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.

Aim As the global population has reached 7 billion and the baby boom generation reaches old age, thrombosis has become the major contributor to the global disease burden. It has been reported that, in moderate doses, beer may protect against thrombosis. Xanthohumol (XN), an antioxidant, is found at high concentrations in hop cones (Humulus lupulus L.) and is a common ingredient of beer. Here, the aim of the present work was to investigate the effects of XN on antithrombotic and antiplatelet activities, and study its mechanism. Approach and Results Using ferric chloride-induced carotid artery injury, inferior vena cava ligation model, and platelet function tests, we demonstrated that XN uniquely prevents both venous and arterial thrombosis by inhibiting platelet activation. Interestingly, in tail bleeding time studies, XN did not increase bleeding risk, which is recognized as a major limitation of current antithrombotic therapies. We also demonstrated that XN induces Sirt1 expression and thereby decreases reactive oxygen species (ROS) overload, prevents mitochondrial dysfunction, and reduces activated platelet-induced mitochondrial hyperpolarization, respiratory disorders, and associated membrane damage at low concentrations. In mitochondrial function assays designed to detect amounts of extracellular mitochondrial DNA (mtDNA), we found that XN prevents mtDNA release, which induces platelet activation in a DC-SIGN-dependent manner. Conclusions XN exemplifies a promising new class of antiplatelet agents that are highly effective at inhibiting platelet activation by decreasing ROS accumulation and platelet mtDNA release without incurring a bleeding risk. This study has also provided novel insights into mechanisms of thrombotic diseases with possible therapeutic implications.

BackgroudCardiac surgery with cardiopulmonary bypass (CPB) may cause inflammatory responses, which can deteriorate the outcomes. Inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6,–8 and -10, can act as both the effector and the predictor for post-operative inflammatory responses. Plasma mitochondrial DNA (mtDNA) was found as a pro-inflammatory agent recently, which was released when cells were insulted.MethodsIn the present study, we included 38 patients undergoing coronary artery bypass graft (CABG) to analyze their perioperative plasma mtDNA and levels of inflammatory cytokines. Blood samples were collected before aortic cross-clamping (T1), at the end of CPB (T2), 6 h post-CPB (T3), 12 h post-CPB (T4), and 24 h post-CPB (T5). Rt-PCR and specific ELISA kits were used to quantify the plasma mtDNA and inflammatory cytokines, respectively. Bivariate correlations analysis was used to check the correlations between plasma mtDNA and inflammatory cytokines respectively.ResultsResults shown that plasma mtDNA elevated significantly at T2 and peaked at T4. Furthermore, plasma TNF-α, IL-6 and IL-8 levels significantly increased at T2 and peaked at T3 while IL-10 elevated and peaked at T2. Bivariate correlations analysis showed that the peak plasma mtDNA were positively correlated with the peak TNF-α (r = 0.677, P < 0.001), the peak IL-6 (r = 0.706, P < 0.001), the peak IL-8 (r = 0.584, P < 0.001) and the peak IL-10 (r = 0.565, P < 0.001).ConclusionWe found that plasma mtDNA might play a key role in CPB-induced post-operative inflammatory responses.

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BackgroundMitochondrial DNA (mtDNA) was reported as a pro-inflammatory agent. In our previous study, elevation of plasma mtDNA was revealed after cardiac surgery with cardiopulmonary bypass (CPB). Platelets were activated during the cardiac surgery and recent study revealed its ability to release mtDNA. Our present study postulated that the elevated plasma mtDNA comes from activated platelets, which plays a critical role in post-CPB inflammatory responses.MethodsSixty-eight patients who underwent coronary artery bypass graft (CABG) with CPB were enrolled in our study. Blood samples were collected before induction of anaesthesia (T1), at the end of CPB (T2), 12 h post-CPB (T3), 24 h post-CPB (T4), 48 h post-CPB (T5) and 72 h post-CPB (T6). Blood samples were analyzed for the routine blood test and prepared for plasma isolation. MtDNA concentration was measured by rt-PCR, and TNF-α and IL-6 were examined by specific ELISA kits. Subgroup study was analyzed by activation levels of platelet. Basic information, mtDNA level, TNF-α level and IL-6 level were all carefully studied in each quartile.ResultsActivation level of platelets increased and peaked at T2, which decreased gradually from T3 to T6 (P < 0.05). MtDNA increased after CPB, peaked at T3, and then backed from T4 to T6 (P < 0.05). Bivariate correlation between peak activation level of platelets and peak plasma mtDNA level showed a positive correlation between these two parameters (r = 0.683, P < 0.0001). Both TNF-α and IL-6 showed similar patterns as mtDNA, with an increase from T1 to T3 and a decrease from T4 to T6 (P < 0.05). Subgroup analysis further demonstrated that patients with higher activation levels of PLT had higher plasma mtDNA levels and inflammatory level (P < 0.05).ConclusionsOur study revealed the dynamic changes of activation level of platelets and identified the interesting association between platelets activation and plasma mtDNA, suggesting a novel potential mechanism of activated platelets-induced post-CPB inflammatory responses.