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3929 12-HETE Regulates Procoagulant Platelet Formation By Promoting Mitochondrial Depolarization

Program: Oral and Poster Abstracts
Session: 301. Platelets and Megakaryocytes: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Bleeding and Clotting, Diseases, Thrombotic disorders
Monday, December 9, 2024, 6:00 PM-8:00 PM

Hem Kumar Tamang, PhD1, Michael Kamradt, PhD2*, Jessica Seifert1*, Catherine Makarewich, PhD2,3* and Benjamin Tourdot, PhD4,5

1Experimental Hematology, Cincinnati Children's Hospital, Cincinnati, OH
2Molecular Cardiovascular Biology, Cincinnati Children's Hospital, Cincinnati, OH
3Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH
4Department of Pediatrics, Cincinnati Children's Hospital, Cincinnati, OH
5Department of Pediatrics, Cincinnati Children's Hospital, CINCINNATI, OH

12-HETE regulates procoagulant platelet formation by promoting mitochondrial depolarization

Procoagulant platelets, characterized by externalized phosphatidylserine (PS), enhance coagulation and leukocyte recruitment, playing a crucial role in thromboinflammatory disorders such as venous thrombosis and ischemic stroke. Inhibiting procoagulant platelet formation has been shown to significantly decrease the incidence and severity of thromboinflammatory disorders. This underscores the importance of understanding the mechanisms of procoagulant platelet formation, as it could pave the way for the development of more effective drugs to treat thromboinflammatory disorders.

12-lipoxygenase (12-LOX), an oxygenase that produces the bioactive lipid 12-HETE (hydroxyeicosatetraenoic acid) potentiates procoagulant platelet formation in humans and mice. Furthermore, mice deficient in 12-LOX are protected from venous thrombosis, demonstrating the enzyme’s critical role in thromboinflammatory diseases.

Mitochondrial permeability transition pore (mPTP) opening during platelet activation drives mitochondrial depolarization and PS externalization. In endothelial and cancer cells, 12-HETE increased mitochondrial calcium, and mitochondrial membrane depolarization. Although 12-HETE regulates mitochondrial function in other cells, its role in modulating mitochondrial function and procoagulant activities in platelets remains unknown. We hypothesize that 12-HETE potentiates procoagulant platelet formation and thus enhances thromboinflammation by regulating mitochondrial function.

To test this hypothesis, we generated platelet-specific 12-LOX deficient mice and confirmed the complete absence of 12-HETE in their platelets using ELISA. Subsequently, an annexin V binding assay revealed that 12-LOX-/- platelets exhibit decreased PS externalization compared to wild-type (WT) platelets. Given that procoagulant platelets are characterized by increased microparticle formation, we assessed the size of the activated platelets using flow cytometry to determine microparticle formation. Our results indicate that 12-LOX-/- platelets generate fewer microparticles compared to WT platelets. Since the opening of the mPTP is also responsible for the decrease in mitochondrial membrane potential, we measured mitochondrial membrane depolarization. Consistent with a reduction in annexin V binding and microparticle formation, mitochondrial membrane depolarization was decreased in 12-LOX-/- platelets.

Recognizing the importance of mitochondrial membrane potential for mitochondrial respiration, we utilized the Seahorse assay to measure the oxygen consumption rate (OCR). The OCR following the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazon (FCCP), which indicates spare respiratory capacity, was found to be higher in 12-LOX-/- platelets activated with thrombin and convulxin. This demonstrates that 12-LOX-/- platelets have an increased capacity for mitochondrial ATP production during agonist-induced metabolic stress.

These results indicate that 12-LOX possibly through 12-HETE generation induces PS externalization and procoagulant platelet formation by inducing mitochondrial membrane depolarization.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH