Maine Medical Center, Medical Education, Maine Medical Center Research Institute, Surgery, Cardiology
Tranexamic Acid, Endothelial Cells, Cell Nucleus, Mitochondria, Biophysical Phenomena, Gene Expression
Purpose Tranexamic acid (TXA), an anti-fibrinolytic inhibitor of plasmin, is widely used in hemorrhagic trauma patients. We have previously demonstrated that the effects of TXA transcend its inhibition of fibrinolysis by suppressing the release of damage associated molecular patterns (DAMPS), specifically mitochondrial DNA, and decreasing inflammation in a mouse burn model . Additionally, we have demonstrated that TXA increases mitochondrial respiration in human endothelial cells in vitro. Collectively, these results prompted us to hypothesize that TXA has other biologically important molecular targets besides plasmin. Herein we present the initial results of our studies addressing this hypothesis.
Methods To assess the intracellular distribution of TXA in human umbilical endothelial cells (HUVEC), we incubated HUVEC with 3H-TXA, fractionated cell compartments using the QProteomics kit (Qiagene, Germany) and analyzed the distribution of TXA by scintillation counting. To define HUVEC proteins binding TXA, we applied the Drug Affinity Responsive Target Stability (DARTS) method. Cell lysates were incubated with TXA, then treated with pronase, and mass-spectrometry (MS) analysis was applied to identify the proteins protected by TXA from proteolysis.
Results Cell fractionation studies have shown that 60-70% of TXA internalized by HUVEC are localized in the nuclear fraction, the rest being almost equally distributed between the membrane, cytosol and cytoskeleton fractions. DARTS studies demonstrated that among 10 proteins most strongly protected by TXA three are proteins involved in the regulation of gene expression and three are metabolic enzymes. Interestingly, among the latter is a component of the oxoglutarate dehydrogenase complex, which can demonstrate both mitochondrial and nuclear localization where it is apparently involved in the regulation of gene expression.
Conclusions Cell fractionation data showed preferential localization of TXA in the nuclei of endothelial cells. The results of studies using DARTS method identified several nuclear and mitochondrial proteins as potential targets of TXA. Further research is needed to elucidate the regulation of these proteins functions by TXA, and to determine TXA effects on gene expression and mitochondrial metabolism.
Prudovsky, Igor; Kacer, Doreen; Zucco, Victoria; Wynne, Keiran; Carter, Damien; Palmeri, Monica; Vary, Calvin; Kramer, Robert; and Rappold, Joseph, "Tranexamic acid preferentially localizes in the nuclei of endothelial cells and binds proteins involved in gene expression and mitochondrial metabolism" (2020). Costas T. Lambrew Research Retreat 2020. 33.