Fenofibrate Attenuates Hyperhomocysteinemia-potentiated Thrombosis by Restoring Platelet Fatty Acid β-oxidation

Figures

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  Fig. 1. Hcy suppresses platelet fatty acid β-oxidation (FAO) and triggers mitochondrial dysfunction. (A) Volcano plot of platelet proteomics after Hcy (100 μM) or vehicle (PBS) treatment for 2 h. DEPs: |log2FC| > 0.67, P < 0.01 (yellow: upregulated; blue: downregulated; gray: no change). (B, C) GO biological process enrichment of upregulated (B) and downregulated (C) DEPs. (D) GSEA showing enrichment of fatty acid metabolism pathways in Hcy-treated platelets. (E) Heatmap of proteins involved in fatty acid catabolism and biosynthesis. (F) Lipidomics analysis of medium and long-chain fatty acids in Hcy-treated platelets. (G) Neutral lipid accumulation measured by BODIPY 493/503 flow cytometry. (H-J) ROS (H), mitochondrial ROS (I), and mitochondrial membrane potential (ΔΨm) (J) measured by DCFH-DA, MitoSOX, and TMRM by flow cytometry, respectively. (K) Western blot analysis of PPARα, ACOX1, and CPT1a expression. Data are presented as mean ± SEM, n = 3 - 5, **P < 0.01, ***P < 0.001.

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  Fig. 2. Activation of FAO by fenofibrate remodels platelet lipid metabolism and alleviates mitochondrial dysfunction. (A) Platelets were treated with vehicle, fenofibrate (Feno, 20 μM), homocysteine (Hcy, 100 μM), or Hcy + Feno (20 μM). K-means clustering of proteomics profiles with GO enrichment analysis. (B) Heatmap of proteins involved in fatty acid catabolism and peroxisome function. (C) Lipidomics analysis of medium and long-chain fatty acids under the indicated treatments. (D) Heatmap of acylcarnitine (AC) levels. (E, F) ROS levels (E) and mitochondrial membrane potential (ΔΨm) (F) measured by flow cytometry. (G) Principal component analysis (PCA) of proteomics profiles. (H) Schematic of lipid metabolic pathways and quantitative analysis of indicated lipid classes, including ceramide (Cer), sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and acylcarnitine. Data are presented as mean ± SEM, n = 3; *P < 0.05, **P < 0.01.

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  Fig. 3. Fenofibrate ameliorates HHcy-induced platelet hyperactivation and reduces thrombus formation. (A) Tail bleeding time in mice treated with Hcy (1.8 g/L in drinking water for 3 weeks) ± fenofibrate (Feno, 100 mg/kg, p. o. every other day starting from week 1). (B, C) Occlusion time (B) and representative images (C) of FeCl3-induced thrombosis in mesenteric arterioles. (D) Platelet aggregation measured by turbidimetry after treatment with Hcy (100 μM) ± Feno (20 μM). (E, F) Platelet spreading area on fibrinogen after treatment with Hcy (100 μM) ± Feno (20 μM) (E) or ± gemfibrozil (Gem, 50 μM) (F). (G) Phosphatidylserine (PS) exposure assessed using Annexin V staining by flow cytometry (representative histograms and quantification). (H) Western blot analysis of p-AKTSer473 and p-SRCTyr416 in platelets stimulated with thrombin (0.01 U/mL) ± Hcy (100 μM) ± Feno (20 μM). Data are presented as mean ± SEM, n = 5; *P < 0.05, **P < 0.01.

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  Fig. 4. Acylcarnitine enhances platelet activation and promotes thrombosis. (A) Tail bleeding times were measured in C57 mice 2 h after an intravenous injection of 18:1 acylcarnitine (4 μg in 100 μL saline). (B) Occlusion times were determined using a FeCl3-induced thrombosis model in the carotid artery following acylcarnitine administration. (C) Similarly, occlusion times were assessed in the mesenteric arterioles using the FeCl3-induced model. (D) Platelet aggregation was monitored turbidimetrically with 25 μM acylcarnitine in the presence of the indicated thrombin concentrations. (E) ATP secretion was measured in platelets stimulated with the indicated doses of acylcarnitine. (F) P-selectin exposure was quantified in platelets stimulated with the indicated acylcarnitine concentrations. (G) Platelet spreading was assessed following stimulation with the indicated doses of acylcarnitine or fenofibrate. Data are presented as mean ± SEM, n = 3 - 8, *P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 5. Perhexiline, a CPT1/2 antagonist, abolishes fenofibrate's protective effect against HHcy-induced thrombosis. (A) The experimental schematic for the in vivo study is shown. (B) Tail bleeding time and (C) blood loss based on hemoglobin (HGB) quantification were measured. (D) Occlusion times were recorded in mesenteric arterioles, and (E) representative images depict thrombi induced by FeCl3. (F-G) Platelet function in vitro was assessed by measuring PS exposure (F), P-selectin exposure (G) with a Cytation 3 Cell Imaging Multi-Mode Reader. (H) Platelet spreading was assessed following stimulation with thrombin. (I) Western blot analysis determined the levels of p-AKTSer473 and p-SRCTyr416 in platelets treated with Hcy, fenofibrate, or perhexiline. Data are presented as mean ± SEM (n = 4 - 5), *P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 6. CPT1/2 inhibition by perhexiline reverses fenofibrate-induced remodeling of platelet FAO and phospholipid metabolism. Platelets were isolated from the mice in Fig. 5 for lipidomics analysis. (A) Principal component analysis (PCA) of the platelet lipidome, with each point representing an independent biological replicate colored by treatment group. (B) Heatmap displaying the relative abundance of individual lipid species in the platelet lipidomic profile across treatment groups. (C) Bar graph showing the normalized relative total concentration of free fatty acids (FFA), phosphatidylglycerol (PG), diacylglycerol (DAG), and triacylglycerol (TAG). (D) Bar graph showing the normalized relative abundance of FFA across different chain lengths. (E) Bar graph showing the normalized relative total concentration of key phospholipid and sphingolipid classes: ceramide (Cer), sphingomyelin (SM), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). (F) Heatmap displaying the normalized relative levels of individual acylcarnitine (AC) species relative to the HHcy group, with color intensity indicating fold-change (red: upregulation; blue: downregulation). Data are presented as mean ± SEM…