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There is a critical need for cerebro-protective interventions to improve the suboptimal outcomes of patients with ischemic stroke who have been treated with reperfusion strategies. We found that nuclear pyruvate kinase muscle 2 (PKM2), a modulator of systemic inflammation, was upregulated in neutrophils after the onset of ischemic stroke in both humans and mice. Therefore, we determined the role of PKM2 in stroke pathogenesis by using murine models with preexisting comorbidities. We generated novel myeloid cell–specific PKM2−/− mice on wild-type (PKM2fl/flLysMCre+) and hyperlipidemic background (PKM2fl/flLysMCre+Apoe−/−). Controls were littermate PKM2fl/flLysMCre– or PKM2fl/flLysMCre–Apoe−/− mice. Genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps after cerebral ischemia and reperfusion, suggesting that PKM2 promotes neutrophil hyperactivation in the setting of stroke. In the filament and autologous clot and recombinant tissue plasminogen activator stroke models, irrespective of sex, deletion of PKM2 in myeloid cells in either wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell–specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrinogen, platelet [CD41+] deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. To enhance the translational significance, we inhibited PKM2 nuclear translocation using a small molecule and found significantly reduced neutrophil hyperactivation and improved short-term and long-term functional outcomes after stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery after reperfusion.

Supplemental Digital Content is available in the text.

Overweight and obesity are significant risk factors for deep vein thrombosis (DVT). Cellular fibronectin containing extra domain A (Fn‐EDA), an endogenous ligand for toll‐like‐receptor 4 (TLR4), contributes to thrombo‐inflammation. The role of Fn‐EDA in the modulation of DVT is not elucidated yet.

Overweight and obesity are significant risk factors for deep vein thrombosis (DVT). Cellular fibronectin containing extra domain A (Fn‐EDA), an endogenous ligand for toll‐like‐receptor 4 (TLR4), contributes to thrombo‐inflammation. The role of Fn‐EDA in the modulation of DVT is not elucidated yet. To determine whether Fn‐EDA promotes DVT in the context of diet‐induced obesity. Wild‐type (WT) and Fn‐EDA‐deficient mice were either fed control or high‐fat (HF) diet for 12 weeks. DVT was induced by inferior vena cava (IVC) stenosis and evaluated after 48 hours. Cellular Fn‐EDA levels in the plasma of venous thromboembolism (VTE) patients were measured by sandwich ELISA. We found that cellular Fn‐EDA levels were significantly elevated in VTE patients' plasma and positively correlated with body mass index. HF diet‐fed WT mice exhibited increased DVT susceptibility compared with control diet‐fed WT mice. In contrast, HF diet‐fed Fn‐EDA‐deficient mice exhibited significantly reduced thrombus weight and decreased incidence (%) of DVT compared with HF diet‐fed WT mice concomitant with reduced neutrophil content and citrullinated histone H3‐positive cells (a marker of NETosis) in IVC thrombus. Exogenous cellular Fn‐EDA potentiated NETosis in neutrophils stimulated with thrombin‐activated platelets via TLR4. Genetic deletion of TLR4 in Fn‐EDA+mice (constitutively express Fn‐EDA in plasma and tissues), but not in Fn‐EDA‐deficient mice, reduced DVT compared with respective controls. These results demonstrate a previously unknown role of Fn‐EDA in the DVT exacerbation, which may be an essential mechanism promoting DVT in the setting of diet‐induced obesity.

Supplemental Digital Content is available in the text.

Evidence suggests that neutrophils contribute to thrombosis via several mechanisms, including neutrophil extracellular traps (NETs) formation. Integrin α9β1 is highly expressed on neutrophils when compared with monocytes. It undergoes affinity upregulation on neutrophil activation, and stabilizes adhesion to the activated endothelium. The role of integrin α9 in arterial thrombosis remains unexplored. We generated novel myeloid cell-specific integrin α9−/−mice (α9fl/flLysMCre+) to study the role of integrin α9 in arterial thrombosis. α9fl/fllittermates were used as controls. We report that α9fl/flLysMCre+mice were less susceptible to arterial thrombosis in ferric chloride (FeCl3) and laser injury-induced thrombosis models with unaltered hemostasis. Neutrophil elastase-positive cells were significantly reduced in α9fl/flLysMCre+mice concomitant with reduction in neutrophil count, myeloperoxidase levels, and red blood cells in the FeCl3injury-induced carotid thrombus. The percentage of cells releasing NETs was significantly reduced in α9fl/flLysMCre+mouse neutrophils stimulated with thrombin-activated platelets. Furthermore, we found a significant decrease in neutrophil-mediated platelet aggregation and cathepsin-G secretion in α9fl/flLysMCre+mice. Transfusion of α9fl/flneutrophils in α9fl/flLysMCre+mice restored thrombosis similar to α9fl/flmice. Treatment of wild-type mice with anti-integrin α9 antibody inhibited arterial thrombosis. This study identifies the potential role of integrin α9 in modulating arterial thrombosis.

Evidence suggests that neutrophils contribute to thrombosis via several mechanisms, including neutrophil extracellular traps (NETs) formation. Integrin α9β1 is highly expressed on neutrophils when compared with monocytes. It undergoes affinity upregulation on neutrophil activation, and stabilizes adhesion to the activated endothelium. The role of integrin α9 in arterial thrombosis remains unexplored. We generated novel myeloid cell-specific integrin α9−/− mice (α9fl/flLysMCre+) to study the role of integrin α9 in arterial thrombosis. α9fl/fl littermates were used as controls. We report that α9fl/flLysMCre+ mice were less susceptible to arterial thrombosis in ferric chloride (FeCl3) and laser injury-induced thrombosis models with unaltered hemostasis. Neutrophil elastase-positive cells were significantly reduced in α9fl/flLysMCre+ mice concomitant with reduction in neutrophil count, myeloperoxidase levels, and red blood cells in the FeCl3 injury-induced carotid thrombus. The percentage of cells releasing NETs was significantly reduced in α9fl/flLysMCre+ mouse neutrophils stimulated with thrombin-activated platelets. Furthermore, we found a significant decrease in neutrophil-mediated platelet aggregation and cathepsin-G secretion in α9fl/flLysMCre+ mice. Transfusion of α9fl/fl neutrophils in α9fl/flLysMCre+ mice restored thrombosis similar to α9fl/fl mice. Treatment of wild-type mice with anti-integrin α9 antibody inhibited arterial thrombosis. This study identifies the potential role of integrin α9 in modulating arterial thrombosis.

Resting platelets rely on oxidative phosphorylation (OXPHOS) and aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) to generate adenosine triphosphate, whereas activated platelets exhibit a high level of aerobic glycolysis, suggesting the existence of metabolic flexibility in platelets. Mitochondrial pyruvate dehydrogenase kinases (PDK 1-4) play a pivotal role in metabolic flexibility by inhibiting pyruvate dehydrogenase complex. We determined whether metabolic reprogramming, diverting metabolism from aerobic glycolysis back to OXPHOS, would inhibit platelet function. PDKs activity in human and mouse platelets was inhibited with dichloroacetic acid (DCA), a potent inhibitor of all 4 forms of PDK. Human and mouse platelets pretreated with DCA exhibited decreased platelet aggregation to suboptimal doses of collagen, convulxin, thrombin, and adenosine diphosphate concomitant with decreased glucose uptake. Bioenergetics profile revealed that platelets pretreated with DCA exhibited decreased aerobic glycolysis in response to convulxin only. Furthermore, DCA inhibited ATP secretion, thromboxane A2 generation, and tyrosine phosphorylation of Syk and PLC?2 in response to collagen or convulxin in human and mouse platelets (P < .05 vs vehicle treated). In the flow chamber assay, human and mouse blood pretreated with DCA formed smaller thrombi when perfused over collagen for 10 minutes at an arterial shear rate of 1500 s-1 (P < .05 vs control). Wild-type mice pretreated with DCA were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury–induced mesenteric artery thrombosis models (P < .05 vs vehicle control), without altering hemostasis. Targeting metabolic plasticity with DCA may be explored as a novel strategy to inhibit platelet function.

Supplemental Digital Content is available in the text.

Supplemental Digital Content is available in the text.

Supplemental Digital Content is available in the text.

Supplemental Digital Content is available in the text.

Supplemental Digital Content is available in the text.

Supplemental Digital Content is available in the text.

Supplemental Digital Content is available in the text.

Introduction:Current treatment regimen for deep vein thrombosis (DVT) includes anticoagulants or surgical intervention. While effective, anticoagulants exhibit a bleeding risk that limits their use. Activated platelets release pro-thrombotic chemokines such as platelet factor 4 (PF4) from their α-granules and are known to potentiate DVT via promoting neutrophil extracellular traps (NETs). Recently, we have shown that dimeric pyruvate kinase M2 (PKM2) regulates platelet function and arterial thrombosis without affecting hemostasis. However, the role of platelet-specific PKM2 in modulating NETosis and DVT remains unexplored.Methods:Susceptibility to DVT was evaluated in the flow-restricted inferior vena cava (IVC) stenosis model in PKM2fl/flPF4Cre+/-mice. The littermate PKM2fl/flPF4Cre-/-(abbreviated as PKM2fl/fl) was used as control. Next, we evaluated whether wild-type mice infused with a specific inhibitor of dimeric-PKM2, ML265, reduced DVT susceptibility in the IVC stenosis model.Results:The platelet-specific PKM2-/-mice were less susceptible to DVT in the IVC stenosis model. The PF4 secretion from α-granules was significantly inhibited in agonist-stimulated platelets from PKM2fl/flPF4Cre+ mice compared to the platelets from PKM2fl/flmice. SNAP-23 phosphorylation regulates SNARE protein complex-mediated mediated secretion from α granules. Therefore, we determined the mechanistic role of PKM2 in modulating SNAP-23-mediated PF4 secretion in platelets. In agonist-stimulated PKM2-/- platelets, SNAP23 phosphorylation and PF4 secretion were reduced compared to agonist-stimulated WT platelets. In addition, neutrophils treated with stimulated platelet releasates from PKM2fl/flPF4Cre+ mice exhibited significantly reduced NET formation in contrast to PKM2fl/flmice. Finally, we demonstrate that ML265-treated mice showed reduced susceptibility to DVT in the IVC stenosis model.Conclusions:Our results suggest an essential role of PKM2 in regulating SNAP-23 mediated α-granule release, NETosis, and subsequent development of DVT.

Rationale:The glycolytic enzyme pyruvate kinase muscle 2 (PKM2) is upregulated in monocytes/macrophages of patients with atherosclerotic coronary artery disease. However, the role of cell type-specific PKM2 in the setting of atherosclerosis remains to be defined.Objective:We determined whether myeloid cell-specific PKM2 regulates efferocytosis and atherosclerosis.Methods and Results:We generated novel myeloid cell-specific PKM2-/-mice on Ldlr-deficient background (PKM2mye-KOLdlr-/-). Controls were littermate PKM2WTLdlr-/-mice. To rule out sex-based differences, male and female mice were placed on a high-fat "Western" diet for 14 weeks, starting at eight weeks. PKM2 was upregulated in macrophages of Ldlr-/-mice fed the Western diet compared with a control chow diet. Myeloid cell-specific deletion of PKM2 led to a significant reduction in lesions in the whole aorta and aortic sinus despite high cholesterol and triglyceride levels. Furthermore, we found decreased macrophage content in the lesions of myeloid cell-specific PKM2-/-mice associated with decreased MCP-1 levels in plasma, reduced transmigration of macrophages in response to MCP-1, and impaired glycolytic rate. Macrophages isolated from myeloid-specific PKM2-/-mice fed the Western diet exhibited reduced expression of pro-inflammatory genes, including MCP-1, IL-1β, and IL-12. Myeloid cell-specific PKM2-/-mice exhibited reduced apoptosis concomitant with enhanced macrophage efferocytosis and upregulation of LRP1 in macrophages in vitroand atherosclerotic lesions in vivo. Silencing LRP1 in PKM2-deficient macrophages restored inflammatory gene expression and reduced efferocytosis. As a therapeutic intervention, inhibiting PKM2 nuclear translocation using a small molecule reduced glycolytic rate, enhanced efferocytosis, and reduced atherosclerosis in Ldlr-/-mice.Conclusion:Genetic deletion or limiting PKM2 nuclear translocation in myeloid cells reduces atherosclerosis by suppressing inflammation and enhancing efferocytosis.

Objective:Obesity is a significant risk factor for deep vein thrombosis (DVT). The mechanisms of increased DVT in preexisting comorbid condition of obesity remain poorly understood. Cellular fibronectin containing extra domain A (Fn-EDA), an endogenous ligand for toll-like-receptor 4 (TLR4), is known to contribute to thrombo-inflammation in the experimental models. However, the role of Fn-EDA in modulation of venous thrombosis in context of obesity is not elucidated yet.

There is a critical need for cerebroprotective interventions to improve the suboptimal outcomes of patients with ischemic stroke treated with reperfusion strategies. We found that nuclear pyruvate kinase muscle 2 (PKM2), a modulator of systemic inflammation, was upregulated in neutrophils after the onset of ischemic stroke both in humans and in mice. Therefore, we determined the role of PKM2 in stroke pathogenesis utilizing murine models with preexisting comorbidities. We generated novel myeloid cell-specific PKM2-/-mice on wild-type (PKM2fl/flLysMCre+) and hyperlipidemic background (PKM2fl/flLysMCre+Apoe-/-). Controls were littermate PKM2fl/flLysMCre-or PKM2fl/flLysMCre-Apoe-/-mice. Genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps following cerebral ischemia/reperfusion, suggesting PKM2 promotes neutrophil hyperactivation in the setting of stroke. In the filament and autologous clot/rtPA stroke models, irrespective of sex, deletion of PKM2 in myeloid cells either in wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell-specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrin(ogen), platelet (CD41-positive) deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. To enhance the translational significance, we inhibited PKM2 nuclear translocation using a small molecule and found significantly reduced neutrophil hyperactivation and improved short-term and long-term functional outcomes following stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery following reperfusion.

No relevant conflicts of interest to declare.

Background:Coordinated interactions between neutrophils, platelets and endothelial cells contribute towards the development of arterial thrombosis. Neutrophils along with platelets are the first immune cells that are recruited at the site of endothelial activation/injury or infection. Recent studies have suggested that neutrophils modulate thrombosis via several mechanisms, including NETosis (formation of neutrophil extracellular traps). The integrin α9 is highly expressed on neutrophils while platelets do not express it. The integrin α9 up-regulated upon neutrophil activation and is implicated in stable adhesion and transmigration. The mechanisms underlying the role of integrin α9 towards the progression of arterial thrombosis has not been explored yet.