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

Figures

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  Figure 1.. PPARα-deficient mice display impaired hemostasis and thrombosis. (A) Genotyping results of wild-type (WT), Ppara+/-and Ppara-/- mice using polymerase chain reaction. Immunoblot analysis of PPARα, PPARβ and PPARγ expression in platelets from WT, Ppara+/-, Ppara-/- mice and humans. (B) Bleeding times for WT (•), Ppara+/- (■) and Ppara-/- (▲) mice. Means are indicated by horizontal lines. Statistical significance was evaluated with a paired t test (*P<0.05; ***P<0.001; ns: not significant). Percentages of WT, Ppara+/- and Ppara-/- mice bleeding times that exceeded 15 min (D) or were within 15 min (■). Results were obtained from 26 WT, 26 Ppara+/- and 26 Ppara-/- mice. (C) An injury to the carotid artery was induced by FeCl3. The dot plot shows occlusion times for carotid arterioles as a result of FeCl3-induced thrombosis in WT (•, n=6), Ppara+/- (■, n=9) and Ppara-/- mice (▲, n=6). Means are indicated by horizontal lines. Statistical significance was evaluated with a two-tailed Mann-Whitney test (*P<0.05; **P<0.01; ns: not significant). (D) Representative images and time courses of thrombus formation induced by FeCl3 injury to mesenteric arterioles in WT (top row), Ppara+/- (middle…

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  Figure 2.. Ppara-/- platelets show impaired aggregation, ATP secretion, spreading and delayed clot retraction. (A) Aggregation and ATP release of washed wild-type (WT), Ppara+/- and Ppara-/- platelets stimulated with thrombin (0.025 U/mL) or collagen (0.8 mg/mL). Aggregation and ATP release were assessed with a Chrono-log lumiaggregometer under stirring at 1,200 rpm. Traces are representative of at least three independent experiments. Statistical significance was evaluated with a two-tailed Mann-Whitney test and a paired t test (*P<0.05; **P<0.01; ***P<0.001). (B) Aggregation and ATP release of washed WT, Ppara+/- and Ppara-/-platelets stimulated with thrombin (0.025 U/mL) or collagen (0.8 mg/mL) in the presence of vehicle or apyrase (1 U/mL) incubated for 5 min. Traces are representative of at least three independent experiments. (C) Aggregation of washed WT, Ppara+/- and Ppara-/- platelets stimulated with thrombin (0.025 U/mL) or (0.8 mg/mL) in the presence of a low concentration of ADP (1 mM). Traces are representative of at least three independent experiments. (D) Spreading of WT, Ppara+/- and Ppara-/- platelets on immobilized fibrinogen in the presence or absence of apyrase (1 U/mL) or…

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  Figure 3.. PPARα promotes platelet activation through a p38/ROS/Akt signal axis. (A) Immunoblot analysis of wild-type (WT), Ppara+/-and Ppara-/- platelets, stimulated with thrombin (0.025 U/mL) and collagen (0.8 mg/mL) for 5 min, with antibodies recognizing phosphorylated Akt Thr308, phosphorylated Akt Ser47 3, total Akt, phosphorylated p38 Thr180/Tyr182 (T180/Y182), and total p38. Representative immunoblots from at least three independent experiments. (B) Washed WT, Ppara+/- and Ppara-/- platelets (2×108/mL) were incubated with dimethyl sulfoxide (DMSO), SH-6 (10 mM), SB203580 (10 mM) for 10 min, then stimulated with thrombin (0.025 U/mL) or collagen (0.8 mg/mL), respectively. Aggregation and ATP release were assessed with a Chrono-log lumiaggregometer under stirring at 1,200 rpm. Traces are representative of at least three independent experiments. (C) Generation of reactive oxygen species (ROS) analyzed by flow cytometry. H2DCFDA-loaded (50 mM) mice platelets were stimulated with thrombin (0.025 and 0.05 U/mL) or convulxin (50 and 100 ng/mL) for 5 min. Samples were analyzed immediately. Statistical significance was evaluated with a two-tailed Mann-Whitney test (*P<0.05; **P<0.01; ns: not…

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  Figure 4.. PPARα mediates hyperlipidemia-associated prothrombotic status and oxLDL-evoked platelet activation. (A) Ppara+/+/Apoe-/- and Ppara-/-/Apoe-/- mice were fed a high-fat diet (HFD) or control diet (CD) for 8 weeks before undergoing in vivo thrombosis experiments. Platelets were labeled by direct tail vein injection of DiOC6 (10 mM, 100 mL). Dot plot showing occlusion times for arterioles as a result of FeCl3-induced thrombosis in Ppara+/+/Apoe-/- and Ppara-/-/Apoe-/- mice. Means are indicated by horizontal lines. Statistical significance was evaluated with a two-tailed Mann-Whitney test (*P<0.05; **P<0.01). (B) Platelets were stimulated with 1-(palmitoyl)-2-(5-keto-6-octenedioyl) phosphatidylcholine (KODiA-PC, 15 mM). Aggregation was assessed with a Chrono-log lumiaggregometer under stirring at 1,200 rpm. Traces are representative of at least three independent experiments. (C) Immunoblot analysis of wild-type (WT), Ppara+/- and Ppara-/- platelets stimulated with KODiA-PC for 5 min, with antibodies recognizing phosphorylated Src Tyr418 (Y418), total Src, phosphorylated p40phox, b-actin, phosphorylated ERK5 Thr218/Tyr220 (T218/Y220), total ERK5, phosphorylated p38, total p38,…

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  Figure 5.. Increased platelet PPARα expression correlates with platelet hyperreactivity in hyperlipidemic mice and patients with hyperlipidemia. (A) Immunoblot analysis of PPARα, PPARβ and PPARγ expression in platelets from Apoe-/- mice fed a high-fat diet (HFD) or control diet (CD) for 8 weeks with PPARα, PPARβ and PPARγ antibodies. Statistical significance was evaluated with a two-tailed Mann-Whitney test (**P<0.01; ns: not significant). (B) Aggregation and ATP release of platelets from Apoe-/- mice fed with a HFD or CD were stimulated with thrombin (0.015 U/mL) or collagen (0.6 mg/mL). Aggregation was assessed with a Chrono-log lumiaggregometer under stirring at 1,200 rpm. Traces are representative of at least three independent experiments. (C) Immunoblot analysis of PPARα, PPARβ and PPARγ expression in platelets from patients with hypertriglyceridemia (HTG) and hypercholesterolemia (HTC) with PPARα, PPARβ and PPARγ antibodies. Representative immunoblots of platelet PPARα and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from four healthy subjects, six patients with HTG and three patients with HTC. Representative immunoblots of platelet PPARβ, PPARγ and GAPDH from four healthy…

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  Figure 6.. Oxidized low-density lipoproteins and lipids upregulate megakaryocyte but not platelet PPARα. (A) Immunoblot analysis of PPARα, PPARβ and PPARγ expression in Meg-01 cells cultured with fatty acids (oleic acid [OA], 400 mM and palmitic acid [PA], 200 mM), cholesterol (CHO, 2.5 mg/mL, 5.0 mg/mL, 7.5 mg/mL) or oxidized low-density lipoproteins (oxLDL, 10 mg/mL, 50 mg/mL) for 24 h with PPARα, PPARβ and PPARγ antibodies. Representative immunoblots from at least three independent experiments. (B) PPARA mRNA expression in Meg01 cells cultured with fatty acids (OA, 400 mM and PA, 200 mM), cholesterol (CHO, 2.5 mg/mL, 5.0 mg/mL, 7.5 mg/mL) or oxLDL (10 mg/mL, 50 mg/mL) for 24 h was analyzed by quantitative real-time polymerase chain reaction. Statistical significance was evaluated with a two-tailed Mann-Whitney test (*P<0.05; ns: not significant). (C) Immunoblot analysis of PPARα and phosphorylated IκBα level in Meg-01 cells cultured with fatty acids (OA, 400 mM and PA, 200 mM), cholesterol (CHO, 2.5 mg/mL, 5.0 mg/mL, 7.5 mg/mL) or oxLDL (10 mg/mL, 50 mg/mL) in the absence or presence of BAY11-7082 (10 mM) for 24 h. Representative immunoblots from at least three independent experiments.…