Thrombosis Research 132 (2013) 702–711

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Regular Article

Oxidized low-density lipoprotein-induced CD147 expression and its inhibition by high-density lipoprotein on platelets in vitro☆ Sheng-Hua Yang, Yun-Tian Li ⁎, Da-Yong Du Coronary Heart Disease Diagnosis and Treatment Center of the Chinese People’s Liberation Army, the 305th Hospital of Chinese People’s Liberation Army, Wenjin Street, Beijing, 100017, PR China

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Article history: Received 19 March 2013 Received in revised form 26 September 2013 Accepted 1 October 2013 Available online 12 October 2013 Keywords: CD147 EMMPRIN ox-LDL HDL Platelet Plaque

a b s t r a c t Introduction: Matrix metalloproteinases (MMPs) are believed to progressively degrade the collagenous components of the protective fibrous cap, leading to atherosclerotic plaque rupture or destabilization. Oxidized lowdensity lipoprotein (ox-LDL) enhances the release of CD147, known as the extracellular MMP inducer, from coronary smooth muscle cells. However, whether ox-LDL can induce platelet CD147 expression is unknown. Therefore, we investigated the influence of ox-LDL and high-density lipoprotein (HDL) on CD147 expression on human platelets. Materials and Methods: Washed platelets were incubated with ox-LDL (or native LDL) and HDL or anti-LOX-1 monoclonal antibody prior to incubation with ox-LDL. In parallel, buffer (PBS) was added to washed platelets as a control. The expression levels of CD147, CD62P, CD63 and Annexin V were assessed by flow cytometry, and soluble CD147 from the platelets was assessed by an enzyme-linked immunosorbent assay. Laser scanning microscopy (LSM) and transmission electron microscopy (TEM) were used to visualize the morphological changes and granule release, respectively, from the platelets. Results: Platelets treated with ox-LDL exhibited a significant increase in the expression of CD147 (or Annexin V), followed by increases in CD62P and CD63, compared with the control group. In contrast, HDL or anti-LOX-1 monoclonal antibody decreased these effects. The expression of soluble CD147 increased as the concentration of ox-LDL used to treat the platelets increased. After exposure to ox-LDL, morphological changes and granule release in the platelets were visualized by LSM and TEM. Additionally, the TEM revealed that HDL inhibits alpha-granule release. Conclusions: In platelets, ox-LDL stimulates the release of CD147 via binding to LOX-1, whereas HDL inhibits this effect. This finding could provide new insights concerning the influence of ox-LDL and HDL on plaque stability by the up-regulation of CD147 on platelets. © 2013 Elsevier Ltd. All rights reserved.

Introduction Most acute coronary syndromes (ACS), such as unstable angina, myocardial infarction, and sudden death, are triggered by plaque rupture and the subsequent thrombus [1–3]. CD147, an extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN), can up-regulate MMPs, and the up-regulation of MMPs leads to atherosclerotic plaque rupture by degrading the extracellular matrix (ECM), which is the main component of fibrous caps [4–6]. CD147 was first identified as a surface protein Abbreviations: Ox-LDL, oxidized low-density lipoprotein; HDL, high-density lipoprotein; MMPs, matrix metalloproteinases; EMMPRIN, extracellular matrix metalloproteinase inducer; ECM, extracellular matrix; ACS, acute coronary syndromes; CAD, coronary artery disease; PRP, platelet-rich plasma; OCS, open canalicular system. ☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⁎ Corresponding author at: Cardiology Department of the 305th Hospital of Chinese People’s Liberation Army, No.13, Wenjin Street, Xi-cheng District, Beijing, 100017, PR China. Tel.: +86 1066799271; fax: +86 1063093137. E-mail address: [email protected] (Y.-T. Li). 0049-3848/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.thromres.2013.10.003

on tumor cells [7] and was found to be expressed constitutively on monocytes, granulocytes, and lymphocytes [8]. CD147, as a novel receptor, was recently reported to be localized in the open canalicular system (OCS) of platelets and α granules, and CD147 activates platelets and stimulates MMP-9 synthesis in monocytes [9]. Platelet CD147 expression is up-regulated after washed platelets are exposed to various stimuli (e.g., thrombin, ADP, and collagen) in vitro [9]. Importantly, in vivo studies have shown that platelet CD147 expression is significantly greater in patients with coronary artery disease (CAD) compared with that in a control population and demonstrates a stronger association with age [10]. Furthermore, CD147 is able to enhance platelet-monocyte interactions in vivo and to promote monocyte recruitment to the arterial wall [11]. Ox-LDL is thought to be involved in the initiation of atherosclerotic lesions, mainly by leading to foam cell formation and vascular endothelial damage [12]. However, more importantly, a growing body of evidence suggests that elevated levels of circulating oxidized LDL serve as a sensitive marker for CAD [13], independently associate with the carotid intima-media thickness [14], display a significant positive correlation with the severity of acute coronary syndromes [15], and even serve as a

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dominant receptor involved in ox-LDL binding to activated platelets. However, whether blocking LOX-1 affects the expression of CD147 in platelets is still unclear. Ox-LDL has been shown to increase CD62P expression in platelets [22,24]. Moreover, ox-LDL has been reported to enhance serotonin release from platelet-dense granules [28] and to induce CD147 expression on coronary artery smooth muscle cells [29]. However, whether ox-LDL stimulates platelet CD147 expression is unknown. In addition, HDL is capable of inhibiting platelet activation [30]. Therefore, we examined whether HDL has an inhibitory effect on platelet CD147 expression. In this study, we evaluated the effects of ox-LDL, HDL and anti-LOX-1 monoclonal antibody (mAb) in the expression of CD147 in platelets in vitro.

strong predictor for acute coronary heart disease events in apparently healthy, middle-aged men [16]. Because ox-LDL is present in the circulation [17,18], it can make contact with platelets. Thus, research on the ox-LDL interaction with platelets is necessary. Previous studies have shown that mildly oxidized LDL enhances platelet aggregation via the activation of phospholipase A2 [19,20], whereas heavily oxidized LDL becomes a platelet aggregation inhibitor [21]. Although some studies reported that a low concentration of ox-LDL inhibited ADP-induced platelet aggregation [22,25], ox-LDL is generally thought to be bound to several receptors on platelets, including SRA [23], CD36 [24], LOX-1 [21], platelet activating factor (PAF) receptor [26], and SR-PSOX/ CXCL16 [27], thereby leading to platelet activation. LOX-1 serves as a

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Fig. 1. Ox-LDL induces platelet CD147 expression. Flow cytometry was used to detect the expression of CD147 on platelets. A, Percentages of CD147-positive platelets treated with ox-LDL (0, 50 or 100 μg/ml); B, Change in the platelet CD147 relative mean fluorescence intensity (rMFI = monoclonal antibody/corresponding isotype control) following ox-LDL treatment. C, Representative of 5 experiments; the overlain histograms on the right show the expression levels of each platelet marker in PBS-treated and ox-LDL-treated platelets (PBS-treated, red; 50 μg/ml ox-LDL, blue; 100 μg/ml ox-LDL, green) ⁎P = 0.032, ⁎ ⁎P = 0.006, &$P b 0.001, NS = not significant. In D, NS vs. Control, ⁎P b 0.01 vs. control, #P b 0.01 vs. Ox-LDL (25 μg/ml). The data represent the means ± SD of 5 independent experiments.

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were resuspended and washed twice with modified Tyrode’s solution (137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.4 mM NaH2PO4, 5 mM HEPES, 0.1% glucose, and 0.35% bovine serum albumin, pH 7.4). The washing procedure was also performed after the addition of 500 nM PGE1 to the platelet suspension. The washed platelets were adjusted to a concentration of 3x108 cells/ml and finally suspended in Tyrode’s solution containing 0.35% bovine serum albumin. Preparation of Ox-LDL and HDL

Fig. 2. Soluble CD147 (sCD147) in platelet supernatant. Platelets stimulated with ox-LDL were centrifuged at 750 x g for 20 minutes, and the sCD147 from the supernatant was detected using ELISA. The results of 5 independent experiments are shown. The error bars represent the means ± SE. #P = 0.048, NS = not significant.

Materials and Methods Blood Collection Whole fresh blood was drawn from healthy, non-smoking human volunteers who were free from hemostatic disorders, diabetes, and infectious, hepatic, renal, cardiovascular, and malignant diseases and who did not ingest alcohol in the previous 24hours or take any medicine during the preceding two weeks. The blood samples were collected after overnight fasting via forearm venipuncture performed by a trained nurse using a 21G needle without a tourniquet. After discarding the initial 4 ml, 24 ml of blood was injected gently into two 15-ml polypropylene tubes containing 1 volume of acid-citrate-dextrose (2.5% sodium citrate, 1.4% citric acid, and 2% glucose) for 6 volumes of blood, and the tubes were mixed by gentle inversion. The study was approved by the committee on the Ethics of Research in Human Experimentation of the 305th Hospital of the Chinese People's Liberation Army, and written informed consent was obtained from every volunteer donator. Platelet Isolation Platelets were prepared as previously described with some modifications [31–33]. Briefly, anti-coagulated blood was centrifuged at 100 x g for 15 minutes at 24°C without braking to generate platelet-rich plasma (PRP). By centrifuging the PRP at 500 x g for 8 minutes in the presence of 500 nM PGE1, the platelets were isolated. The platelets

Ox-LDL and HDL were obtained from Beijing Union Biotechnology (Beijing, China). LDL was purified by ultracentrifugation (1.019– 1.063 g/ml) and oxidized with 10 μmol/l CuSO4 at 37 °C for 24 hours. The oxidation was terminated by adding excess EDTA-Na2. Each lot was analyzed by agarose gel electrophoresis for migration versus LDL. Copper and EDTA were removed by dialysis. The concentration of the ox-LDL was 1.0-2.0 mg protein/ml. Thiobarbituric acid–reactive substances were determined colorimetrically using malondialdehyde as the standard. HDL was isolated by a series of ultracentrifugation steps, and the obtained density was 1.063-1.21 g/ml. In parallel, ox-LDLs were prepared by oxidization with 10 μmol/l CuSO4 at 37°C for 4 or 6 hours. Ox-LDL (10 μmol/l CuSO4 at 37°C for 24 hours) was used in the flow cytometry analysis, phosphatidylserine (PS) exposure assay, enzyme-linked immunosorbent assay (ELISA), laser scanning microscopy (LSM) and transmission electron microscopy (TEM), while ox-LDLs (10 μmol/l CuSO4 at 37°C for 4 or 6 hours) were used only in the flow cytometry analysis. The ox-LDL and HDL were stored at 4°C and placed at room temperature for 30 minutes before use. Flow Cytometry Analysis The washed platelets were incubated with ox-LDL (25μg/ml, 50μg/ml or 100 μg/ml), native LDL (100 μg/ml) or HDL alone (100 μg/ml) for 25 minutes at 37°C. In parallel, washed platelets were treated with PBS as a control. In addition, six other groups of washed platelets were pre-incubated with 100 μg/ml HDL or anti-LOX-1 mAb (Abcam, Cambridge, UK; 50 μg/ml) for 15 minutes prior to incubation with oxLDL at concentrations of 25 μg/ml, 50 μg/ml or 100 μg/ml for 25 minutes at 37 °C. Then, all of the samples were incubated with anti-CD62P-PE (e-Bioscience, San Diego, CA, USA), anti-CD147-PE (e-Bioscience, San Diego, CA, USA) or anti-CD63-PE (BioLegend, San Diego, CA, USA) for 20 minutes at 24 °C. To minimize in vitro artifacts, a fixation procedure was omitted [34,35]. Acquisition was performed using an EPICS XL flow cytometer (Beckman Coulter, Miami, FL, USA). The platelets were gated on the basis of light scatter and CD61-FITC (e-Bioscience, San Diego, CA, USA) expression. The evaluated platelet population was ≥ 98% positive for CD61, and 50,000 events were collected per sample. The isotype-matched control for nonspecific binding was

Fig. 3. Effect of ox-LDL on platelet morphology using laser scanning microscopy. The platelets were visualized using anti-CD61-FITC. A, B, and C represent the PBS-treated platelets, 50 μg/ml ox-LDL-stimulated platelets, and 100 μg/ml ox-LDL-stimulated platelets, respectively. Compared with the control group, the ox-LDL-stimulated platelets form pseudopods. This figure is representative of 4 independent experiments. Bars: 10 μm.

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Fig. 4. Morphological changes in the platelets induced by ox-LDL or HDL visualized under transmission electron microscopy (TEM). The platelets were prepared as described in the Materials and Methods section. A and B represent the control group (treated with PBS). Compared with the control group, the 50 μg/ml ox-LDL-treated platelets (C and D) and 100 μg/ml ox-LDL-treated platelets (E and F) both exhibited platelet aggregation and degranulation. G and H represent the platelets treated with 50 μg/ml ox-LDL following incubation with 100 μg/ml HDL. Compared with the 50 μg/ml ox-LDL-treated platelets, HDL had an inhibitory effect on platelet degranulation. A, x 50,000; B, x 15,000; C, x 25,000; D, x 10,000; E, x 30,000; F, x 7,000; G, x 40,000; H, x 12,000.

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utilized to establish positive events based on a gate ratio of 2% background expression [36]. The results were expressed as the percentage of antibody-positive platelets and CD147 relative mean fluorescence intensity (CD147rMFI = monoclonal antibody/corresponding isotype control) and analyzed with FCS Express 4 De Novo flow cytometry analysis Software (Los Angeles, CA, USA, 2012). Phosphatidylserine (PS) Exposure Assay Washed platelets (3 × 108) were incubated with LDL (100 μg/ml), ox-LDL (50 μg/ml or 100 μg/ml) or PBS (control) for 25 minutes at 37°C, and then resuspended at a final concentration of 5x107/ml. The treated platelets were mixed with Annexin V binding buffer incubated with Annexin V-PE (e-Bioscience, San Diego, CA, USA) for 10 minutes in the dark at room temperature and immediately analyzed by flow cytometry. Enzyme-linked Immunosorbent Assay (ELISA) After incubation of the washed platelets with or without ox-LDL (50 μg/ml or 100 μg/ml) for 25 minutes at 37 °C, the samples were centrifuged at 750 x g for 20 minutes. The supernatants were collected to assess the soluble EMMPRIN (sCD147) levels using a commercially available ELISA kit, according to the manufacturer’s instructions (Abcam, Cambridge, UK). Laser Scanning Microscopy (LSM) and Transmission Electron Microscopy (TEM) The washed platelets either treated or not with ox-LDL (50 μg/ml or 100 μg/ml) for 25 minutes at 37 °C were incubated with anti-CD61-FITC for 20 minutes. Then, the samples were viewed using a LSM710 laser scanning microscope (CarlZeiss, Jena, Germany). In parallel, the other ox-LDL-treated platelets were fixed with 0.1 M cacodylate buffer (pH 7.4) containing 0.1% glutaraldehyde and 2% paraformaldehyde for 2 hours at 24°C and then washed twice with 0.1 M cacodylate buffer (pH 7.4). The fixed samples were dehydrated with alcohol, embedded, sectioned, and then double-stained with uranyl acetate and lead citrate. The samples were observed with a transmission electron microscope (TEM, Hitachi, H-7650, Japan).

C). However, no significant change in the expression of CD147 on platelets was identified between the two groups of ox-LDL-treated platelets (50 μg/ml vs. 100 μg/ml) (Fig. 1A). Similarly, the platelets treated with ox-LDL (50μg/ml or 100μg/ml) had a significantly higher CD147 relative mean fluorescence intensity (rMFI) than the untreated platelets (control group) (P b 0.001), whereas there was no significant difference between the ox-LDL-treated platelets (50 μg/ml or 100 μg/ml) (Fig. 1B). Furthermore, CD147 rMFI from the ox-LDL (25 μg/ml) group was higher than that from the control group and lower than that from the ox-LDL (50 μg/ml) group (P b 0.01 for both; Fig. 1D). Additionally, neither native LDL nor HDL alone showed a statistically significant difference in the platelet CD147 expression level compared with the control group (PN0.05 for both; Fig. 1D). To determine whether the washing procedure could cause platelet CD147 expression, platelet-rich plasma (PRP) obtained by centrifugation at 100 x g for 15 minutes at 24 °C was used. We observed that ox-LDL (50 μg/ml, 100 μg/ml) failed to up-regulate platelet CD147 expression in resting PRP in a short period (1 hour) (data not shown). However, PRP incubated with ox-LDL (50 μg/ml or 100 μg/ml) for 3 days showed an increased expression of platelet CD147 compared with untreated PRP (data not shown). Additionally, the soluble EMMPRIN (sCD147) levels increased in the 100 μg/ml ox-LDL-treated platelets compared with the untreated platelets (P = 0.048); the difference between the 50 μg/ml ox-LDL-treated and untreated platelet sCD147 levels was not significant (Fig. 2). Platelet CD147 was reported to be localized within the open canalicular system (OCS) and α-granules of platelets [9]. Thus, we observed the platelets in the presence or absence of ox-LDL (50 μg/ml or 100 μg/ml) via LSM and TEM. The morphology of the untreated platelets was different from that of the ox-LDL-treated platelets (Figs. 3 and 4). In particular, more intracellular α-granules were observed in the untreated platelets than in the ox-LDL-treated platelets by TEM, indicating that ox-LDL promoted platelet α-granule release (Fig. 4).

Statistical Analysis The Kolmogorov-Smirnov test was used to check the normality of the data. Those data with normally distributed variables were presented as the means ± SD and assessed using one-way ANOVA (LSD, S-N-K, Dunnet) or a paired Student’s t test. When the data were not normally distributed, we applied the Kruskal-Wallis H test or Wilcoxon’s matched pair signed-rank test. The statistics were analyzed using SPSS for Windows 13.0 (SPSS13.0 Inc. Chicago, IL, USA). A value of P b 0.05 was considered statistically significant. All experiments were performed at least five times with platelets from different donors. Results Platelet CD147 Expression is Up-regulated by Ox-LDL To evaluate the effect of ox-LDL on the expression of CD147 on platelets, washed platelets were incubated for 25minutes with different concentrations of ox-LDL (25 μg/ml, 50 μg/ml or 100 μg/ml). Ox-LDL significantly increased the platelet CD147 expression. The percentages of platelets treated with 50μg/ml or 100μg/ml ox-LDL that were positive for CD147 expression (8.06 ± 3.90%, 3.22-12.90% range and 10.70 ± 3.33%, 6.57-14.82% range, respectively) were considerably higher than those of platelets untreated with ox-LDL (0.58 ± 0.31%, 0.20-0.97% range), being approximately 13-fold and 17-fold greater (Fig. 1A and

Fig. 5. Reduction of ox-LDL-induced CD147 release by anti-LOX-1 monoclonal antibody. Washed platelets were pre-incubated with 50 μg/ml anti-LOX-1 monoclonal antibody for 15 minutes prior to incubation with ox-LDL at concentrations of 50 μg/ml or 100 μg/ml for 25 minutes at 37 °C. rMFI = monoclonal antibody/corresponding isotype control. *P b 0.01 vs. anti-LOX-1 + ox-LDL (50 μg/ml), **P b 0.01 vs. anti-LOX-1 + ox-LDL (100 μg/ml). The data represent the means ± SD of 5 independent experiments.

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82.66%, and 37.78-76.81% for untreated, 50 μg/ml and 100 μg/ml oxLDL platelets, respectively). Similarly, ox-LDL (50 μg/ml or 100 μg/ml) increased the expression of CD63 on the platelets (Fig. 6B and C). Interestingly, platelets treated with 100μg/ml ox-LDL demonstrated a significant decrease in CD63 expression compared with platelets treated with 50 μg/ml ox-LDL (Fig. 6B and C).

Blocking LOX-1 Reduces Ox-LDL-induced CD147 Expression in Platelets To examine whether the increasing release of CD147 by platelets treated with ox-LDL is associated with LOX-1, washed platelets were pre-incubated with anti-LOX-1 mAb and then treated the platelets with ox-LDL. By flow cytometry analysis, we found that the platelet CD147 rMFI from the anti-LOX plus ox-LDL groups (50 μg/ml or 100 μg/ml) decreased compared with those from the ox-LDL groups (50 μg/ml or 100 μg/ml), with P-values b0.01 for anti-LOX-1 plus ox-LDL (50 μg/ml) (0.96 ± 0.08) vs. ox-LDL (50 μg/ml) (1.18 ± 0.07) and for anti-LOX-1 plus ox-LDL (100 μg/ml) (1.05 ± 0.07) vs. ox-LDL (100 μg/ml) (1.24 ± 0.08; Fig. 5).

HDL Inhibits the Ox-LDL-induced Expression of Platelet CD147, CD62P, and CD63 To determine whether HDL (1.063-1.21 g/ml) inhibits the ox-LDLinduced up-regulation of platelet CD147, platelets pre-incubated with HDL (100 μg/ml) were stimulated with ox-LDL (50 μg/ml or 100 μg/ml). Flow cytometry analysis suggested that HDL (100 μg/ml) decreased CD147 expression not only in the 50 μg/ml ox-LDL-stimulated platelets (P = 0.027) but also in the 100 μg/ml ox-LDL-stimulated platelets (P = 0.017) (Fig. 7A). With respect to the CD147 rMFI, there was a significant decrease in the platelets pre-incubated with HDL (100 μg/ml) (P = 0.031 for the 50μg/ml ox-LDL-stimulated platelets [Fig. 7B], and Pb 0.05 for the 100 μg/ml ox-LDL-stimulated platelets [data not shown]). In parallel with the reduction in platelet CD147 expression, pretreatment with HDL (100 μg/ml) also attenuated the expression of CD62P and CD63 on platelets stimulated with ox-LDL (50 μg/ml or 100 μg/ml) (Fig. 8A and

Ox-LDL Induces Platelet CD62P and CD63 Expression The percentage of CD62P-positive platelets was significantly higher in ox-LDL-stimulated platelets (50 μg/ml or 100 μg/ml) than in nonstimulated platelets (P b 0.001 and P = 0.004, respectively) (Fig. 6A and B). Meanwhile, as the concentration of ox-LDL increased from 50 to 100 μg/ml, the CD62P surface expression on the platelets tended to decrease, although not to a significant level (Fig. 6A). In addition, there was a high degree of variation between subjects with respect to the level of platelet CD62P expression (ranges of 1.07- 4.50%, 58.07-

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Fig. 6. Ox-LDL increases the percentage of platelets expressing CD62P and CD63. The bars indicate the means ± SD of 7 experiments. ⁎&P b 0.001, ⁎⁎&P = 0.004. &⁎P b 0.001, &⁎⁎P = 0.009, &&⁎ P = 0.006, NS = not significant. C, Representative of 5 experiments. The overlain histograms on the right show the expression levels of each platelet marker in PBS-treated and ox-LDLtreated platelets (PBS-treated, red; 50 μg/ml ox-LDL, blue; 100 μg/ml ox-LDL, green). The data represent the means ± SD of 5 independent experiments.

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Fig. 7. HDL inhibits platelet CD147 expression. The platelets were pre-incubated for 15 minutes with HDL (100 μg/ml) followed by incubation with ox-LDL (50 μg/ml or 100 μg/ml) for 25 minutes. CD147 expression was detected by flow cytometry. The platelet CD147 (%) and rMFI (rMFI = monoclonal antibody/corresponding isotype control) are expressed as the means ± SD (n = 5). #*P = 0.027, ##*P = 0.017, #$P = 0.031, #$$P b 0.01.

B). Moreover, the TEM images supported the inhibition of the ox-LDLinduced platelet degranulation by HDL, thereby suppressing the expression of CD147, CD62P, and CD63 (Fig. 4). Ox-LDL Induces Platelet Apoptosis To examine the effect of ox-LDL on platelet viability, we detected the expression of Annexin V in the platelets treated with ox-LDL (50 μg/ml or 100 μg/ml) or LDL (100 μg/ml). The percentage of platelets positive for Annexin V from the 50 μg/ml ox-LDL group (2.32% ± 0.27%) was higher than that from the control group (0.18% ± 0.05%, P b 0.001) and lower than that from the 100 μg/ml ox-LDL group (3.37% ± 0.28%, P b 0.001); no significant difference was found between the control and LDL groups (P N 0.05; Fig. 9). Discussion The major findings of this study were that ox-LDL increases platelet CD147 expression and that HDL decreases ox-LDL-induced platelet CD147 expression. In this study, ox-LDL (25 μg/ml, 50 μg/ml and 100 μg/ml) induced the expression of CD147 on platelets in a concentration-dependent manner, whereas native LDL or HDL alone did not. To reduce the effect of paraformaldehyde fixation on platelet activation [34,35], the washed platelets in this study were not fixed and all samples were analyzed by flow cytometry within two hours. However, unfixed platelets would activate spontaneously with time, so we could not performance time-dependent experiments. Ox-LDL

Fig. 8. HDL decreases platelet CD62P and CD63 expression. Washed platelets were incubated with HDL (100 μg/ml) for 15 minutes at 37 °C, followed by incubation with ox-LDL (50 μg/ml or 100 μg/ml) for 25 minutes. A, CD62P expression was detected by flow cytometry. The data are expressed as the means ± SD (n = 5). @#P = 0.002, @##P = 0.018. B, CD63 expression was detected by flow cytometry. The data are expressed as the means ± SD (n = 5). &&P = 0.038, $$P = 0.014.

induced the expression of CD147 on washed platelets in a short period (b1 hour), whereas PRPs incubated with ox-LDL required a long period (approximately 3 days) to increase their expression of CD147. This observation may be due to the interference of ox-LDL binding to CD36 on the platelets with the binding of soluble fibrinogen in PRPs to αIIbβ3 [21], which affects platelet activation within a short period (b1 hour), whereas with the incubation time prolonged, the ox-LDLinduced platelet activation might predominate. In addition, we showed that the level of soluble CD147 isolated from platelet supernatants was higher in 100 μg/ml ox-LDL-induced platelets compared with untreated platelets, whereas the level did not increase in the 50 μg/ml ox-LDLinduced platelets. We hypothesize that this result may be explained by the higher concentration of ox-LDL causing the CD147 expressed on the platelet surface to be released into solution. Additionally, we repeated the experiments with less oxidized LDL (10 μM CuSO4 for 4 h or 6 h); we obtained the same results found with ox-LDL-induced platelet CD147 expression (Table 1). In this study, the CD147 MFI was quantified by the rMFI (see Methods), as this measurement is more reproducible. Ox-LDL, as an independent platelet activator, can induce the aggregation of isolated platelets [35–38]. Similarly, we observed ox-LDLinduced platelet aggregation via LSM. Moreover, in this study, we also demonstrated that ox-LDL (50 μg/ml or 100 μg/ml) induced the secretion of alpha-granule membrane protein (CD62P) and lysosome intact membrane protein (CD63). These results are consistent with those of past studies [24,26,39]. However, in our study, ox-LDL increased

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Fig. 9. Ox-LDL induces phosphatidylserine (PS) exposure in platelets. (A) Representative dot plots and the percentage annexin V-positive cells are shown for platelets treated for 25 minutes at 37 °C with control buffer, LDL or ox-LDL. (B) Means ± SD for 5 experiments. ⁎P b 0.001 vs. control, #P b 0.001 vs. ox-LDL (50 μg/ml).

CD62P and CD63 more markedly than anticipated, possibly due to the oxidation concentration of ox-LDL (10 μM CuSO4). Interestingly, we did not observe an increase in CD62P and CD63 expression levels when the concentration of ox-LDL was 100 μg/ml instead of 50 μg/ml. On the contrary, CD63 expression levels exhibited an apparent reduction at the 100 μg/ml concentration. We speculate that the increasing ox-LDL concentration might cause CD62P and CD63 expressed on the platelet surface to be released into the supernatant or platelet death

Table 1 Expressions of CD147 on platelets treated with different concentrations of ox-LDL with different extent of lipid oxidation. (%, mean ± SD). oxidation conditions (37°C) 10 μM CuSO4 for 4 h 10 μM CuSO4 for 6 h 10 μM CuSO4 for 24 h

ox-LDL (μg/ml) 0 (control)

50

100

0.22 ± 0.05 0.35 ± 0.08 0.58 ± 0.31

1.22 ± 0.11⁎ 1.91 ± 0.10⁎ 8.06 ± 3.90#

2.66 ± 0.17⁎ 3.52 ± 0.15⁎ 10.69 ± 3.33#

⁎P b 0.001 vs. Control, #P b 0.01 vs Control. n = 6.

due to cytotoxicity. Thus, we detected Annexin V, a marker of platelet apoptosis. The results demonstrate that ox-LDL induces platelet apoptosis in a concentration-dependent manner, while LDL does not. Platelet apoptosis can be induced by chemical and physical stimuli, including calmodulin antagonists [40], thrombin [41], high pathological shear stresses [42] or hyperthermia [43]. The present study demonstrates that ox-LDL also triggers platelet apoptosis. This might explain why the platelets showed lower CD62P or CD63 expression when treated with 100 μg/ml ox-LDL instead of 50 μg/ml ox-LDL. Recently, Schmidt et al. [9] reported that platelet CD147 expression is up-regulated when washed platelets are exposed to agonists (including ADP, thrombin, and collagen) with a similar kinetic response to that of CD62P. In our study, when the washed platelets were stimulated with ox-LDL, the expression levels of CD147, CD62P and CD63 were markedly increased, which suggests that ox-LDL might at least partially lead to atherosclerotic plaque instability by up-regulating the expression of platelet CD147. In contrast, some studies have reported that a low concentration of ox-LDL inhibited agonist-induced platelet aggregation [21,22,25]. Although the reason for this discrepancy is unclear, the results may have been affected by methodological variations in the

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isolation, oxidization, dosage of lipoproteins employed and duration of the platelet-ox-LDL contact. Many of the effects of ox-LDL are mediated via the expression and activation of LOX-1, such as endothelial injury and the formation of foam cells; the induction of adhesion molecules [44]; and the stimulation of collagen formation [45] and platelet activation. LOX-1 is an activationdependent receptor on platelets [46]. In activated platelets, LOX-1 expression can be induced by ox-LDL. By using ox-LDL to stimulate the platelets pretreated with anti-LOX-1 mAb, we found that ox-LDLinduced platelet CD147 expression decreased. Hence, the results show that ox-LDL induces platelet CD147 expression via LOX-1, at least in part. Mehta JL et al. [47] reported that the deletion of LOX-1 ameliorated ox-LDL-mediated endothelial dysfunction and inhibited atherogenesis in an LDL receptor-deficient mouse model of atherosclerosis. HDL not only inhibits agonist-stimulated platelet aggregation, fibrinogen binding, and the secretion of alpha-granules and dense granules but also reduces the liberation of thromboxane A2 and 12-hydroxyeicosatetraenoic acid (12-HETE) [48,49]. Thus, we further examined whether HDL inhibits ox-LDL-induced platelet CD147 expression. As expected, HDL inhibits ox-LDL-induced platelet CD147 expression at the two different concentrations of ox-LDL (50 μg/ml or 100 μg/ml). Similarly, HDL also decreases the expression of CD62P and CD63 on ox-LDL-induced platelets. Recently, HDL has been demonstrated to inhibit platelet activation via the scavenger receptor BI (SR-BI) [30]. Additionally, SR-BI deficiency leads to platelet hyper-reactivity as a consequence of dyslipidemia and platelet cholesterol overload [50,51]. However, whether the inhibitory effect of HDL on the ox-LDL-induced platelet CD147 expression also occurs via SR-BI awaits further delineation. Because ox-LDL has been found to be exposed to circulating blood [17,18], platelets can make contact with ox-LDL and be activated through the binding of ox-LDL to some receptors expressed on the platelets, such as SR-A [23], CD36 [23,24], LOX-1 [21], PAF-receptor [26], and SR-PSOX/CXCL16 [27]. The levels of circulating ox-LDL have been shown to increase with age [52]. Moreover, Pennings et al. reported that platelet CD147 expression increased with age [10]. Here, we found that ox-LDL increases platelet CD147 expression. Therefore, our findings might partially explain why the expression of platelet CD147 increases with age, namely that the increased ox-LDL with increased age might enhance the production of CD147 on platelets. However, this hypothesis needs to be confirmed by further study. More studies have demonstrated that circulating ox-LDL is associated with preclinical atherosclerosis, coronary artery disease (CAD), acute coronary syndromes (ACS), restenosis, and vulnerable plaques [13,15,53,54,17]. In addition, several studies have suggested that elevated levels of oxidized LDL are a prognostic indicator of cardiovascular outcomes [16,55]. CD147 on the platelet surface can induce platelet degranulation and MMP-9 production in monocytes by augmenting NF- kB [9]. Consequently, circulating oxLDL, either from atherosclerotic plaques or from an imbalance between oxidants and antioxidants, might bind to platelets, causing plaque “activity” and destabilization by increasing CD147 expression on the platelet surface. A recent study showed that platelet-bound ox-LDL in patients with acute coronary syndromes (ACS) increases [56], indicating the ox-LDL-platelet interaction may play a crucial role in coronary thrombosis in ACS. Although our study demonstrated that ox-LDL promotes the expression of CD147 on platelets and that HDL inhibits this effect, our data were obtained from in vitro experiments. The results from in vitro studies do not necessarily reflect the in vivo situation. In addition, the mechanism involved in the interaction of HDL with the platelets is still not entirely clear. Thus, further studies are required. Conclusions In conclusion, our findings demonstrate that ox-LDL stimulates CD147 expression on platelets via LOX-1, at least in part, and that the

ox-LDL-stimulated effect is inhibited by HDL. CD147 expressed by oxLDL-stimulated platelets may activate the platelets and induce MMP-9 expression by monocytes, thereby promoting plaque instability. These findings provide new insights into the relationship between ox-LDL/ HDL and CD147 in platelets within the context of plaque instability. However, further studies should be performed to determine whether circulating ox-LDL can also induce platelet CD147 expression, with HDL having the opposite effect, in vivo. Conflict of Interest Statement The authors declare that they have no competing interests. Acknowledgements The Medical and Healthy Science Foundation from the Chinese People's Liberation Army (12BJZ29) supported this study. References [1] Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med 1992;326:242–50. [2] Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation 1995;92:657–71. [3] Schaar JA, Muller JE, Falk E, Virmani R, Fuster V, Serruys PW, et al. Terminology for high-risk and vulnerable coronary artery plaques. Report of a meeting on the vulnerable plaque, June 17 and 18, 2003, Santorini, Greece. Eur Heart J 2004;25:1077–82. [4] Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994;94:2493–503. [5] Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation 2001;104:365–72. [6] Schmidt R, Bültmann A, Ungerer M, Joghetaei N, Bülbül Ö, Thieme S, et al. Extracellular Matrix Metalloproteinase Inducer Regulates Matrix Metalloproteinase Activity in Cardiovascular Cells Implications in Acute Myocardial Infarction. Circulation 2006;113:834–41. [7] Biswas C. Tumor cell stimulation of collagenase production by fibroblasts. Biochem Biophys Res Commun 1982;109:1026–34. [8] Kasinrerk W, Fiebiger E, Stefanova I, Baumruker T, Knapp W, Stockinger H. Human leukocyte activation antigen M6, a member of the Ig superfamily, is the species homologue of rat OX-47, mouse basigin, and chicken HT7 molecule. J Immunol 1992;149:847–54. [9] Schmidt R, Bültmann A, Fischel S, Gillitzer A, Cullen P, Walch A, et al. Extracellular Matrix Metalloproteinase Inducer (CD147) Is a Novel Receptor on Platelets, Activates Platelets, and Augments Nuclear Factor kB-Dependent Inflammation in Monocytes. Circ Res 2008;102:302–9. [10] Pennings GJ, Yong ASC, Kritharides L. Expression of EMMPRIN (CD147) on circulating platelets in vivo. J Thromb Haemost 2010;8:472–81. [11] Schulz C, von Brühl M-L, Barocke V, Cullen P, Mayer K, Okrojek R, et al. EMMPRIN (CD147/basigin) mediates platelet–monocyte interactions in vivo and augments monocyte recruitment to the vascular wall. J Thromb Haemost 2011;9:1007–19. [12] Steinberg D, Parthasarathy S, Carew T, Khoo J, Witztum J. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989;320:915–24. [13] Holvoet P, Mertens A, Verhamme P, Bogaerts K, Beyens G, Verhaeghe R, et al. Circulating Oxidized LDL Is a Useful Marker for Identifying Patients With Coronary Artery Disease. Arterioscler Thromb Vasc Biol 2001;21:844–8. [14] Liu M-L, Ylitalo K, Salonen R, Salonen JT, Taskinen M-R. Hyperlipidemia Families Intima-Media Thickness in Asymptomatic Members of Familial Combined Circulating Oxidized Low-Density Lipoprotein and Its Association With Carotid. Arterioscler Thromb Vasc Biol 2004;24:1492–7. [15] Ehara S, Ueda M, Naruko T, Haze K, Itoh A, Otsuka M, et al. Elevated Levels of Oxidized Low Density Lipoprotein Show a Positive Relationship With the Severity of Acute Coronary Syndromes. Circulation 2001;103:1955–60. [16] Meisinger C, Baumert J, Khuseyinova N, Loewel H, Koenig W. Plasma Oxidized LowDensity Lipoprotein, a Strong Predictor for Acute Coronary Heart Disease Events in Apparently Healthy, Middle-Aged Men From the General Population. Circulation 2005;112:651–7. [17] Anselmi M, Garbin U, Agostoni P, Fusaro M, Pasini AF, Nava C, et al. Plasma levels of oxidized-low-density lipoproteins are higher in patients with unstable angina and correlated with angiographic coronary complex plaques. Atherosclerosis 2006;185:114–20. [18] Podrez EA, Byzova TV, Febbraio M, Salomon RG, Ma Y, Valiyaveettil M, et al. Platelet CD36 links hyperlipidemia, oxidant stress and a prothrombotic phenotype. Nat Med 2007;13:1086–95. [19] Weidtmann A, Scheithe R, Hrboticky N, Pietsch A, Lorenz R, Siess W. Mildly oxidized LDL induces platelet aggregation through activation of phospholipase A2. Arterioscler Thromb Vasc Biol 1995;15:1131–8. [20] Akkerman JW. From low-density lipoprotein to platelet activation. Int J Biochem Cell Biol 2008;40:2374–8.

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