Recruited platelets enhance glycolytic program in Kupffer cells.

(A) KEGG pathway enrichment analysis of differentially upregulated genes in Kupffer cells (KCs) isolated from mice treated with APAP for 12 hours versus 0 hours (n=3 mice/group), based on RNA-seq. (B) Heatmap depicting the expression of glycolysis and oxidative phosphorylation (OXPHOS) genes in KCs following APAP treatment. (n=3). (C) Gene Set Variation Analysis (GSVA) of metabolic and immune pathways in KCs from mice treated with control IgG (α-IgG) or platelet-depleting (α-CD41) antibodies. The glycolysis pathway was significantly downregulated following platelet depletion (p=0.038). (D) Heatmap of glycolysis- and OXPHOS-related gene expression in KCs from mice treated with α-IgG or α-CD41 antibodies followed by APAP for 12 hours. (E) Extracellular acidification rate (ECAR) of KCs cultured alone or co-cultured with mouse platelets for 2 hours, measured in response to glucose, oligomycin, and 2-deoxy-D-glucose (2-DG) (n=3). (F) Basal and maximal respiration of KCs cultured alone or with platelets for 2 hours (n=3). p-value was calculated using an unpaired two-tailed Student’ s t-test.

Kupffer cell glycolysis promotes APAP-induced liver injury.

(A) Experimental timeline to assess the role of Kupffer cell glycolysis in APAP-induced liver injury. Wild-type C57BL/6J mice were divided into three treatment regimens: i) Vehicle Control: Mice were treated with PBS or 2-deoxy-D-glucose (2-DG), followed 0.5 hours later by PBS for 12 hours. ii) KC-intact + APAP: Mice were pre-treated with control (PBS-loaded) liposomes for 8.5 hours, followed by PBS or 2-DG for 0.5 hours, and then challenged with APAP for 12 hours. iii) KC-depleted + APAP: Mice were pre-treated with clodronate liposomes (CLDN) to deplete Kupffer cells for 8.5 hours, followed by PBS or 2-DG for 0.5 hours, and then challenged with APAP for 12 hours. (B) Representative H&E-stained liver sections and quantification of necrotic area from mice in (A). Necrotic regions are outlined in black, and the percentage of necrotic area was quantified in each picture. Scale bars: 100 µm. (C) Serum alanine aminotransferase (ALT) levels from mice in (A). (D) mRNA expression levels of inflammation-related genes in Kupffer cells (KCs) isolated from mice in (A), measured by qPCR. Data in (B-D) were analyzed by one-way ANOVA followed by Tukey’ s multiple-comparisons test.

Platelets-derived extracellular vesicles promote glycolysis in Kupffer cells.

(A) mRNA expression levels of glycolysis and inflammation-related genes in Kupffer cells (KCs) treated for 2 hours with supernatant from in vitro-cultured platelets (PLT sup). (B) Western blot analysis confirming the expression of extracellular vesicle (EV) markers (TSG101, CD63) in platelet-derived extracellular vesicles (PEVs). (C) Representative transmission electron microscopy (TEM) image of isolated PEVs. Scale bar, 200 nm. (D) Representative nanoparticle tracking analysis (NTA) profile of the isolated PEVs. (E) Extracellular acidification rate (ECAR) in KCs cultured alone or with PEVs for 2 hours, followed by sequential injection of glucose, oligomycin, and 2-deoxy-D-glucose (2-DG). (F) Basal and maximal respiration rates in KCs cultured alone or with PEVs for 2 hours. Data in (A) and (F) were analyzed using an unpaired Student’s t-test.

Platelet-derived extracellular vesicles promote APAP-induced liver injury.

(A) Experimental timeline to assess the role of PEVs in APAP-induced liver injury following platelet depletion. Wild-type C57BL/6J mice were depleted of platelets using an anti-CD41 antibody (α-CD41 Ab) and then treated with either Vehicle or PEVs concurrently with APAP for 3 hours (n=3 mice/group). (B) Immunofluorescence analysis confirming platelet depletion efficiency in liver sections from mice in (A), stained for CD41 (red) and counterstained with DAPI (blue) for nuclei. Scale bar, 50 µm. (C) Serum alanine aminotransferase (ALT) levels from mice in (A). (D) Representative H&E-stained liver sections and quantification of necrotic area from mice in (A). Necrotic regions are outlined in black, and the percentage of necrotic area was quantified in each picture. Scale bars: 100 µm. (E) Analysis of PEVs uptake by Kupffer cells in vivo. Mice were intravenously (i.v.) injected with PKH67-labeled PEVs for 3 hours. Liver sections were immunostained for Kupffer cells (F4/80, red) and nuclei (DAPI, blue). The number of PEVs-positive Kupffer cells was quantified. Data in (C-E) were analyzed by an unpaired two-tailed Student’s t-test.

ALDOA from platelet-derived extracellular vesicles induces a glycolytic switch in Kupffer cells.

(A) Schematic workflow for identifying PEV-specific protein mediators. Proteins secreted by hepatocyte-derived EVs (HEVs) and those related to cytoskeleton and coagulation were excluded from the mass spectrometry analysis. Candidate proteins were defined as those appearing in all three independent replicates with more than two unique peptides. (B) Western blot analysis of ALDOA expression in isolated PEVs. CD63 and TSG101 were used as EV markers. (C) Extracellular acidification rate (ECAR) in Kupffer cells (KCs) treated with vehicle or recombinant ALDOA (rALDOA) for 2 hours, followed by sequential injection of glucose, oligomycin, and 2-deoxy-D-glucose (2-DG). (D) Basal and maximal respiration rates in KCs treated with vehicle or rALDOA for 2 hours. (E) Western blot analysis of ALDOA expression in mouse serum at different time points (0, 3, 6, 12 h) after APAP treatment (n=3). (F) Western blot analysis of ALDOA expression in serum from mice under various conditions: untreated (0 h), 12 h post-APAP, and 12 h post-APAP following platelet depletion with either a control IgG antibody (α-IgG Ab) or an anti-CD41 antibody (α-CD41 Ab) (n=3). Data in (D) were analyzed by an unpaired two-tailed Student’s t-test.

ALDOA serve as a potential therapeutic target for acute liver injury.

(A) Serum ALT levels were measured in Aldoafl/fl and PF4ΔAldoa mice treated with 210mg/kg APAP for 24 hours. (n=9 mice/group) (B) Hematoxylin & Eosin (H&E) was performed to evaluate liver morphology in liver sections of mice in (A) (left). Necrotic areas were outlined in black, and the percentage of necrotic area was quantified (right) in each picture. (C) Experimental strategy for inhibiting ALDOA in vivo during AILI. Wild-type C57BL/6J mice were starved overnight (5 PM-9 AM), followed by an intraperitoneal injection of APAP at 9 AM. Mice were treated with 2 mg/kg Aldometanib one hour after APAP injection, and samples were collected 12 hours post-APAP. n=6 mice/group. (D) Serum ALT levels were measured in mice in (C). (E) H&E staining was performed to evaluate liver morphology in liver sections of mice in (C). Necrotic areas were outlined in black, and the percentage of necrotic area was quantified in each picture. (F) Platelets were isolated from healthy donors, and proteins were identified in the alpha granule fractions using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described by Maynard et al., 2007. (G) Extracellular vesicles (EVs) were isolated from serum of patients with polycythemia vera (Fel et al. (2019)) or Alzheimer’s Disease (Nielsen et al. (2021)). Protein content within the EVs was characterized using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Among the identified proteins, ALDOA intensity was quantified. (H) The correlation between ALDOA protein levels and serum ALT levels in patients with ALI (the detailed patients information is in Table S3). Correlations were evaluated by the Pearson correlation coefficient. (n=16) p-values were calculated using an unpaired two-tailed Student’s t-test (A, B, D, E, G).

Platelets promote acute liver injury by transferring ALDOA to Kupffer cells via extracellular vesicles.

Platelet-derived extracellular vesicles (PEVs) deliver Aldolase A (ALDOA) to Kupffer cells, enhancing their glycolytic activity and driving acute liver injury. Targeting the ALDOA pathway in platelets represents a promising therapeutic strategy.