Understanding the protective effects of tranexamic acid in trauma: beyond anti-fibrinolysis
Figure 1: Tranexamic acid increases mitochondrial respiration and ATP production in endothelial cells: study using a Seahorse analyzer.
Tranexamic acid (TXA) is a popular anti-fibrinolytic drug widely used in hemorrhagic trauma, cardiovascular surgery and in orthopedic surgery patients. TXA binds plasminogen and prevents its maturation to the fibrinolytic enzyme plasmin. A number of studies demonstrated broad life-saving effects of TXA in trauma, superior to those of other anti-fibrinolytic agents. Besides preventing fibrinolysis and blood loss, TXA has been reported to suppress post-traumatic inflammation and edema. Although the efficiency of TXA transcends simple inhibition of fibrinolysis, very little is known about cell and molecular mechanisms underlying its extensive activities. Understanding these mechanisms would improve the methods of TXA application and aid in the development of novel more efficient derivatives of TXA. The major events taking place after severe trauma, are metabolic shock, release to the bloodstream of pro-inflammatory Damage Associated Molecular Patterns (DAMP), and shedding of endothelial glycocalyx. In our recent studies in collaboration with Drs. Joseph Rappold, Damien Carter and Robert Kramer (all surgeons from Maine Medical Center), we have found that TXA enhances the efficiency of mitochondrial respiration in endothelial cells (Figure 1), suppresses the release of a major DAMP, mitochondrial DNA, from endothelial cells and granulocytes, and decreases the burn-induced lung inflammation in mice and rats. Our hypothesis is that enhancement of mitochondrial respiration by TXA suppresses DAMP release and metabolic stress, resulting in decreased inflammation and endothelial damage, and improved outcomes after severe trauma. We work on cell culture and animal models to understand the molecular mechanisms of TXA activity.
Defining the molecular mechanisms of obesity suppression by CTHRC1
Figure 2. Genetic knockout of CTHRC1 enhances adipogenesis. Adipocyte precursors from CTHRC1 null mice more efficiently differentiate to adipocytes (Oil Red staining) than preadipocytes from wild type mice.
Recent population studies indicate that obesity prevalence among the US adult population is nearing 40%. Obesity presents medical complications such as cardiovascular disease, diabetes, and certain types of cancer, as well as financial burden. The obesity epidemic stresses the importance of studies focused on mechanisms responsible for adiposity regulation. The laboratory of our colleague Volkhard Lindner found that mice with knockout of the CTHRC1 gene, coding for secreted protein involved in tissue repair, exhibit increased adiposity. Because of our long-term interest in the mechanisms of tissue repair, especially the repair-related decrease of fat tissue, we started a collaboration aimed to understand the molecular mechanisms of CTHRC1 activities. We have found that CTHRC1 suppresses the differentiation of preadipocytes (precursors of fat cells) to adipocytes (mature fat cells)(Figure 2). We have also found that CTHRC1 strongly decreases the expression of major transcription factors participating in adipogenesis. Our aim is to understand the molecular mechanisms of CTHRC1 signaling underlying its anti-adipogenic effect.
Stressed-Induced Nonclassical Export of Growth Factors and Cytokines
Unlike other secreted proteins, the potent pro-angiogenic factor FGF1 and pro-inflammatory cytokine IL1alpha lack a signal peptide for classical secretion, and are released through Golgi-independent pathways. Cell stress induces the export of FGF1 and IL1 alpha. At stress, FGF1 and IL1alpha translocate to the vicinity of cell membrane, where they co-localize with cortical actin cytoskeleton to be further exported to the extracellular milieu. We found that the export of FGF1 relies on copper-dependent formation of a complex containing several secreted signal-peptide-less proteins: S100A13, sphingosine kinase 1 (SphK1), 40 kDa form of synaptotagmin 1 (p40 Syt1) and, apparently, annexin II (Anx II). A similar complex is required for stress-induced export of IL1alpha. The export of FGF1 release complex depends on transmembrane translocation of acidic phospholipids (Figure 3).
Figure 3. Stress-induced FGF1 export.
Our current aims are to understand: 1.how the release complex assembles; 2. what is the role of actin cytoskeleton in the export of FGF1 and IL1α; 3.what is the molecular mechanism of transmembrane translocation of the release complex. In addition, to study the regulation of FGF1 export in vivo, we produced transgenic mice with FGF1 expression in endothelial cells and macrophages.
Regulation of Nonclassical Protein Export by Notch Signaling
FGF, IL1 and Notch are important regulators of angiogenesis and cancer growth. We found that the inhibition of Notch signaling results in strong spontaneous release of FGF1 (Figure 3) and IL1, and the increase of their expression. Fibroblasts with inhibited Notch pathway acquire transformed phenotype, which is dependent on FGF signaling. Our aim is to understand the transcriptional regulation of nonclassical protein export by Notch signaling.
Restricted Proliferation of FGF-Stimulated Cells
We found that several cell types of mesenchymal origin respond to FGF presented as a single mitogen by a strictly limited proliferation. They undergo one cell cycle and then get blocked in the non-proliferating state characterized by dramatic accumulation of cell cycle inhibitors (Figure 4) and active FGFR/Erk signaling. We hypothesize that the restricted character of FGF-induced proliferative response prevents hyperplasia in damaged tissue, where FGF is released under stress conditions. We are currently studying the molecular mechanisms, which limit the growth of FGF-stimulated cells.
Figure 4. Unlike serum-stimulated cells, FGF1 treated fibroblasts express large amounts of cell cycle inhibitor p21. Red – nuclei. Green – p21.
Prudovsky I. Cellular Mechanisms of FGF-Stimulated Tissue Repair. Cells. 2021, 10 (7):1830.
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Prudovsky I, Carter D, Kacer D, Palmeri M, Soul T, Kumpel C, Pyburn K, Barrett K, DeMambro V, Alexandrov I, Brandina I, Kramer R, Rappold J. Tranexamic acid suppresses the release of mitochondrial DNA, protects the endothelial monolayer and enhances oxidative phosphorylation. J Cell Physiol. 2019, 234, 19121-19.
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Liaw L, Prudovsky I, Koza RA, Anunciado-Koza RV, Siviski ME, Lindner V, Friesel RE, Rosen CJ, Baker PR, Simons B, Vary CP. Lipid Profiling of In Vitro Cell Models of Adipogenic Differentiation: Relationships with Mouse Adipose Tissues. J Cellular Biochemistry. 2016, 117, 2182-93.
Talukder HMA, Preda M, Rhyzova L, Prudovsky I, Pinz IM. Heterozygous caveolin-3 mice show increased susceptibility to palmitate-induced insulin resistance. Physiology Reports. 2016,4, pii: e12736. doi: 10.14814/phy2.12736.
Prudovsky I, Kacer D, Davis J, Shah V, Jayanthi S, Huber I, Dakshinamurthy R, Ganter O, Soldi R, Neivandt D, Guvench O, Suresh Kumar TK. Folding of Fibroblast Growth Factor 1 Is Critical for Its Nonclassical Release. Biochemistry. 2016: 1159-67.
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