubated with oxLDL (31% to 60% oxidation) and adhesion to immobilized fibrinogen was measured at a shear rate of 300 s–1. OxLDL increased adhesion with about 35%, probably as a result of activation through the p38MAPK pathway because the p38MAPK inhibitor SB203580 strongly reduced adhesion (Figure 5B). At a higher shear rate (1200 s–1), resembling the shear rates encountered in the arterial circulation, oxLDL induced a similar increase (24%) in platelet adhesion to fibrinogen (data not shown). Although the anti-CD36 antibody FA6.152 alone had no effect on oxLDL-induced p38MAPK activation, it inhibited platelet adhesion to fibrinogen by 37% to the level found in the absence of oxLDL. The presence of fucoidan alone had no effect, but in combination with FA6.152 there was a reduction of 63% (Figure 5B). Thus, adhesion to fibrinogen under flow is enhanced by oxLDL signaling in which both CD36 and SR-A participate and which appears to be a direct result of increased p38MAPK activation through these receptors.


Figure 5
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  		Figure 5. CD36 and SR-A mediate oxLDL-induced platelet adhesion to immobilized fibrinogen. Reconstituted blood containing platelets was treated with vehicle or (A, B) FA6.152, fucoidan, SB203580 (anti-p38MAPK; 10 µmol/L, 30 minutes, 37°C), or (C) AR-C69931MX (anti-P2Y12 receptor, 1 µmol/L, 5 minutes, 37°C) before stimulation with partially oxidized LDL (0.2 g/L, 5 minutes, 37°C). The blood was perfused over immobilized fibrinogen for 5 minutes at a shear rate of 300 s–1. Original magnification x400. B and C, Platelet adhesion was expressed as the percentage of the surface covered with platelets compared with nontreated platelets (100%). Means±SEM, n=3. *P<0.05, **P<0.01.

To investigate the role of feedback activation by secreted ADP, the experiment was repeated in the presence of the P2Y12 receptor antagonist AR-C69931MX. This treatment reduced adhesion from 100% to 50%, confirming earlier observations.22 In the presence of oxLDL, platelet adhesion was reduced from 135% to 75%. Thus, also in platelets with blocked P2Y12 signaling, the stimulation of adhesion by oxLDL was preserved (Figure 5C).


*    Discussion
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 up arrowResults
 *Discussion
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The present study shows that oxLDL activates p38MAPK through the combined stimulation of CD36 and SR-A thereby increasing platelet adhesion to fibrinogen under flow. This property functions concurrently with platelet activation by the LPA content of oxLDL because both pathways are mutually independent.17 Platelet activation by oxLDL is also independent of apoER2', which is the receptor through which nLDL increases the sensitivity to platelet activating agents.13 Because under physiological conditions LDL oxidation is probably far from complete, oxLDL particles will change platelet behavior through at least 3 independent signaling routes which are initiated by apoER2' (nLDL), LPA1/LPA3 receptors (LPA in oxLDL), and CD36/SR-A (oxLDL).

Earlier studies showed that the ability of LDL to act as a platelet agonist increases on oxidation,23–25 and involves LPA-dependent3,11,12,17 and LPA-independent17 signaling. LPA-independent signaling was mediated through p38MAPK activation, which increased with increasing oxidation of LDL. Because p38MAPK is also an intermediate in nLDL-induced platelet activation, a simple explanation emerged in which nLDL activates a receptor and oxidation enhances this process. The discovery of apoER2' as the exclusive receptor for platelet activation by nLDL made it possible to study whether this receptor recognizes oxLDL. The present data showed that nLDL and partially oxidized LDL shared a similar activation of apoER2'. In contrast, both preparations induced different degrees of p38MAPK activation with partially oxidized LDL inducing more activation at lower concentrations than nLDL. These data are best explained by assuming that LDL oxidized for <60% contains an nLDL fraction responsible for apoER2' activation with concurrent p38MAPK phosphorylation and an oxidized fraction that triggers more p38MAPK activation through a different receptor. This second pathway is unaffected by RAP which blocks ligand binding to LDL receptor family members, chondroitinase ABC which interferes with the binding of nLDL to apoER2' mediated by proteoglycans, and L-NASPA, which blocks ligand binding to LPA receptors. It is therefore independent of LDL receptor family members or LPA receptors. In addition, this pathway remains intact after prolonged incubation with nLDL offering a means to clarify oxLDL signaling without interference of the apoER2' pathway.

Many receptors become inaccessible to ligand binding after prolonged ligand exposure. Examples are glycoprotein Ib and PAR-1 which are internalized after platelet activation and sorted to lysosomes rather than recycled to the plasma membrane.26,27 After 30 minutes incubation with nLDL, activation of apoER2' was fully reversible showing no signs of irreversible ligand binding, receptor inactivation, or internalization. Surprisingly, this treatment almost completely abolished a second activation of p38MAPK by nLDL revealing a block between apoER2' activation and p38MAPK activation generated on prolonged nLDL contact. The cause of this blockade is unclear but may involve mechanisms known to downregulate p38MAPK such as the inhibitory receptor PECAM-1.28

OxLDL binds to nucleated cells through SRs expressed on their cell surface. SRs are glycoproteins that recognize a broad variety of ligands, such as oxidized- and glycosylated lipoproteins, anionic phospholipids, apoptotic cells, and fatty acids.19 SRs identified on platelets so far include CD36,29 SR-BI,30 CD68,31 and LOX-1.32 Earlier reports described that CuSO4-oxidized LDL binds to platelets and that binding to CD36 accounted for about 75% of total binding.21,33 Binding was disturbed by maleylated human serum albumin, which inhibits ligand binding to SRs of classes A and B. Other receptors might also contribute to oxLDL binding to platelets such as LOX-1, the function of which appears to be restricted to activated platelets.32 The present study shows that p38MAPK phosphorylation induced by oxLDL was unaffected by separate addition of inhibitors of CD36 and SR-A. A combination of inhibitors to both scavenger receptors effectively reduced oxLDL-induced activation of p38MAPK to the level found with nLDL. Consistent with these findings were observations in mice deficient in either CD36 or SR-A that responded normally to oxLDL, but the absence of both receptors either by genetic targeting or addition of inhibitors blocked signaling by oxLDL. These findings indicate that the LPA-independent platelet activation by oxLDL is mediated through the combined involvement of CD36 and SR-A.

In platelet suspensions, oxLDL binding to CD36 interferes with fibrinogen binding to integrin {alpha}IIbβ3 thereby inhibiting TRAP-induced aggregation.17 The present study shows that oxLDL increases platelet adhesion to immobilized fibrinogen under flow and that this effect is independent of ADP secretion. Binding of soluble fibrinogen is known to depend on prior activation of {alpha}IIbβ3, but surface-coated fibrinogen binds to the closed conformation present on resting platelets.34 At the low shear rate used in the present study (300 s–1), binding to immobilized fibrinogen exclusively involves {alpha}IIbβ3.35,36 Hence, binding of oxLDL to CD36 might interfere with the activation of {alpha}IIbβ3 thereby interfering with binding of soluble but not with surface-coated fibrinogen. There is a 63% reduction of the stimulation by oxLDL in the presence of inhibitors of CD36 and SR-A. A similar inhibition is seen at the level of p38MAPK activation. This suggests that the better adhesion induced by oxLDL is the result of combined activation of CD36 and SR-A and enhanced signaling to p38MAPK, which is an upstream regulator of thromboxane A2 formation.37


*    Acknowledgments
 
We thank M. Bezemer (Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, the Netherlands) and R.B. Hildebrand (Division of Biopharmaceutics, Leiden Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, the Netherlands) for their technical assistance.

Sources of Funding

This study was supported in part by grants 1999B061 (to S.J.A.K.), 2001T041 (to M.V.E.), and 2003B134 (to R.O.) of the Netherlands Heart Foundation, and the Netherlands Thrombosis Foundation (to J.-W.N.A.).

Disclosures

None


*    Footnotes
 
Original received July 31, 2006; final version accepted August 2, 2007.


*    References
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 *References
 

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