OC 19.2 - Metabolic reprogramming is essential for myeloid cell-dependent haemostatic activity

Background: Myeloid cell activation leading to enhanced tissue factor (TF) expression, decryption and procoagulant activity represents a key event in the initiation of thrombo-inflammatory disease. Macrophage polarisation promotes rewiring of cellular metabolism to favour enhanced aerobic glycolysis, which allows for rapid generation of the energy required for phagocytosis and cytokine production.

Aims: To investigate whether metabolic reprogramming in response to inflammatory stimuli impacts myeloid cell procoagulant and antifibrinolytic activity.

Methods: Mouse bone marrow-derived macrophages or human monocytes were skewed towards pro-inflammatory (M1) or anti-inflammatory (M2) polarisation states. Myeloid cell gene and protein expression was analysed using ELISA, RT-PCR, flow cytometry and confocal microscopy. Additionally, we developed new myeloid cell-based thrombin and plasmin generation assays to assess myeloid cell procoagulant function.

Results: We demonstrate that inhibition of inflammation-associated metabolic reprogramming of myeloid cells ablates activated TF-dependent thrombin generation. Treatment of macrophages or monocytes with 2-Deoxy-D-glucose (2-DG; hexokinase inhibitor) reduced F3 gene expression and significantly extended TF-dependent lag-time in a novel assay of myeloid cell-dependent thrombin generation. We next assessed how inhibition of glycolysis restricts TF-dependent procoagulant activity by measuring the impact of 2-DG on cellular determinants of cell surface TF decryption. Notably, flow cytometric assessment of lactadherin binding to M1-skewed macrophages revealed significantly reduced phosphatidylserine externalisation upon 2-DG treatment. Furthermore, membrane localisation of acid sphingomyelinase, which degrades membrane sphingomyelin to promote TF procoagulant decryption, was significantly impaired by 2-DG, suggesting a new role for activated macrophage metabolic reprogramming in facilitating TF decryption. Finally, we examined the role of glycolysis in controlling macrophage-dependent plasmin generation. Enhanced M1-skewed macrophage antifibrinolytic activity via increased plasminogen activator inhibitor-1 gene expression and secretion was significantly attenuated by inhibition of glycolysis, leading to restoration of plasmin generation in 2-DG treated, M1-skewed macrophages.

Conclusion(s): The discovery of immunometabolic regulation of myeloid cell hypercoagulability opens new therapeutic possibilities for the mitigation of thrombo-inflammatory disease.