miR-146a mimic therapy protects against platelet-mediated thrombosis

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  Figure 1. rs2431697-T allele promoted platelet activation in humans The scheme shows decreased miR-146a levels by ∼50% in cells from rs2431697-T allele carriers. Basal platelet activation from the cohort of patients with community-acquired pneumonia (CAP) (n = 163) was measured by flow cytometry on days (A) 1 (33 CC and 117 CT+TT), (B) 4–5 (25 CC and 82 CT+TT), and (C) 30 (24 CC and 85 CT+TT) after hospital admission. Median fluorescence intensity (MFI) of P-selectin or CD62P was represented. A two-tailed t test was used to calculate statistical significance (∗p < 0.05). (D and E) Platelets from healthy blood donors were activated using different agonists (10 μM ADP, 25 μM TRAP-6, 10 μg/mL CRP, and 100 nM PMA), in which (D) fibrinogen binding and (E) P-selectin exposure were determined by flow cytometry (n = 8/group). The total thrombus formation analysis system (T-TAS) was used to study platelet aggregation under shear conditions (n = 10–11/group). Whole blood was perfused over a collagen chip, registering pressure changes. (F) Occlusion start time (OST), (G) area under the curve (AUC), and (H) occlusion time (OT) were measured. The statistical analysis for these assays was performed by a…

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  Figure 2. miR-146a deficiency caused platelet hyperactivation Platelet activation was assessed in diluted blood from WT and miR-146a−/− mice with different agonists (collagen-related peptide [CRP], PAR4-agonist peptide [PAR4-AP], phorbol-12-myristate-13-acetate [PMA], and adenosine diphosphate [ADP]) and concentrations. (A) Fibrinogen binding and (B) P-selectin exposure were determined by flow cytometry. (C) The main receptor’s expression was measured by flow cytometry. (D) Platelet phosphatidylserine (PS) exposure over time following thrombin (Thr) activation and (E) ROS (H2DCFDA) generation at basal and stimulated state (Thr). For all assays, n = 4–9 mice/group were used (mean ± SD). Statistical analyses were performed using a two-tailed t test, a Mann-Whitney U test, or a one-way ANOVA (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001).

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  Figure 3. α-Granules and cytoskeleton alterations in miR-146a-deficient platelets Washed platelets from WT and miR-146a-deficient mice were characterized by transmission electron microscopy (TEM). (A) α-Granule length and (B) α-granule count were quantified in ≥100 platelets; scale bar: 2 μm. Spreading assays: F-actin (green) and β-tubulin (red) were labeled, (C) number of adhered platelets at different time points and (D) phenotype of spread platelets at 45 min were quantified in 2–3 images (1,870 × 1,860), and statistical significance was determined by multiple unpaired t tests; scale bar 2 μm. (E) Platelet F-actin content after thrombin (Thr) activation for 2 min. (F) Clot retraction assay; clot weight was measured after 150 min. The results are represented as mean ± SD (n = 3–8 mice/group in all experiments), and the significances were calculated by a two-tailed t test, a Mann-Whitney U test, or a one-way ANOVA (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001) depending on the assay.

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  Figure 4. miR-146a−/− platelets presented greater mitochondrial respiration Oxygen consumption rate (OCR) was determined by Seahorse Analyzer using the Cell Mito Stress Test kit. OCR, maximal respiratory capacity (MRC), and spare capacity (SC) in platelets from WT vs. miR-146a−/− were calculated at the (A) basal and (B–E) activated states with (B) Thr (0.5 IU/mL), (C) PAR-4AP (500 μM), (D) PMA (200 nM), and (E) CRP (20 μg/mL) (pool of 6 mice/group, mean ± SD). The significance was calculated by a two-tailed t test or a Mann-Whitney U test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001).

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  Figure 5. Metabolomics showed enhanced fatty acid β-oxidation for miR-146a-deficient platelets Untargeted metabolomics was performed in WT vs. miR-146a−/− platelets ± agonist 0.5 IU/mL Thr by Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer (2–3 pools of 3 mice/group and technical triplicates). (A) Principal-component analysis (PCA) and (B) volcano plot of platelet metabolome. Main intermediates or products of (C) pentose phosphate pathway (PPP), (D) glycolysis, and (E) tricarboxylic acid cycle (TCA). (F) Acylcarnitine/carnitine (AC/C) ratio, (G) fatty acid levels, and (H) coenzyme A and carnitine levels in resting or stimulated (Thr) platelets. (I) Expression of Cpt1a mRNA was measured by qPCR (n = 6–7 pools of 3–4 mice/group), and the significance was determined by a two-tailed t test (∗p < 0.05). For the remaining assays, statistical significance was calculated by a two-way ANOVA (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001); the genotype statistical significance is represented in graphs.

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  Figure 6. Fatty acid metabolism supported higher respiration in miR-146a−/− platelets Oxygen consumption rate (OCR) was determined by Seahorse Analyzer using the Cell Mito Stress Test kit. WT or miR-146a−/− platelets were incubated with palmitate conjugated with BSA (Pal, 150 μM), a long-chain fatty acid, BSA as a control, or etomoxir (Eto; 5 μM), a specific Cpt1a inhibitor. (A) OCR profile in the presence or absence of palmitate for resting platelets. (B) Basal respiration, non-mitochondrial respiration, ATP production, proton-leak, maximal respiratory capacity (MRC), and spare capacity (SC) were calculated. (D) OCR profile in the presence or absence of Eto of WT vs. miR-146a−/− resting platelets. (E) Basal respiration, non-mitochondrial respiration, ATP production, proton leak, MRC, and SC were calculated. For stimulated (0.5 IU/mL Thr) platelets, basal respiration, non-mitochondrial respiration, ATP production, proton leak, MRC, and SC were determined in the presence or absence of palmitate (C) or following Eto (F) incubation without Thr (n = 2–3 mice/group and condition; values are mean ± SD). The significance was calculated by one-way ANOVA (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and…

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  Figure 7. miR-146a mimic therapy led to survival after pulmonary embolism in miR-146a−/− mice Pulmonary embolism (PE) was induced by injecting collagen and epinephrine in WT and miR-146a−/− mice (n = 17–21 mice/group). (A) Percentage of survival of these mice and miR-146a−/− mice following miR-146a mimic administration (n = 9 mice/group) for 3 days before PE induction was monitored 30 min after PE challenge. Log-rank Mantel-Cox test was applied to determine the statistical significance. (B) Perfusion defect score of lungs was calculated by Evans Blue injection in the heart of WT and miR-146a−/− mice following the respiratory arrest. A Mann-Whitney U test was used for statistical analysis. (C) Quantification of integrated density of platelet aggregates (CD41) in lungs from miR-146a-deficient mice treated with miRNA therapy (C-scrRNA or C-miR-146a mimic) after PE induction. At least 3 lung sections were analyzed per mouse (mean ± SD, n = 4–6 mice/group, two-tailed t test). (D) Representative immunofluorescence images (1,875 × 980) of the previous lung sections stained with FITC anti-CD41 antibody and DAPI for the nucleus. Scale bar 20 μm. (E) Platelets from WT and miR-146a−/− mice + miRNA…