Metformin Uniquely Prevents Thrombosis by Inhibiting Platelet Activation and mtDNA Release

Guang Xin, Zeliang Wei, Chengjie Ji, Huajie Zheng, Jun Gu, Limei Ma, Wenfang Huang, Susan L Morris-Natschke, Jwu-Lai Yeh, Rui Zhang, Chaoyi Qin, Li Wen, Zhihua Xing, Yu Cao, Qing Xia, Yanrong Lu, Ke Li, Hai Niu, Kuo-Hsiung Lee, Wen Huang, Guang Xin, Zeliang Wei, Chengjie Ji, Huajie Zheng, Jun Gu, Limei Ma, Wenfang Huang, Susan L Morris-Natschke, Jwu-Lai Yeh, Rui Zhang, Chaoyi Qin, Li Wen, Zhihua Xing, Yu Cao, Qing Xia, Yanrong Lu, Ke Li, Hai Niu, Kuo-Hsiung Lee, Wen Huang

Abstract

Thrombosis and its complications are the leading cause of death in patients with diabetes. Metformin, a first-line therapy for type 2 diabetes, is the only drug demonstrated to reduce cardiovascular complications in diabetic patients. However, whether metformin can effectively prevent thrombosis and its potential mechanism of action is unknown. Here we show, metformin prevents both venous and arterial thrombosis with no significant prolonged bleeding time by inhibiting platelet activation and extracellular mitochondrial DNA (mtDNA) release. Specifically, metformin inhibits mitochondrial complex I and thereby protects mitochondrial function, reduces activated platelet-induced mitochondrial hyperpolarization, reactive oxygen species overload and associated membrane damage. In mitochondrial function assays designed to detect amounts of extracellular mtDNA, we found that metformin prevents mtDNA release. This study also demonstrated that mtDNA induces platelet activation through a DC-SIGN dependent pathway. Metformin exemplifies a promising new class of antiplatelet agents that are highly effective at inhibiting platelet activation by decreasing the release of free mtDNA, which induces platelet activation in a DC-SIGN-dependent manner. This study has established a novel therapeutic strategy and molecular target for thrombotic diseases, especially for thrombotic complications of diabetes mellitus.

Figures

Figure 1. Metformin suppresses the release of…
Figure 1. Metformin suppresses the release of platelet mtDNA via inhibition of complex I.
(A,B) Metformin influences mtDNA escape from activated platelets in vitro (metformin: 1 mM, 6 h) and in vivo (metformin: 400 mg/kg/d, 7 d). (C) Transmission electronic microscopy imaging of platelets and mitochondria (black arrows) treated with metformin or without. (D) C11-BODIPY 581/591 (lipid peroxidation sensor), (E) ROS(CM-H2DCFDA fluorescence), (I) ATP, (H) hyperpolarization (JC-1 fluorescence ratio, FL2/FL1) ΔΨ, (J) mitochondrial routine respiration, (K) complex I values in activated platelets treated with metformin (metformin: 1 mM, 6 h). (F) ROS (hydrogen peroxide, 6 h) induces mtDNA escape from platelets. (G) NAC reduce mtDNA release from platelets (NAC: 10 mM, 6 h). (L) Low concentrations of rotenone reduce mtDNA release from platelets (rotenone: 12.5 nM, 6 h). Data are expressed as mean ± SD. n = 8. *P < 0.05 vs control, #P < 0.05 vs ADP.
Figure 2. mtDNA induced platelet activation may…
Figure 2. mtDNA induced platelet activation may through DC–SIGN dependent pathway.
(A,C) mtDNA affects aggregation of platelets with or without metformin treatment in vivo (mtDNA: 50 μg/kg/d, 7 d) and in vitro (mtDNA: 40 ng/μL, 6 h). (B,D) mtDNA influences αIIbβ3 level with or without metformin treatment in vivo and in vitro. (E,F) DNase decreases inhibition of metformin on platelet aggregation and αIIbβ3 level induced by ADP in vitro (Pre-incubated for 0.5 h at 20 μg/mL DNase I). (G) mtDNA increases DC-SIGN expression in vitro. (H,I) Anti-DC-SIGN (Pre-incubated for 0.5 h at 25 μg/mL) agent decreases platelet aggregation and αIIbβ3 level induced by mtDNA in vitro. Data are expressed as mean ± SD. n = 8. *P < 0.05 vs control, #P < 0.05 vs mtDNA.
Figure 3. Metformin inhibits platelet activation in…
Figure 3. Metformin inhibits platelet activation in vivo and in vitro.
(A,B) Influence of metformin on aggregation of platelets induced by ADP, AA, and thrombin in vitro (metformin: 1 mM, 6 h) and in vivo (metformin: 400 mg/kg/d, 7 d). (C,D) Effect of metformin on platelet adhesion to collagen-coated surfaces (phalloidin-labelled platelets). (E,F) αIIbβ3 expression in ADP-activated platelets with metformin in vitro and in vivo. (G,H) P-selectin expression in ADP-activated platelets with treatment in vitro and in vivo. (I,J) Influence of metformin on the elevation of cytosolic calcium levels in ADP-stimulated platelets in vitro and in vivo. Data are expressed as mean ± SD. n = 9–10. *P < 0.05 vs control, #P < 0.05 vs ADP.
Figure 4. Metformin inhibits formation of FeCl…
Figure 4. Metformin inhibits formation of FeCl3-induced carotid arterial thrombosis and partial inferior vena cava ligation induced venous thrombosis in animals.
Decreases weight (A,E) and length (B,E) of arterial thrombus in diabetic and normal rats (pre-treated with 400 mg/kg/d metformin for 7 d). Decreases weight (C,F) and length (D,F) of venous thrombus in normal and diabetic rats. (G) Reduces mortality from pulmonary thromboembolism in diabetic and normal mice (metformin: 400 mg/kg/d, 7 d). (H) Thromboelastogram of whole blood from rats treated with or without metformin. (I) Does not significantly prolong bleeding times in normal and diabetic C57/BL6 mice (metformin: 400 mg/kg/d, 7 d). (J) Reduces bleeding risk (increased bleeding time %/inhibited thrombosis %) in diabetic and normal rats compared with the use of aspirin. (K) Metformin decreases the incidence of gastric ulcer in normal rats compared with the use of aspirin (metformin: 400 mg/kg/d, 60 d. Aspirin: 30 mg/kg/d, 60 d). Data are expressed as mean ± SD. n = 9–10. *P < 0.05 vs control, #P < 0.05 vs aspirin.

References

    1. Cho N. H. et al.. The IDF Diabetes Atlas 7th edition (2015).
    1. Mackman N. Triggers, targets and treatments for thrombosis. Nature 451, 914–918 (2008).
    1. Tang W. H. et al.. Aldose reductase-mediated phosphorylation of p53 leads to mitochondrial dysfunction and damage in diabetic platelets. Circulation 129, 1598–1609 (2014).
    1. Creager M. A., Lüscher T. F., Cosentino F. & Beckman J. A. Diabetes and Vascular Disease Pathophysiology, Clinical Consequences, and Medical Therapy: Part I. Circulation 108, 1527–1532 (2003).
    1. Vazzana N., Ranalli P., Cuccurullo C. & Davì G. Diabetes mellitus and thrombosis. Thromb. Res. 129, 371–377 (2012).
    1. Randriamboavonjy V. et al.. Metformin reduces hyper-reactivity of platelets from patients with polycystic ovary syndrome by improving mitochondrial integrity. Thromb Haemost. 114, 569–578 (2015).
    1. Cannegieter S. C. et al.. Risk of venous and arterial thrombotic events in patients diagnosed with superficial vein thrombosis: a nationwide cohort study. Blood 125, 229–235 (2015).
    1. Inzucchi S. E. Metformin and heart failure. Diabetes Care. 28, 2585–2587 (2005).
    1. Roussel R. et al.. Metformin use and mortality among patients with diabetes and atherothrombosis. Arch. Intern. Med. 170, 1892–1899 (2010).
    1. Madiraju A. K. et al.. Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase. Nature 150, 542–546 (2014).
    1. Lu D. Y. et al.. Metformin use in patients with type 2 diabetes mellitus is associated with reduced risk of deep vein thrombosis: a non-randomized, pair-matched cohort study. BMC Cardiovasc Disord. 14, 187 (2014).
    1. DeFronzo R. A. et al.. Effects of exenatide (Exendin-4) on glycemic control and weight over 30 weeks in metformin-treated patients with type 2 diabetes. Diabetes Care. 28, 1092–1100 (2005).
    1. El-Mir M. Y. et al.. Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. J Biol Chem. 275, 223–228 (2000).
    1. Owen M. R., Doran E. & Halestrap A. P. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex I of the mitochondrial respiratory chain. Biochem J. 348, 607–614 (2000).
    1. Barile C. J. et al.. Inhibiting platelet-stimulated blood coagulation by inhibition of mitochondrial respiration. PNAS USA 109, 2539–2543 (2012).
    1. Yamagishi S. I., Edelstein D., Du X. L. & Brownlee M. Hyperglycemia potentiates collagen-induced platelet activation through mitochondrial superoxide overproduction. Diabetes 50, 1491–1494 (2001).
    1. Cardenes N. et al.. Platelet bioenergetic screen in sickle cell patients reveals mitochondrial complex V inhibition, which contributes to platelet activation. Blood 123, 2864–2872 (2014).
    1. Boudreau L. H. et al.. Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation. Blood 124, 2173–2183 (2014).
    1. Murphy M. P. How mitochondria produce reactive oxygen species. Biochem J. 417, 1–13 (2009).
    1. Kwiecien S. et al.. Lipid peroxidation, reactive oxygen species and antioxidative factors in the pathogenesis of gastric mucosal lesions and mechanism of protection against oxidative stress-induced gastric injury. J Physiol Pharmacol. 65, 613–622 (2014).
    1. Hottz E. D. et al.. Dengue induces platelet activation, mitochondrial dysfunction and cell death through mechanisms that involve DC-SIGN and caspases. J Thromb Haemost. 11, 951–962 (2013).
    1. Caparrós E. et al.. DC-SIGN ligation on dendritic cells results in ERK and PI3K activation and modulates cytokine production. Blood 107, 3950–3958 (2006).
    1. Geijtenbeek T. B. et al.. Identification of DC-SIGN, a novel dendritic cell–specific ICAM-3 receptor that supports primary immune responses. Cell 100, 575–585 (2000).
    1. King S. M. et al.. Platelet dense-granule secretion plays a critical role in thrombosis and subsequent vascular remodeling in atherosclerotic mice. Circulation 120, 785–791 (2009).
    1. Wang X. et al.. Murine model of ferric chloride-induced vena cava thrombosis: evidence for effect of potato carboxypeptidase inhibitor. J Thromb Haemost. 4, 403–410 (2006).
    1. Zhang S. et al.. Nucleotide-binding oligomerization domain 2 receptor is expressed in platelets and enhances platelet activation and thrombosis. Circulation 131, 1160–1170 (2015).
    1. Ballinger S. W. et al.. Hydrogen peroxide and peroxynitrite-induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circ Res. 86, 960–966 (2000).
    1. Inzucchi S. E., Masoudi F. A. & McGuire D. K. Metformin in heart failure. Diabetes Care. 28, 2585–2587 (2007).
    1. Grant P. J. Beneficial effects of metformin on haemostasis and vascular function in man. Diabetes Metab. 29, 6S44–6S52 (2003).
    1. Jy W. et al.. Comparison of pharmacokinetics and hemostatic efficacy of red cell microparticles (RMP) in rabbits using different infusion regimens. Blood 124, 2811–2811 (2014).
    1. Musumeci L. et al.. Dual-specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis. Circulation 131, 656–668 (2015).
    1. Wallace J. L. et al.. A diclofenac derivative without ulcerogenic properties. Eur J Pharmacol. 257, 249–255 (1994).
    1. Dayal S. et al.. Hydrogen peroxide promotes aging-related platelet hyperactivation and thrombosis. Circulation 127, 1308–1316 (2013).
    1. Zhou Q. et al.. Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis. Thromb Res. 133, 57–65 (2014).
    1. Piel S., Ehinger J. K., Elmér E. & Hansson M. J. Metformin induces lactate production in peripheral blood mononuclear cells and platelets through specific mitochondrial complex I inhibition. Acta Physiol. 213, 171–180 (2015).
    1. Qin C. et al.. Variation of perioperative plasma mitochondrial DNA correlate with peak inflammatory cytokines caused by cardiac surgery with cardiopulmonary bypass. J Cardiothorac Surg. 10, 85 (2015).
    1. Lal I., Dittus K. & Holmes C. E. Platelets, coagulation and fibrinolysis in breast cancer progression. Breast Cancer Res. 15, 207 (2013).
    1. West A. P. et al.. Mitochondrial DNA stress primes the antiviral innate immune response. Nature 520, 553–557 (2015).
    1. Fitzgerald J. R., Foster. T. J. & Cox D. The interaction of bacterial pathogens with platelets. Nat Rev Microbiol. 4, 445–457 (2006).
    1. Milne J. C. et al.. Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature 450, 712–716 (2007).
    1. Brill A. et al.. von Willebrand factor-mediated platelet adhesion is critical for deep vein thrombosis in mouse models. Blood 117, 1400–1407 (2011).
    1. Anil-Kumar K. S. et al.. Synthesis and identification of chiral aminomethylpiperidine carboxamides as inhibitor of collagen induced platelet activation. Eur. J Med Chem. 81, 456–472 (2014).
    1. Wang S. B. et al.. Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation. Free Radic Biol Med. 83, 41–53 (2015).
    1. Bae O. N. et al.. Trivalent methylated arsenical-induced phosphatidylserine exposure and apoptosis in platelets may lead to increased thrombus formation. Toxicol Appl Pharmacol. 239, 144–153 (2009).
    1. Schoenwaelder S. M. et al.. Bcl-xL-inhibitory BH3 mimetics can induce a transient thrombocytopathy that undermines the hemostatic function of platelets. Blood 118, 1663–1674 (2011).
    1. Shirakabe A. et al.. Drp1-dependent mitochondrial autophagy plays a protective role against pressure overload-induced mitochondrial dysfunction and heart failure. Circulation 133, 1249–1263 (2016).
    1. Bridges H. R., Jones A. J., Pollak M. N. & Hirst J. Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria. Biochem J. 462, 475–487 (2014).
    1. Ouseph M. M. et al.. Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis. Blood 126, 1224–1233 (2015).
    1. Gong G. H., Qin Y. & Huang W. Anti-thrombosis effect of diosgenin extract from Dioscorea zingiberensis CH Wright in vitro and in vivo. Phytomedicine 18, 458–463 (2011).
    1. Priestley E. S. et al.. Structure-based design of macrocyclic coagulation Factor VIIa inhibitors. J Med Chem. 58, 6225–6236 (2015).
    1. Citirik M., Beyazyildiz E., Simsek M., Beyazyildiz O. & Haznedaroglu I. C. MPV may reflect subcinical platelet activation in diabetic patients with and without diabetic retinopathy. Eye 29, 376–379 (2015).
    1. Nakahira K. et al.. Circulating mitochondrial DNA in patients in the ICU as a marker of mortality: derivation and validation. PLoS Med. 10, e1001577 (2013).
    1. Lamkanfi M. et al.. Caspase-7 deficiency protects from endotoxin-induced lymphocyte apoptosis and improves survival. Blood 113, 2742–2745 (2009).

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