Critical role of peroxisome proliferator-activated receptor a in promoting platelet hyperreactivity and thrombosis under hyperlipidemia

Platelet hyperreactivity and increased atherothrombotic risk are specifically associated with dyslipidemia. Peroxisome proliferator-activated receptor alpha (PPARα) is an important regulator of lipid metabolism. It has been suggested to affect both thrombosis and hemostasis, yet the underlying mechanisms are not well understood. In this study, the role and mechanism of PPARα in platelet activation and thrombosis related to dyslipidemia were examined. Employing mice with deletion of PPARα (Pparα-/-), we demonstrated that PPARα is required for platelet activation and thrombus formation. The effect of PPARα is critically dependent on platelet dense granule secretion, and is contributed by p38MAPK/Akt, fatty acid b-oxidation, and NAD(P)H oxidase pathways. Importantly, PPARα and the associated pathways mediated a prothrombotic state induced by a high-fat diet and platelet hyperactivity provoked by oxidized low density lipoproteins. Platelet reactivity was positively correlated with the levels of expression of PPARα, as revealed by data from wild-type, chimeric (Pparα+/-), and Pparα-/- mice. This positive correlation was recapitulated in platelets from hyperlipidemic patients. In a lipid-treated megakaryocytic cell line, the lipid-induced reactive oxygen species-NF-kB pathway was revealed to upregulate platelet PPARα in hyperlipidemia. These data suggest that platelet PPARα critically mediates platelet activation and contributes to the prothrombotic status under hyperlipidemia.


Hematologic analysis
Complete blood count and hematocrit were conducted by Animal hospital affiliated to Zhejiang University with ProCyteDx™ hematology analyzer (IDEXX Laboratories, Westbrook,Maine, USA).

Tail-bleeding time
As previously described, 1 tails of anesthetized mice were cut 0.5 cm from the tip and immediately immersed in saline (37°C). The time taken for the bleeding to stop (no blood flow for 1 minute) was recorded. Tail-bleeding assays were stopped at 900 seconds if the bleeding did not stop.

FeCl3-induced murine carotid artery thrombosis model
As previously described, 2, 3 8-to 10-week-old mice were anesthetized, a midline incision was made in the neck, and the right carotid artery was isolated from surrounding tissues. Carotid artery thrombosis was induced by treating the exposed artery with a piece of filter paper (1 ˟ 2 3 mm) soaked in 10% FeCl3 for 3 minutes, and blood flow through the carotid artery was monitored with a laser-speckle blood flow imaging system (RFLSI Ⅲ; RWD Life Science, Shenzhen, China). The end points were: 1) blood flow perfusion was less than 50 for >1 minute; or 2) occlusion is not seen after 15 minutes of FeCl3 injury. In this case, the time will be recorded as 15 minutes for statistical analysis.

FeCl3-induced mesenteric arteriole thrombosis model
Fluorescently labeled platelets (10 8 platelets) were injected through the tail vein. The intestines were exposed, and the mesentery was spread on the translucent stage of the fluorescence microscope. Injury of mesenteric arterioles (60-100 μm in diameter) was induced by topical application of 10% FeCl3. Arterioles were monitored for 60 minutes or until complete occlusion occurred (blood flow stopped for > 1 minute).

Flow restriction model
Mice were anesthetized and placed in a supine position. After gentle separation from aorta, IVC was ligated by a 6.0 polypropylene suture immediately below the renal veins to obtain complete blood stasis. For partial flow restriction (stenosis), IVC ligation was performed over a 30-gauge needle and then the needle was removed. The needle was placed outside the vessel so that piercing or any other injury to the IVC wall was completely avoided. After surgery, peritoneum and skin were closed by monofilament absorbable suture and 6.0 silk, respectively.
Mice were euthanized after 48 h, and thrombi developed in the IVC below the suture (toward the tail) were taken for analysis. 4

In vitro thrombus formation under flow conditions
Thrombus formation was evaluated in a microfluidic whole-blood perfusion assay on a fibrillar collagen matrix under arterial shear conditions (a shear rate of 1000 s -1 ) using a Bioflux-200 system (Fluxion, South San Francisco, CA). Bioflux plates were coated with fibrillar collagen at 50 μg/mL overnight, and after blocking with 0.5% bovine serum albumin in PBS for 15 minutes, plates were placed on an inverted microscope and 0.4 mL of mepacrine-labeled blood applied to the inlet well. A shear force of 40 dynes/cm 2 was applied, and the platelets were allowed to adhere to collagen for 5 minutes. Adherent platelets were viewed with an inverted fluorescence microscope using an S Plan Fluor lens (× 10/0.4 numerical aperture objectives).
Images were acquired with a Nikon DS-Qi1-U3 CCD camera. The platelet-covered area was measured using Bioflux software (Fluxion, South San Francisco, CA).

Platelet aggregation and ATP secretion
Platelet aggregation and secretion were measured using a lumi-aggregometer (Chrono-Log) at 37°C with stirring (1200 rpm). Washed platelets (2 × 10 8 /mL) in modified Tyrode buffer were stimulated with thrombin, collagen, U46619, and ADP (in the presence of fibrinogen). Platelet secretion was monitored in parallel with aggregation as adenosine triphosphate (ATP) release with the addition of luciferin/luciferase reagent to the platelet suspension. Inhibitor was incubated with the platelets for 10 minutes prior to stimulation.

Platelet spreading on fibrinogen
Glass coverslips were coated with 20 μg/mL fibrinogen in 0.1 M NaHCO3 (pH 8.3) at 4°C overnight. Washed platelets (2 × 10 7 /mL, 200 μL) or platelets preincubated with 1 U/mL apyrase or 1 μM ADP for 10 minutes at 37°C were allowed to spread on the fibrinogen-coated surfaces at 37°C for 60 minutes. After 3 washes with phosphate-buffered saline (PBS), the platelets were fixed, permeabilized, and stained with fluorescein-labeled phalloidin (Molecular Probes, Eugene, OR). Adherent platelets were viewed with an inverted fluorescence microscope (Nikon Ti-S, Japan). Images were acquired using a Nikon DS-Qi1-U3 camera. The platelet-covered area was measured using NIS-D software (Nikon).

Clot retraction
Murine platelets were resuspended using citrated human platelet-depleted plasma to a concentration of 4 × 10 8 /mL. Recombined plasma was induced to coagulate by stimulation with thrombin (0.4 U/mL). The clots were allowed to retract at room temperature and photographed at various time points.

Measurement of intracellular ROS
Intracellular ROS was measured according to a previously reported method. 5 Briefly, washed platelets (1 × 10 8 /mL) were incubated for 15 minutes at 37 °C with fluorogenic probe 2',7'dichlorofluorescein diacetate (H2DCF-DA, 50 µM) in the dark, then pretreated with or without designated inhibitors for 10 minutes, followed by stimulation with convulxin or thrombin for 10 minutes at 37 °C in the darkness. Samples were diluted with 10-fold PBS containing 50 µM H2DCF-DA and analyzed by flow cytometry immediately.

Serum lipid profile analysis
Serum lipid profile (TC and TG) was evaluated by enzymatic analysis using four commercial kits (Nanjing Jiancheng Bioengineering Institute, China).

Platelets culture
As previously described, 6 washed platelets were resuspended at 10 8 /mL in serum-free M199 medium, placed in round-bottom polypropylene tubes (BD Biosciences, USA), and cultured in a 37°C humidified incubator (HF90, Heal Force, China).

Cell Culture
The human megakaryocytic cell line (Meg-01) was cultured in RPMI-1640 medium (Gibco) supplemented with 1% heat-inactivated fetal bovine serum, 20 mM L-glutamine for at least 5 passages allowing for their adaptation prior to use. Cells were treated in medium with 8 cholesterol or fatty acid (oleic acid (OA, 400 μM) and palmitic acid (PA, 200 μM)) for 24 hours.
For experiments with pharmacological inhibition of intracellular reactive oxygen species (ROS) or NF-κB, 1 mM N-acetyl-L-cysteine (NAC), 1 mM DL-dithiothreitol (DTT) or 10 μM BAY 11-7082 was added. At the end of the treatment, the cells were collected and processed for RNA and protein extraction.

RNA extraction and real-time polymerase chain reaction (PCR)
Total RNA was extracted from megakaryocytes using TRIzol reagent (Invitrogen, 15596026), following the manufacturer's instructions. RNA samples (1 µg) were subsequently reversetranscribed into cDNA with a Reverse Transcription Reagent Kit (TOYOBO, FSQ-101), and the resulting cDNA was amplified by semi-quantitative RT-PCR using SYBR Green Mix (CWBIO, CW0957M). PCR products were confirmed by a single band of the expected size on 2% agarose gels. All the primers used for RT-PCR are shown in Supplemental table 4.

Chromatin Immunoprecipitation (ChIP) Assay
ChIP assays were performed using the Simple ChIP enzymatic chromatin immunoprecipitation kit (Beyotime Biotechnology) according to the manufacturer's protocol with minor modification. Briefly, Meg-01 cells were cross-linked with 1.5% (v/v) formaldehyde for 10 minutes and then stopped with 0.125 M glycine. Subsequently, nuclei were collected and chromatin sonicated to desired chromatin length (300bp-500bp). Sonicated lysates were then diluted to 2 mL, with protease inhibitor cocktail, and 20 μL of this solution was removed for PCR analysis (Input). After preclearing with Protein A/G Plus Agarose (Beyotime 9 Biotechnology) for 1 hour at 4°C, antibody specific to nuclear factor NF-κB p65 subunit was added and incubated at 4°C overnight on a rocking platform. Immune complexes were collected by the addition of Protein A/G Plus Agarose for 1 hour at 4°C, and the agarose beads were extensively washed with solutions of increasing ionic strength. Bound immune complexes were eluted and cross-links were reversed by incubating at 65°C for 4 hours. Samples were then treated with proteinase K, and DNA was purified using DNA purification spin columns.
DNA was amplified for detection of the binding motif of transcription factors in PPARα regulation region by PCR using the primers in Supplemental table 5.

Statistical analysis
Results are expressed as mean ± standard error of the mean (SEM). Statistical significance was evaluated with Paired t-test, 2-tailed Mann-Whitney U tests and 2-way ANOVA tests using statistical software GraphPad Prism (GraphPad Software, La Jolla, CA, USA).

Supplemental tables
Supplemental RBC, red blood cell; WBC, white blood cell; MPV, mean platelet volume; WT, wild type.
Statistical significance was evaluated with 2-tailed Mann-Whitney test (ns, no significance).

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Results are expressed as mean ± SEM.
(G) Platelets aggregates formed when stimulated with low doses of thrombin and collagen.
Washed WT, Pparα +/and Pparα -/platelets were stimulated with thrombin (0.025 U/mL) and collagen (0.8 μg/mL) with a Chrono-log lumiaggregometer under stirring at 1200 rpm for 5 minutes. Platelets were then fixed with 1.5% paraformaldehyde for 30 minutes at room temperature, and observed by differential interference contrast (Nikon TE-