Adipose Triglyceride Lipase Deficiency Attenuates In Vitro Thrombus Formation without Affecting Platelet Activation and Bleeding In Vivo

Figure 1. Neutral lipases are expressed in mouse and human platelets. mRNA expression of (A) adipose triglyceride lipase (Atgl), monoglyceride lipase (Mgl), hormone-sensitive lipase (Hsl), and (B) ATGL cofactors in megakaryocytes (n = 6) relative to the expression of hypoxanthine phosphoribosyltransferase 1 (Hprt) as a housekeeping gene. (C) RT-PCR of neutral lipases in purified platelets (lane 1). Whole blood RNA from wt (lane 2) and Atgl−/− mice (lane 3) as well as ddH2O (lane 4) were used as controls. (D) Lysates of platelets (pooled from 6 wt mice) were separated by SDS-Page and analyzed for HSL, ATGL, and MGL protein expression (lane 1) by Western blotting. Protein lysate of white adipose tissue (WAT) from a wt mouse was used as a positive control. (E) To confirm platelet purity, platelet RNA was analyzed with primers specific for platelets (Cd41), leukocytes (Cd45), and erythrocytes (Cd235a). (F) Human platelet mRNA expression data from 154 healthy subjects determined by microarray (http://www.plateletomics.org/plateletomics/, accessed on 10 January 2022, Copyright © 2022–2013. Bray Laboratory, Thomas Jefferson University. Shaw Laboratory, Baylor College of Medicine) [32] revealed…

Figure 2. Sporadic lipid droplet formation but unchanged overall morphology in Atgl−/− platelets. (A) Representative electron micrographs of platelets from wt and Atgl−/− mice. The asterisk indicates a cytosolic LD. Scale bar, 200 nm (upper panel) and 1 µm (lower panel). (B) Platelet spreading was visualized by scanning electron microscopy. Scale bar, 1 µM. Lipids were extracted from platelets (pooled from 6 mice) and analyzed by UPLC-MS to quantify (C) triglyceride (TG) and (D) cholesteryl ester (CE) concentrations and determine (E) phosphatidylcholine (PC) species. Data are shown as mean + SEM (n = 5). ***, p < 0.001.

Figure 3. Reduced mitochondrial respiration in whole-body Atgl−/− mice. Oxygen consumption rate (OCR) in isolated platelets from (A) Atgl−/− and (B) platAtgl−/− mice and their respective controls determined on a Seahorse XF Analyzer. Ten million cells were seeded in XF assay medium supplemented with sodium pyruvate (1 mM), L-glutamine (2 mM), and glucose (25 mM) per 96-well. Cells were treated with 2 μM oligomycin (Oligo), 0.2 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and 2.5 μM antimycin A. Values were normalized to protein content using the PierceTM BCA protein assay kit according to manufacturer’s instructions. Data are presented as mean values ± SEM of sextuplicate from 5 independent experiments. *, p < 0.05; **, p < 0.01. Significance was calculated by ANOVA followed by Bonferroni post hoc test. Blood from (C,D) Atgl−/− and (E,F) platAtgl−/− mice was activated with ADP (50 µM), protease-activated receptor 4 agonist peptide (PAR4-AP, 75 µM), collagen-related peptide (CRP, 10 µg/mL), and convulxin (CVX, 125 ng/mL) in the presence of PE-Cy7-conjugated anti-mouse P-selectin antibody and JON/A-PE antibody directed against the activated form of integrin αIIbβ3. Data…

Figure 4. Unchanged hemostatic function in Atgl−/− mice. Platelet aggregation in (A) Atgl−/− and (B) platAtgl−/− blood was measured using a Multiplate® analyzer. Data are expressed as mean arbitrary units (AU) (n = 10). (C) Bleeding time, (D) blood volume loss, and (E) hemoglobin (Hgb) concentration as absorbance at 540 nm were determined in isolated blood of Atgl−/− mice. Data are shown as mean ± SEM (n = 6–10).

Figure 5. Reduced in vitro thrombus formation in Atgl−/− blood. (A) Platelet reactivity was determined by in vitro thrombus formation, in which platelets were stained in whole blood and perfused over collagen-coated channels. (B) In vitro thrombus formation of wt and Atgl−/− blood every 30 sec up to 3 min of perfusion was recorded by fluorescence microscopy. (C) The thrombus-covered area was calculated by computerized image analysis. Iloprost, which inhibits thrombus formation in wt blood, was used as a positive control. Data are expressed in arbitrary units (AU) (mean ± SEM) (n = 10). ** p ≤ 0.01. (D) Mean platelet volume (MPV) from whole blood was determined by automated cell counting. Data represent mean values + SEM (n = 3–9). (E) Whole blood (resting and CRP-stimulated) was stained with an FITC-conjugated anti-GPVI antibody, and platelets were analyzed by flow cytometry. Data are shown as geometric mean of fluorescence intensity (MFI) + SEM (n = 5). (F) Plasma TXB2 concentrations were measured by ELISA (mean + SEM) (n = 6). (G) Thrombus formation was induced by applying a drop of 10% FeCl3, and occlusion time was recorded by intravital microscopy. Data are presented as mean + SEM (n =…