Journal: Blood Advances

Article Title: Deleting ACC1 in platelets alters phospholipidome and reduces platelet activation and thrombosis in mice ∗

doi: 10.1182/bloodadvances.2025015796

Figure Lengend Snippet: Characterization of ACC1 flx/flx , GPIbα-Cre +/− , and ACC1 pKO mice. (A) Washed murine platelets or leukocytes were lysed and subjected to western blot analysis for ACC1 and ACC2 expression. Liver extract was used as positive control for ACC2. GAPDH served as loading control for platelet, leukocyte, and liver samples. (B-C) Platelet counts and MPV were determined by Cell-Dyn Emerald Hematology Analyzer (Abbott) in a single measurement conducted during the usage period of mice (8-16 weeks old). Data are expressed as mean ± standard error of the mean (SEM; n = ≥27). # P ≤ .0001 relative to ACC1 flx/flx mice, $ P ≤ .0001 relative to GPIbα-Cre +/− mice, and § P ≤ .0001 between ACC1 flx/flx and GPIbα-Cre +/− mice. One-way analysis of variance (ANOVA) was used for data analysis. (D) Alexa 488–GPIbβ antibody was injected into ACC1 flx/flx , GPIbα-Cre +/− , and ACC1 pKO mice to analyze platelet lifespan. Blood samples were collected on the day of the injection (day 0), as well as on days 3 and 5 after injection. Platelets were stained ex vivo with PE-conjugated anti-CD41 antibody, and the percentage of Alexa 488+ platelets among total CD41 + platelets was quantified by flow cytometry. Data are expressed as mean ± SEM (n ≥ 3). # P ≤ .05 relative to ACC1 flx/flx mice, $ P ≤ .01 relative to GPIbα-Cre +/− mice. Data were subjected to 2-way ANOVA analysis. (E) Platelet surface expression of αIIbβ3, GPIbα, and GPVI was measured by flow cytometry and normalized to MPV. Data are expressed as MFI adjusted to MPV (fL) ± SEM (n ≥ 8). # P ≤ .05 relative to ACC1 flx/flx mice, $ P ≤ .01 relative to GPIbα-Cre +/− mice, and § P ≤ .01 between ACC1 flx/flx and GPIbα-Cre +/− mice. Data were subjected to 1-way ANOVA analysis. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPVI, glycoprotein VI; MFI, median fluorescence intensity; MPV, mean platelet volume.

Article Snippet: Platelet-specific ACC1 knockout mice were generated by crossing ACC1 flx/flx females (The Jackson Laboratory) with GPIbα-Cre +/− males, yielding ACC1 flx/flx × GPIbα-Cre +/− mice and ACC1 flx/flx control mice.

Techniques: Western Blot, Expressing, Positive Control, Control, Injection, Staining, Ex Vivo, Flow Cytometry, Fluorescence

Journal: Blood Advances

Article Title: Deleting ACC1 in platelets alters phospholipidome and reduces platelet activation and thrombosis in mice ∗

doi: 10.1182/bloodadvances.2025015796

Figure Lengend Snippet: Lipidomic analysis revealed changes in lipid profiles of ACC1 pKO platelets. Lipid extraction was performed on washed murine platelets from 6 samples for 1 genotype, each sample consisting of platelets pooled from 2 or 3 mice of the same genotype. (A) Platelet lipidome overview of ACC1 pKO, and ACC1 flx/flx , and GPIbα-Cre +/− controls by principle component analysis. The colors and symbols distinguish the 3 genotypes. The first and second dimensions with their associated percentage of explained variance are displayed on the x-axes and y-axes, respectively. (B-F) Lipidomic analysis comparing ACC1 pKO and ACC1 flx/flx platelets. (B) Box and scatter plot representations of the modulation (log 2 FC) of 446 identified lipid species categorized into 5 (sub)classes, each represented by a distinct color (red, FA; orange, GL; yellow, PL; green, SL; and blue, ST). Lipid species are represented as black or gray dots based on their statistical significance (black dots representing a significant adjusted P value, FDR ≤ 0.05; whereas gray dots correspond to FDR ≥ 0.05). Lipids above the horizontal dotted line are upregulated, whereas lipids below are downregulated. Log FCs and FDR were calculated from the multivariate regression model. (C-E) Dot plots displaying the length and unsaturation levels of the 2 fatty acyl chains from PC (C), PE (D), and PEP (E). Color bars indicate the logFC from red (upregulation) to blue (downregulation). The adjusted P value is represented by the circle diameter (the largest circle corresponds to FDR ≤ 0.05). (F) Forest plot of AA-containing PC, PE, and PEP comparing ACC1 flx/flx and ACC1 pKO mice. Data are presented as logFC (squares) relative to ACC1 flx/flx platelets. (G) Radar plot showing the relative intensity of PC, PE, and PEP containing the 20:4 fatty acyl chains. The dark gray area corresponds to ACC1 flx/flx mice, whereas the orange area corresponds to ACC1 pKO mice. CE, cholesterol ester; DG, diacylglycerol; FA, fatty acid; FC, fold change; GL, glycerolipid; HCER, hydroxyceramide; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PL, phospholipid; SL, sphingolipid; SM, sphingomyelin; ST, sterol; TG, triacylglycerol.

Article Snippet: Platelet-specific ACC1 knockout mice were generated by crossing ACC1 flx/flx females (The Jackson Laboratory) with GPIbα-Cre +/− males, yielding ACC1 flx/flx × GPIbα-Cre +/− mice and ACC1 flx/flx control mice.

Techniques: Extraction

Journal: Blood Advances

Article Title: Deleting ACC1 in platelets alters phospholipidome and reduces platelet activation and thrombosis in mice ∗

doi: 10.1182/bloodadvances.2025015796

Figure Lengend Snippet: ACC1 deficiency in platelets had an impact on both mitochondrial reserve capacity and glycolytic activity. OCR was measured in washed murine platelets from ACC1 flx/flx or pKO mice. (A-B) OCR was assessed under basal conditions, after stimulation with 1 U/mL Thr, and after sequential treatment with 1 μM oligomycin (O), 0.4 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (F), and a mix of 1 μM AtA/R. (A) Representative OCR profiles are shown as mean ± SEM (n = 4). (B) Quantification of mitochondrial respiratory parameters including basal (baseline OCR – R/AtA-sensitive OCR), Thr (Thr response − baseline OCR), ATP-linked (thrombin response − oligomycin sensitive OCR), maximal (FCCP [carbonyl cyanide-p-trifluoromethoxyphenylhydrazone]-sensitive – R/AtA-sensitive OCR), reserve capacity (FCCP-sensitive – Thr-responsive OCR), proton leak (oligomycin-sensitive – R/AtA-sensitive OCR), and nonmitochondrial (R/AtA-sensitive OCR). Results are expressed as mean ± SEM (n = 4). (C-D) ECAR, used as an indirect indicator of glycolytic activity, was measured in the same experimental conditions. (C) Representative ECAR profiles are shown as mean ± SEM (n = 4). (D) Quantification of basal and Thr-induced ECAR. Results are expressed as mean ± SEM (n = 4). ∗ P ≤ .05 relative to unstimulated conditions. # P ≤ .05 relative to ACC1 flx/flx mice. Statistical analysis was performed using 2-way ANOVA. Ata, antimycin A; DMEM, Dulbecco’s modified eagle medium; NS, nonstimulated; R, rotenone; Thr, thrombin.

Article Snippet: Platelet-specific ACC1 knockout mice were generated by crossing ACC1 flx/flx females (The Jackson Laboratory) with GPIbα-Cre +/− males, yielding ACC1 flx/flx × GPIbα-Cre +/− mice and ACC1 flx/flx control mice.

Techniques: Activity Assay, Modification

Journal: Blood Advances

Article Title: Deleting ACC1 in platelets alters phospholipidome and reduces platelet activation and thrombosis in mice ∗

doi: 10.1182/bloodadvances.2025015796

Figure Lengend Snippet: ACC1 pKO platelets displayed reduced TxA 2 generation, dense granule secretion, and aggregation upon agonist stimulation. (A-G) Washed murine platelets from ACC1 floxed or pKO mice were stimulated with various Thr or CRP concentrations. (A) Platelets were stimulated with 100 mU/mL Thr or 0.3 μg/mL CRP for 5 minutes, and TxB 2 was measured in the supernatant using an enzyme-linked immunosorbent assay kit. Aspirin-treated platelets were included as a positive control for TxB 2 generation inhibition in the CRP condition. Data are expressed as mean ± SEM (n = 4). ∗ P ≤ .05 relative to unstimulated conditions. # P ≤ .05 relative to ACC1 flx/flx mice. Data were analyzed via 2-way ANOVA. (B) Aggregation was analyzed by turbidimetry (Chrono-Log). Data are expressed as mean ± SEM (n ≥ 4). ∗ P ≤ .05 relative to the first agonist concentration. # P ≤ .05 relative to ACC1 flx/flx mice. Two-way ANOVA was used for analysis. (C) Aggregation profiles are shown. (D) Activated αIIbβ3 (JON/A) and (E) p-selectin (CD62P) exposure was detected by flow cytometry. Data are expressed as fold change ± SEM (n = 4) and mean ± SEM (n = 4) respectively. ∗ P ≤ .05 relative to unstimulated conditions. # P ≤ .05 relative to ACC1 flx/flx mice. Two-way ANOVA was used for analysis. (F) Dense granule secretion was assessed via addition of luciferase-luciferin reagent (Chrono-log). Data are expressed as mean ± SEM (n ≥ 3). ∗ P ≤ .05 relative to the first agonist concentration. # P ≤ .05 relative to ACC1 flx/flx mice. Two-way ANOVA was used for analysis. (G) Profiles are shown. ASA, aspirine; Thr, thrombine.

Article Snippet: Platelet-specific ACC1 knockout mice were generated by crossing ACC1 flx/flx females (The Jackson Laboratory) with GPIbα-Cre +/− males, yielding ACC1 flx/flx × GPIbα-Cre +/− mice and ACC1 flx/flx control mice.

Techniques: Enzyme-linked Immunosorbent Assay, Positive Control, Inhibition, Concentration Assay, Flow Cytometry, Luciferase

Journal: Blood Advances

Article Title: Deleting ACC1 in platelets alters phospholipidome and reduces platelet activation and thrombosis in mice ∗

doi: 10.1182/bloodadvances.2025015796

Figure Lengend Snippet: Platelet-specific ACC1 deletion affects platelet spreading and actin cytoskeleton organization upon CRP stimulation. (A-E) Washed murine platelets were stimulated with or without CRP 0.3 μg/mL for 30 minutes. Platelets were stained with phalloidin-fluorescein isothiocyanate for 45 minutes at room temperature. (A) Panel of representative pictures. Scale bar, 5 μm. (B-C) Quantification of the mean surface area covered by (B) unstimulated or (C) CRP-stimulated platelets independently of the platelet morphology. Data are expressed as mean ± SEM (n = 4). # P ≤ .05 relative to ACC1 flx/flx mice. Data underwent 1-way ANOVA. (D-E) Proportion of the surface area covered by (D) unstimulated or (E) CRP-stimulated platelets, being inactivated (discoid), extending filopodia, forming lamellipodia or fully spread after activation. Data are expressed as means ± SEM (n = 4). # P ≤ .05 relative to ACC1 flx/flx mice. $ P ≤ .05 relative to GPIb-Cre +/− mice. Data underwent 2-way ANOVA.

Article Snippet: Platelet-specific ACC1 knockout mice were generated by crossing ACC1 flx/flx females (The Jackson Laboratory) with GPIbα-Cre +/− males, yielding ACC1 flx/flx × GPIbα-Cre +/− mice and ACC1 flx/flx control mice.

Techniques: Staining, Activation Assay

Journal: Blood Advances

Article Title: Deleting ACC1 in platelets alters phospholipidome and reduces platelet activation and thrombosis in mice ∗

doi: 10.1182/bloodadvances.2025015796

Figure Lengend Snippet: ACC1 deficiency in platelets impaired their activation and thrombus formation under flow conditions. (A-G) Whole blood from ACC1 flx/flx , GPIbα-Cre +/− , and ACC1 pKO mice was perfused over collagen 1–coated surface (50 μg/mL) at a shear rate of 1000/s. (A) Representative images of multilayered platelet thrombi, and activated αIIbβ3, CD62P, and phosphatidylserine (PS) stainings. (B-D) Platelet deposition, thrombus formation and contraction score were assessed on BF images. (E) αIIbβ3 activation was evaluated using JON/A Ab, (F) P-selectin exposure was evaluated via staining with anti-CD62P antibody, and (G) PS exposure was determined using annexin V. The results are expressed as mean ± SEM (≥4 mice per group). # P ≤ .05 relative to ACC1 flx/flx mice. $ P ≤ .05 relative to GPIbα-Cre +/− mice. One-way ANOVA was used for analysis. BF, brightfield; JON/A Ab, JON/A antibody; SAC, surface area covered.

Article Snippet: Platelet-specific ACC1 knockout mice were generated by crossing ACC1 flx/flx females (The Jackson Laboratory) with GPIbα-Cre +/− males, yielding ACC1 flx/flx × GPIbα-Cre +/− mice and ACC1 flx/flx control mice.

Techniques: Activation Assay, Shear, Staining

Journal: Blood Advances

Article Title: Deleting ACC1 in platelets alters phospholipidome and reduces platelet activation and thrombosis in mice ∗

doi: 10.1182/bloodadvances.2025015796

Figure Lengend Snippet: ACC1 pKO mice exhibited reduced arterial thrombosis while maintaining normal hemostasis. (A) Tail bleeding times of ACC1 flx/flx , GPIb-Cre +/− , and ACC1 pKO mice in saline solution preheated at 37°C. Results are expressed as mean ± SEM (n ≥ 23). Data were analyzed via 1-way ANOVA. (B-D) Mice were subjected to thrombosis induced in vivo by FeCl 3 application to the carotid artery (10% of FeCl 3 during 5 minutes). Thrombus formation was visualized using intravital microscopy analyzing exogenous platelet accumulation stained with carboxyfluorescein succinimidyl ester. (B-C) Thrombus growth area quantification over time. Data are expressed as mean ± SEM (n ≥ 6). # P ≤.05 relative to ACC1 flx/flx mice. Data were analyzed using unpaired t test comparing area under the curve. (D) Representative pictures of thrombus formation over time (1, 5, 9, 15, and 19 minutes) after FeCl 3 injury.

Article Snippet: Platelet-specific ACC1 knockout mice were generated by crossing ACC1 flx/flx females (The Jackson Laboratory) with GPIbα-Cre +/− males, yielding ACC1 flx/flx × GPIbα-Cre +/− mice and ACC1 flx/flx control mice.

Techniques: Saline, In Vivo, Intravital Microscopy, Staining