doi: 10.1172/JCI186065.
eCollection 2025 Aug 1.
TNF-α impairs platelet function by inhibiting autophagy and disrupting metabolism via syntaxin 17 downregulation
Affiliations
- PMID: 40493419
- PMCID: PMC12321402
- DOI: 10.1172/JCI186065
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TNF-α impairs platelet function by inhibiting autophagy and disrupting metabolism via syntaxin 17 downregulation
J Clin Invest.
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Abstract
Platelets play a dual role in hemostasis and inflammation-associated thrombosis and hemorrhage. Although the mechanisms linking inflammation to platelet dysfunction remain poorly understood, our previous work demonstrated that TNF-α alters mitochondrial mass, platelet activation, and autophagy-related pathways in megakaryocytes. Here, we hypothesized that TNF-α impairs platelet function by disrupting autophagy, a process critical for mitochondrial health and cellular metabolism. Using human and murine models of TNF-α-driven diseases, including myeloproliferative neoplasms and rheumatoid arthritis, we found that TNF-α downregulates syntaxin 17 (STX17), a key mediator of autophagosome-lysosome fusion. This disruption inhibited autophagy, leading to the accumulation of dysfunctional mitochondria and reduced mitochondrial respiration. These metabolic alterations compromised platelet-driven clot contraction, a process linked to thrombotic and hemorrhagic complications. Our findings reveal a mechanism by which TNF-α disrupts hemostasis through autophagy inhibition, highlighting TNF-α as a critical regulator of platelet metabolism and function. This study provides potentially new insights into inflammation-associated pathologies and suggests autophagy-targeting strategies as potential therapeutic avenues to restore hemostatic balance.
Keywords: Autophagy; Hematology; Metabolism; Mitochondria; Platelets.
Conflict of interest statement
Figures
(A) Demographics of volunteers providing healthy controls (HCs) and MPN platelets (JAK2 V617F polycythemia vera). (B) The OCRs of washed platelets from HCs (n = 6) and patients with MPN (n = 10) were measured using a Seahorse XFe24 analyzer (mean ± SEM). (C) Basal, maximal, and ATP-linked respiration parameters (n = 6 HCs, n =10 patients with MPN); unpaired t test. (D) PLS-DA of metabolome profiles (n = 18 HCs, n = 8 patients with MPN) was performed using UHPLC-MS. (E) Metabolite enrichment of the top 30 significant hits (see Supplemental Figure 1D). (F) ATP and (G) AMP levels (n = 6 HCs, n = 10 MPNs); unpaired t test with Welch’s correction. (H) Thrombus formation assay in whole blood using the T-TAS analyzer and PL chips (n = 30 HCs, n = 41 patients with MPN); Mann-Whitney U test. (I) Platelet strength assay and (J) peak force (n = 11 each); unpaired t test. (K) Thrombin-induced clot contraction assay (n = 5 HCs, n = 7 MPNs); Mann-Whitney U test. Box plots (C, F, G, H, J, and K) represent the data distribution.
(A) AMP/ATP ratios derived from identified metabolites (Supplemental Figure 1D); unpaired t test. (B) Immunoblot of phosphorylated AMPK (T172) in platelets from HCs (n = 8) and patients with MPN (n = 8); unpaired t test. Noncontiguous gel lanes are indicated. (C) Transmission electron microscopy (TEM) analysis of pooled HC and pooled MPN platelets (n = 5 each). Representative images showing autophagosome-like structures, unpaired t test. Scale bar: 0.5 μm. (D) Guide for interpreting autophagic flux by immunoblotting. Platelets from HCs (n = 14) and patients with MPN (n = 14) were incubated for 2 hours with vehicle (Veh, PBS) or CQ (50 μM). Immunoblots of (E) LC3B-II and (F) TOM20. The graphs show their levels before and after CQ treatment; 2-way ANOVA with Šídák’s test. (G) Summary of the differences in autophagic flux status, mitochondrial respiration, and platelet clot contraction differences between HCs and patients with MPN. Box-and-whisker plots (A–C) represent the data distribution.
(A) Experimental approach. (B) The OCR of washed platelets (n = 3) was measured using a Seahorse XF HS Mini Analyzer (mean ± SEM). (C) Basal, maximal, and ATP-linked respiration parameters with before-and-after graphs (n = 3); paired t test. (D) Thrombus formation assay in whole blood using the T-TAS analyzer and PL chips; before-and-after graph (n = 12); paired t test. (E) Platelet strength assay (n = 11) and (F) peak force, before-and-after graph (n = 11); paired t test. (G) Thrombin-induced clot contraction assay with normalized platelet counts (n = 6), before-and-after graph; paired t test. (H) Summary of the effects of the pharmacological inhibition of autophagy with CQ on platelets from HCs. (I) Methodology. (J) The OCR of washed platelets (n = 4) was measured using a Seahorse XF HS Mini Analyzer (mean ± SEM of 3 independent samples). (K) Basal, maximal, and ATP-linked respiration, before-and-after graph (n = 4); paired t test. (L) Thrombin-induced clot contraction assay with normalized platelet counts (n = 4); before-and-after graph; paired t test.
(A) Immunoblot analysis of STX17 in HCs (n = 5) and MPN (n = 4) platelets; before-and-after graph; 2-way ANOVA, Šídák’s test. Noncontiguous gel lanes are indicated. (B) Methodology. (C) Immunoblotting for LC3B-II and (D) TOM20. Before-and-after graphs (n = 3); paired t test. (E) OCR of washed platelets (n = 5) was measured with a Seahorse XF HS Mini Analyzer (mean ± SEM). (F) Basal, maximal, and ATP-linked respiration parameters with before-and-after graphs (n = 5); paired t test. (G) Thrombus formation assay in whole blood using the T-TAS analyzer and PL chips, before-and-after graph (n = 9); paired t test. (H) Thrombin-induced clot contraction assay using normalized platelet counts, before-and-after graph (n = 8); paired t test. (I) Summary of the effects of the pharmacological inhibition of STX17 on HC platelets. (J) Immunoblot analysis of STX17 expression in platelets from PF4-STX17-KO mice and STX17fl/fl littermate controls, STX17fl/fl (n = 7) and PF4-STX17-KO (n = 9); unpaired t test with Welch’s correction. (K) OCR of washed platelets measured with a Seahorse XF HS Mini Analyzer; (n = 9) and PF4-STX17-KO (n = 7). (L) Basal, maximal, and ATP-linked respiration parameters, STX17fl/fl (n = 9) and PF4-STX17-KO (n = 7); unpaired t test for basal and ATP-linked respiration, unpaired t test with Welch’s correction for maximal respiration. (M) Thrombin-induced clot contraction assay using normalized platelet counts, STX17fl/fl (n = 4) and PF4-STX17-KO (n = 5); unpaired t test. Box-and-whisker plots (J, L, and M) represent the data distribution.
(A) Experimental design. (B) Immunoblot analysis of STX17(n = 4), 1-way ANOVA, Tukey’s post hoc test. Noncontiguous gel lanes are indicated. (C) Immunoblot analysis of LC3B-II and TOM20, (n = 4), 1-way ANOVA, Tukey’s post hoc test. Noncontiguous gel lanes are indicated. (D) The OCR of Meg-01 cells (n = 4) was measured with a Seahorse XF HS Mini Analyzer (mean ± SEM of 3 independent samples). (E) Basal, maximal, and ATP-linked respiration parameters (n = 3); unpaired t test. (F) Experimental design. (G) Immunoblot analysis of STX17 (n = 3); unpaired t test. (H) Immunoblot analysis of LC3B-II and TOM20 (n = 3); unpaired t test. (I) The OCR of Meg-01 cells (n = 4) was measured with a Seahorse XF HS Mini Analyzer (mean ± SEM of 3 independent samples). (J) Basal, maximal, and ATP-linked respiration rates (n = 4); unpaired t test. (K) Summary of the effects of TNF-α and siSTX17 on autophagy, mitophagy, and mitochondrial respiration. Box-and-whisker plots (B, C, E, G, H, and J) represent the data distribution.
(A) Spatial RNA transcriptomics of bone marrow megakaryocytes (MKs); littermate controls (n = 1) and TNFdARE (n = 1) mice. Immunofluorescence for CD41, ACTA2, PECAM1, and DNA; the circles represent the regions of interest (ROIs) selected for the transcriptomic analysis. Scale bar: 2 mm. KEGG analysis of differentially expressed genes in the control (n = 9) and TNFdARE (n = 12) groups. (B) Immunoblot analysis of LC3B-II and TOM20 in platelets from control (n = 5) and TNFdARE (n = 4) mice treated with vehicle (PBS) or CQ (50 μM), before-and-after graph; 2-way ANOVA, Šídák’s test. Noncontiguous gel lanes are indicated. (C) Immunoblot analysis of STX17 in platelets from Ctrl (n = 3) and TNFdARE (n = 3) mice, before-and-after graph; 2-way ANOVA, Šídák’s test. (D and E) Immunoblot analysis of LC3B-II from HCs (n = 8) and patients with RA (n = 5) and TOM20 from HCs (n = 6) and patients with RA (n = 5) treated with vehicle (PBS) or CQ (50 μM). Before-and-after graph; 2-way ANOVA, Šídák’s test, Šídák’s test. (F) Summary of autophagic flux, accumulation of damaged mitochondria, and platelet hemostasis in TNFdARE mice and patients with RA. (G) Summary of autophagic flux.
(A) Experimental approach. Young (3-month-old) C57BL/6 mice were treated daily with vehicle (0.01% BSA in PBS), TNF-α, or TNF-α plus an anti–TNF-α mAb (every other day) for 15 days (n = 4 each). Platelets were incubated with vehicle (PBS) or CQ (50 μM) for 2 hours to analyze autophagic flux. (B) Immunoblotting of STX17, LC3B-II, and TOM20. (C) Analysis of STX17, (D) LC3B-II, and (E) TOM20 levels. Before-and-after graph, n = 4 each; 2-way ANOVA, Šídák’s test. (F) Quantification of intracellular LC3 by FACS, representative histogram with before-and-after graphs, Veh and TNF-α plus anti–TNF-α mAb n = 4, TNF-α n = 3; 2-way ANOVA, Šídák’s test. (G) The OCR of washed platelets (n = 4) was measured with a Seahorse XF HS Mini Analyzer (mean ± SEM of 3 independent samples). (H) Basal, maximal, and ATP-linked respiration (n = 4), Brown-Forsythe and Welch’s ANOVA tests. (I) Thrombin-induced clot contraction assay using normalized platelet counts, Veh (n = 4), TNF-α (n = 5), and TNF-α plus anti–TNF-α mAb (n = 4); Brown-Forsythe and Welch’s ANOVA test. Box-and-whisker plots (H and I) represent the data distribution.