Platelet CD36 links hyperlipidemia, oxidant stress and a prothrombotic phenotype - PubMed (original) (raw)

. 2007 Sep;13(9):1086-95.

doi: 10.1038/nm1626. Epub 2007 Aug 26.

Tatiana V Byzova, Maria Febbraio, Robert G Salomon, Yi Ma, Manojkumar Valiyaveettil, Eugenia Poliakov, Mingjiang Sun, Paula J Finton, Brian R Curtis, Juhua Chen, Renliang Zhang, Roy L Silverstein, Stanley L Hazen

Affiliations

Eugene A Podrez et al. Nat Med. 2007 Sep.

Abstract

Dyslipidemia is associated with a prothrombotic phenotype; however, the mechanisms responsible for enhanced platelet reactivity remain unclear. Proatherosclerotic lipid abnormalities are associated with both enhanced oxidant stress and the generation of biologically active oxidized lipids, including potential ligands for the scavenger receptor CD36, a major platelet glycoprotein. Using multiple mouse in vivo thrombosis models, we now demonstrate that genetic deletion of Cd36 protects mice from hyperlipidemia-associated enhanced platelet reactivity and the accompanying prothrombotic phenotype. Structurally defined oxidized choline glycerophospholipids that serve as high-affinity ligands for CD36 were at markedly increased levels in the plasma of hyperlipidemic mice and in the plasma of humans with low HDL levels, were able to bind platelets via CD36 and, at pathophysiological levels, promoted platelet activation via CD36. Thus, interactions of platelet CD36 with specific endogenous oxidized lipids play a crucial role in the well-known clinical associations between dyslipidemia, oxidant stress and a prothrombotic phenotype.

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Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1

CD36 plays a role in thrombosis in vivo in the setting of hypercholesterolemia. (a–c) Mice of the indicated genotypes were maintained on Western diet (solid bars) and then used for an intravital thrombosis assay. Mesenteric arterioles (a) or venules (b) or carotid arteries (c) were visualized, and in vivo thrombosis times were measured as described in Methods. Mice on chow diet (empty bars) were analyzed in the same way, except that FeCl3 exposure times were increased by 1 min. Data are presented as mean ± s.e.m. for n ≥ 7 for chow diet and n ≥ 8 for Western diet. (d) _Apoe_−/− mice and _Apoe_−/− _Cd36_−/− on a Western diet were depleted of platelets by γ-irradiation, subsequently injected with platelets from either _Apoe_−/− mice (n = 5) or _Apoe_−/− _Cd36_−/− mice (n = 5) and the carotid artery occlusion test was performed as described in Methods. (e) Tail vein bleeding assay was performed as described in Methods. Data are presented as mean ± s.e.m.; numbers of mice used are: wild-type (n = 8), _Cd36_−/− (n = 5), _Apoe_−/− (n = 4) and _Cd36_−/− _Apoe_−/− (n = 4). (f) Total plasma cholesterol concentration is presented as mean ± s.d. mg/dl for at least 6 animals with the indicated genotype on a Western diet. ***P < 0.001 by _t_-test as compared to wild-type. (g) Indicated groups of mice were fed a high-cholesterol diet as described in Methods, and in vivo thrombosis times were assessed as in a–c. n ≥ 7 animals in each group. See Supplementary Methods for details.

Figure 2

Figure 2

CD36 deficiency blunts platelet responses to agonists in hypercholesterolemic plasma. (a,b) Platelet aggregation in platelet-rich plasma from mice of the indicated genotypes on the indicated diets was induced by ADP and optically monitored. Representative aggregation curves are shown. Bar graph shows aggregation results expressed as maximal amplitude of aggregation (mean ± s.e.m.; n = 3 for WT, n = 4 for _Apoe_−/−, n = 6 for _Apoe_−/− _Cd36_−/−, n = 3 for Ldlr−/− and n = 4 for _Ldlr_−/− Cd36−/−). (c) Platelets were isolated by gel filtration of pooled blood from wild-type and _Cd36_−/− mice on a chow diet and _Apoe_−/− mice on a Western diet as described in Methods. Citrated, pooled, platelet-poor plasma from either wild-type or _Apoe_−/− mice on a Western diet was isolated and combined with platelets of the indicated genotype at a 1:1 ratio. Aggregation was induced by ADP and optically monitored. Representative aggregation curves are shown. (d) Bar graph shows aggregation results from c expressed as maximal amplitude of aggregation (mean ± s.e.m.; n = 3 for WT, n = 3 for _Apoe_−/− and n = 4 for _Cd36_−/−). (e) Flow cytometric analysis of integrin αIIbβ3 activation on mouse platelets. Platelets and citrated, platelet-poor plasma were isolated and combined at a 1:2 ratio (vol/vol), stimulated with ADP and integrin αIIbβ3 activation was assessed using JON/A, a monoclonal, phycoerythrin-conjugated antibody for mouse αIIbβ3 in the activated conformation (mean ± s.e.m.; n = 3 for WT, n = 3 for _Apoe_−/−, n = 4 for _Cd36_−/−). *P < 0.05, **P < 0.01 and ***P < 0.001. See Supplementary Methods for details.

Figure 3

Figure 3

Platelet CD36 specifically binds oxPCCD36 and LDL oxidized by MPO−H2O2−NO2− system. (a) [125I]LDL modified by the indicated methods was incubated with platelets isolated from humans in Tyrode’s buffer in the presence of 2 mM Ca2+ at room temperature. Unbound [125I]LDL was separated from platelets and the bound radioactivity was quantified. NO2-LDL, LDL oxidized by the MPO-H2O2-NO2− system; AcetLDL, acetylated LDL; Cu-oxLDL, Cu2+-oxidized LDL. (b) Concentration dependence of [125I]NO2-LDL binding by platelets. Binding of [125I]LDL was used as a control. (c) Human platelets were fixed with paraformaldehyde and incubated with [125I]NO2-LDL in the presence or absence of the indicated antibodies (20 μg/ml), and the bound radioactivity was assessed. (d) Binding of native [125I]LDL and [125I]NO2-LDL by isolated platelets from _Cd36_−/− and wild-type mice was assessed as in a. (e) Binding of [125I]NO2-LDL to human platelets in the presence of various concentrations of the indicated phospholipids was determined as in a. (f) Direct binding of vesicles containing 40 mol % of PAPC (for KOOA-PC) or 40 mol % of PLPC (for KODA-PC), 40 mol % of the indicated specific native phospholipid or oxPCCD36, 20 mol % of cholesterol, and trace amounts of [3H]DPPC (1,2-dihexadecanoyl-_sn_-glycero-3-phosphocholine) were generated and then incubated with platelets alone or, in the case of KODA-PC–containing vesicles, in the presence of either CD36-specific monoclonal antibody (FA6) or nonimmune, isotype-matched antibody (NI IgG). Unbound vesicles were separated and platelet-bound radioactivity was then quantified. Results represent the mean ± s.d. of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. See Supplementary Methods for details.

Figure 4

Figure 4

oxPCCD36 activates platelet fibrinogen receptor integrin αIIbβ3 in a CD36-dependent manner. (a) Human platelets isolated by gel filtration were incubated with increasing concentrations of oxPCCD36 or OV-PC and αIIbβ3 activation was assessed on the basis of the binding of 125I-labeled fibrinogen. (b) Human platelets isolated by gel filtration were incubated with increasing concentrations of oxPCCD36 or OV-PC. After addition of agonist, FITC-labeled PAC-1 mouse monoclonal antibody (specific for activated integrin αIIbβ3) was added at a dilution of 1:100 and αIIbβ3 activation was assessed by FACS analysis. (c) Human platelets were isolated from CD36+/+ or _CD36_−/− donors and analyzed for αIIbβ3 activation as in b. The final concentrations of stimuli used were 10 μM ADP, 20 μM lipid oxidized phospholipids, 0.5 U/ml thrombin and 10 nM PMA. NA, no additions. (d) Washed platelets from wild-type or _Cd36_−/− mice were incubated with 10 μM ADP or 20 μM synthetic oxidized phospholipids, and platelet activation was assessed by 125I-labeled fibrinogen binding. NA, no additions. Results represent the mean ± s.d. of three independent experiments. Statistical significance was determined by _t_-test: *P < 0.05, **P < 0.01, ***P < 0.001. See Supplementary Methods for details.

Figure 5

Figure 5

oxPCCD36 induce platelet P-selectin expression in a CD36-dependent manner. (a) Isolated human platelets were incubated with the indicated stimuli and P-selectin expression was assessed as described in Methods. NA, no additions. Results represent the mean ± s.d. of three independent experiments. 5–144 aa, CD36-GST fusion peptide with amino acids 5–144 of CD36; GST, glutathione _S_-transferase. (b) Binding of 3H-labeled small unilamellar vesicles containing oxPCCD36 (HODA-PC) to recombinant CD36-GST fusion peptides immobilized on glutathione-Sepharose was assessed in the presence or absence of the indicated competitors. 93–120 aa, CD36-GST fusion peptide with amino acids 93–120 of CD36. (c,d) Effect of the Fab fragment of the CD36-blocking monoclonal antibody on platelet activation induced by (c) NO2-LDL and NO2-PAPC and (d) oxPCCD36. NI, nonimmune isotype-matched antibody. (e) Platelet P-selectin expression was determined using FITC-conjugated antibody to mouse P-selectin. (f) Human platelets isolated from CD36+/+ or _CD36_−/− donors were treated with agonists and P-selectin expression was assessed. (b,c,e) Data are presented as mean ± s.d. of three independent experiments; (d,f) data are presented as a typical result of at least three independent experiments. (g) Human platelets from CD36+/+ or _CD36_−/− donors (n = 3) were combined with human citrated plasma from various donors, stimulated with ADP, and P-selectin expression was assessed. Samples were divided into tertiles according to P-selectin expression, the mean plasma sample lipid levels were assigned a relative value of 1 for the lowest P-selectin tertile, and the means ± s.e.m. are shown for all P-selectin tertiles. (h) Human plasma samples (n = 24) were analyzed for levels of multiple oxidized phospholipids, HDL cholesterol and LDL cholesterol. Samples were divided into tertiles according to HDL cholesterol or LDL cholesterol concentrations. The mean oxidized phospholipid levels were assigned relative values of 1 for the lowest lipoprotein tertile, and corresponding mean ± s.e.m. are shown for all lipoprotein tertiles. *P < 0.05, **P < 0.01, ***P < 0.001. See Supplementary Methods for details.

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