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Updated: May 21, 2025
Preparation of Washed Human Platelets for Quantitative Metabolic Flux Studies
Published on: January 10, 2025
BAD-Glucokinase Axis Regulates Platelet Activation and Thrombosis
Mengnan Yang1, Shuang Chen1, Qing Li1
1Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Suzhou Medical College, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China.
Background:
BAD (Bcl2-associated death promoter), a member of the Bcl2 proapoptotic family, promotes cell apoptosis by sequestering the prosurvival proteins Bcl-XL and Bcl2 from the proapoptotic proteins BAK (Bcl2 homologous antagonist/killer) and BAX (Bcl2-associated X protein) in nucleated cells. BAD is also expressed in platelets, playing a role in regulating platelet lifespan, apoptosis, and clearance. However, whether BAD regulates platelet activation and arterial thrombosis remains unclear.
Methods:
The role of BAD in platelet activation and arterial thrombosis was investigated using BAD-deficient mice (Bad-/-), in vitro functional studies, and arterial thrombosis models. The regulatory effect of BAD on platelet energy metabolism was detected using a Seahorse Extracellular Flux Analyzer. The regulatory effect of BAD on glucokinase was investigated by coimmunoprecipitation and activity measurement. The glucokinase heterozygous knockout mice (Gck+/-) and activator were used to study its role in platelet activation.
Results:
BAD-deficient mice (Bad-/-) and wild-type mice transfused with Bad-/- platelets displayed prolonged tail bleeding and arterial occlusion times. Bad-/- platelets exhibited decreased aggregation in response to stimulations by proteinase-activated receptor 4-activating peptide, thrombin, and U46619. Furthermore, BAD ablation suppressed platelet integrin αIIbβ3 activation, granule secretion, and clot retraction induced by these agonists. Mechanistically, BAD interacted with glucokinase, and BAD deficiency resulted in decreased platelet glucokinase activity, mitochondrial oxidative phosphorylation, and mitochondrial ATP production. The partial loss of glucokinase (Gck+/-) phenocopied platelet function defects caused by BAD deficiency, and a glucokinase activator rescued the impaired mitochondrial ATP production and function of Bad-/- platelets. Additionally, the glucokinase activator enhanced human platelet activation.
Conclusions:
Our findings demonstrate the critical role of the BAD-glucokinase axis in platelet activation and thrombosis, suggesting a potential target for antithrombotic therapy.
Insights
The Bcl2-associated death promoter (BAD) protein is crucial for platelet activation and arterial thrombosis by interacting with glucokinase. Targeting this BAD-glucokinase axis may offer new antithrombotic therapies.
Area of Science:
- Hematology
- Molecular Biology
- Cardiovascular Research
Background:
- Bcl2-associated death promoter (BAD) is a proapoptotic protein regulating cell death.
- BAD is expressed in platelets, influencing their lifespan, apoptosis, and clearance.
- The role of BAD in platelet activation and arterial thrombosis is not well understood.
Purpose of the Study:
- To investigate the role of BAD in platelet activation and arterial thrombosis.
- To elucidate the molecular mechanisms by which BAD influences platelet function.
- To explore the potential of the BAD-glucokinase pathway as an antithrombotic target.
Main Methods:
- Utilized BAD-deficient mice and in vitro platelet studies.
- Assessed platelet aggregation, integrin activation, and granule secretion.
- Investigated BAD's effect on platelet energy metabolism and glucokinase activity.
- Employed glucokinase heterozygous knockout mice and a glucokinase activator.
Main Results:
- BAD deficiency prolonged bleeding and arterial occlusion times in mice.
- BAD-deficient platelets showed reduced aggregation, integrin activation, and secretion.
- BAD deficiency decreased platelet glucokinase activity, mitochondrial respiration, and ATP production.
- Glucokinase deficiency phenocopied BAD deficiency, and a glucokinase activator rescued platelet function.
Conclusions:
- The BAD-glucokinase axis is critical for platelet activation and arterial thrombosis.
- Targeting the BAD-glucokinase pathway presents a potential strategy for antithrombotic therapy.
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