Binding of Oxidized Low-Density Lipoprotein on Circulating Platelets Is increased in Patients With Acute Coronary Syndromes and Induces Platelet Adhesion to Vascular Wall In Vivo—Brief Report
Objective—Hyperlipidemia is associated with platelet hyperactivity. In the present study, we evaluated the binding of oxidized low-density lipoprotein (oxLDL) on the surface of circulating platelets in patients with stable coronary artery disease and acute coronary syndromes and its possible association with platelet activation. Furthermore, the role of oxLDL binding on platelet adhesion to collagen and endothelial cells in vitro as well as after carotid ligation in mice was investigated.
Methods and Results—Using flow cytometry, patients with acute coronary syndromes (n=174) showed significantly enhanced oxLDL binding compared with patients with stable coronary artery disease (n=182; P=0.007). Platelet-bound oxLDL positively correlated with the degree of platelet activation (expression of P-selectin and activated fibrinogen receptor; P<0.001 for both). Plasma oxLDL was increased in patients with acute coronary syndromes compared with stable angina pectoris patients. Preincubation of isolated platelets with oxLDL, but not with native LDL, resulted in enhanced platelet adhesion to collagen and activated endothelial cells under high shear stress in vitro, as well as after carotid ligation in C57BL/6J mice and apolipoprotein E−/− mice fed a high cholesterol diet.
Conclusion—Increased platelet-bound oxLDL in patients with acute coronary syndromes may play an important role in atherothrombosis, thus providing a potential future therapeutic target.
Blood platelets and oxidized low-density lipoprotein (oxLDL) are critically involved in atherogenesis and acute coronary syndromes (ACS).1–6 Although their interplay in platelet activation, foam cell formation, and vascular inflammation has been suggested by several experimental studies,4–7 its clinical value remains obscure. oxLDL binds to 5 scavenger receptors expressed on the platelet surface: class A scavenger receptor, CD36, lectin-like oxidized LDL receptor-1, class B scavenger receptor I, and scavenger receptor that binds phosphatidylserine and oxidized lipoprotein/chemokine (C-X-C motif) ligand 16.5,6 We have previously reported that binding of oxLDL on platelets results in enhanced platelet activation and platelet phagocytosis by macrophages and foam cell formation.4,6,7 Therefore, platelet–oxLDL interaction may play a crucial role in the initiation and progression of arteriosclerosis, but its clinical relevance has not been elucidated so far.
The aim of the present study was to assess platelet-bound oxLDL in patients with stable coronary artery disease (CAD) or ACS and its association with clinical presentation of CAD and platelet activation. Furthermore, the impact of oxLDL on platelet adhesion to the vascular wall in vitro and in vivo was investigated.
Patients and Methods
Detailed description of patients, methods, and materials is presented in the online-only Data Supplement.
Three hundred fifty-six consecutive patients with symptomatic CAD undergoing coronary angiography were recruited into the study. Informed written consent was obtained from each patient, and the study was approved by the local ethics committee.
Whole-Blood Flow Cytometry
Whole blood obtained from all patients was studied for platelet-bound oxLDL and glycoprotein Ib (GPIb) (CD42b) by flow cytometry analysis. The surface expression of P-selectin (CD62P) and activated fibrinogen receptor (GPIIb/IIIa) was measured as markers of platelet activation.
Adhesion Assays In Vitro
To evaluate platelet adhesion of human platelets from healthy donors to immobilized collagen or endothelial cells under flow conditions, perfusion experiments were performed at shear rates of 2000 per second (high shear) in a flow chamber (Oligene, Berlin, Germany).
The common carotid artery of C57BL/6J mice or apolipoprotein E−/− mice fed a high cholesterol diet for 6 weeks was dissected free and ligated vigorously for 5 minutes to induce vascular injury. Before and after vascular injury, platelet–endothelium interaction was visualized by in vivo video microscopy. All images were recorded and evaluated off-line.
Data are presented as mean±SD, unless otherwise stated. All tests were 2-tailed, and statistical significance was considered for P<0.05. All statistical analyses were performed using SPSS version 19 for windows (Chicago, IL).
We first analyzed the surface expression of platelet-bound oxLDL in a consecutive cohort of 356 patients with symptomatic CAD, including ACS (n=174) and stable angina pectoris (SAP; n=182) as well as in an elderly group of patients without known or suspected CAD (n=30). The demographic details are given in Table I in the online-only Data Supplement. Platelet-bound oxLDL was significantly enhanced in ACS compared with SAP (platelet-oxLDL in ACS versus SAP [mean±SD]: 124.8 ± 42.1 versus 111.7 ± 41.9 mean fluorescence intensity P=0.007; Figure and Figure I in the onine-only Data Supplement). Furthermore, platelet-bound oxLDL was significantly increased in patients with SAP compared with the elderly control group without known or suspected CAD (SAP versus control: 111.7 ± 41.9 versus 76.6 ± 11.6 mean fluorescence intensity P<0.001; Figure). In plasma, oxLDL was significantly increased in patients with ACS compared with patients with SAP (plasma oxLDL in ACS versus SAP: 65.3 ± 32.2 versus 58.9 ± 32.8; P=0.029; Figure I in the online-only Data Supplement). There was no significant difference in the platelet-bound oxLDL, when we compared patients with non–ST-elevation myocardial infarction versus ST-elevation myocardial infarction (Figure II in the online-only Data Supplement). Interestingly, platelet-bound oxLDL positively correlated with the degree of platelet activation and inversely with platelet number in patients with CAD as well as in patients with SAP or ACS (Figure and Figure IIIA–IIIF in the online-only Data Supplement). The 95% CI for mean (lower bound to upper bound) was 72.27 to 80.93 for control group, 105.62 to 117.88 for SAP, and 118.46 to 131.06 for ACS. Furthermore, platelet-bound oxLDL inversely correlated with total cholesterol (r=−0.221; P=0.005) and LDL (r=−0.166; P=0.041) and positively with troponin I (r=0.144; P=0.024). In a univariate ANOVA, the increase of platelet-bound oxLDL in ACS was influenced only by high-density lipoprotein levels and number of coronary arteries affected (Table).
In vitro, isolated human platelets from healthy donors treated with oxLDL showed increased activation as assessed by the expression of P-selectin and activated GPIIb/IIIa (Figure IV in the online-only Data Supplement). Similarly, murine platelets treated with oxLDL revealed increased expression of activated GPIIb/IIIa (clone JON/A) (Figure IV in the online-only Data Supplement). Binding of human oxLDL to murine platelets was verified by flow cytometry (Figure V in the online-only Data Supplement). To further investigate the impact of oxLDL binding on platelet function, dynamic adhesion assays and intravital microscopy after carotid ligation were performed. Preincubation of isolated platelets with human oxLDL resulted in increased platelet adhesion to collagen (P<0.05) and activated endothelial cells (P<0.05) under high shear stress in vitro (P<0.05; Figure). Interestingly, adhesion of oxLDL-treated platelets to endothelial cells was inhibited by preincubation with an anti-GPIIb/IIIa antibody, but not with an anti-GPIbα antibody, whereas adhesion to immobilized collagen was significantly reduced by soluble GPVI-Fc compared with Fc control (Figure VI in the online-only Data Supplement). In vivo, platelet adhesion to the injured vascular wall after carotid ligation in C57BL/6J mice was significantly increased after treatment of platelets with human oxLDL compared with LDL (Figure). Accordingly, we performed intravital microscopy experiments in atherosclerotic apolipoprotein E−/− mice fed a high cholesterol diet and observed increased adhesion of oxLDL-treated platelets to the vascular lesion compared with LDL-treated platelets (P<0.05; Figure VII in the online-only Data Supplement).
The major findings of the present study are as follows: (1) binding of oxLDL on circulating platelets is elevated in patients with ACS compared with patients with stable CAD; (2) oxLDL binding on the platelet surface correlates with platelet activation; (3) binding of oxLDL, but not of native LDL, on isolated platelets results in enhanced platelet adhesion to immobilized collagen and activated cultured endothelium in vitro and to the injured carotid artery in vivo.
oxLDL plays a key role in CAD and, particularly, in ACS by increasing the vulnerability of coronary atherosclerotic plaques.2,8 OxLDL induces macrophage activation, foam cell generation, smooth muscle cell proliferation, and a decrease in endothelial production of NO. Furthermore, oxLDL promotes thrombogenicity by stimulating the release of tissue factor but, most importantly, by enhancing platelet activation and adhesion to endothelium. Platelet exposure to oxLDL varies depending on the available concentrations of oxLDL either in blood (circulating oxLDL) or at sites of unstable lesions where increased oxLDL concentrations are released after plaque rupture.9 In vitro studies have shown that oxLDL binds to platelet CD36, inducing platelet activation.10 In the present study, we show for the first time that oxLDL binding to platelets is increased in ACS and correlates with the extent of platelet activation in patients with CAD. In conclusion, lipid oxidation may represent the link between dyslipidemia and prothrombotic state. Interactions between oxLDL and platelets enhance platelet reactivity and adhesion and may play a crucial role in coronary thrombosis in ACS. Lipoprotein-platelet interplay may be a promising therapeutic target in patients with atherothrombotic disease.
We thank Jadwiga Kwiatkowska, Sarah Gekeler, and Viktoria Mozes for perfect technical assistance.
Sources of Funding
The study was supported by the Fortüne programme of the University of Tübingen to K.S. and by the Deutsche Forschungsgemeinschaft (TR-SFB19) and the Klinische Forschergruppe KFO 274 Platelets – Molecular Mechanisms and Translational Implications. H.F.L. is supported by the Volkswagen Foundation (Lichtenberg program) and the Wilhelm-Sander foundation.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.111.244707/-/DC1.
- Received December 22, 2011.
- Accepted May 25, 2012.
- © 2012 American Heart Association, Inc.
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