Toll-Like Receptor 9 Signaling Regulates Tissue Factor and Tissue Factor Pathway Inhibitor Expression in Human Endothelial Cells and Coagulation in Mice

OBJECTIVE: Bacterial DNA (CpG DNA) persists in tissues and blood under pathological conditions that are associated with enhanced intravascular coagulation. Toll-like receptor 9 recognizes CpG DNA and elicits innate and adoptive immunity, yet the impact of CpG DNA on coagulation has not been studied. In this study, we investigated the effects of CpG DNA on the expression and activity of tissue factor, a key initiator of coagulation and tissue factor pathway inhibitor in human coronary artery endothelial cells and on coagulation in mice. DESIGN: Controlled in vitro and in vivo studies. SETTING: University research laboratory. SUBJECTS: Cultured human coronary artery endothelial cell, wild-type mice, and TLR9-deficient mice. INTERVENTIONS: Human coronary artery endothelial cell was challenged with CpG DNA, and tissue factor and tissue factor pathway inhibitor expression and activity were assessed. In mice, the effects of CpG DNA on bleeding time and plasma levels of thrombin-antithrombin complexes and tissue factor were measured. MEASUREMENTS AND MAIN RESULTS: We found that CpG DNA, but not eukaryotic DNA, evoked marked nuclear factor-κB-mediated increases in tissue factor expression at both messenger RNA and protein levels, as well as in tissue factor activity. Conversely, CpG DNA significantly reduced tissue factor pathway inhibitor transcription, secretion, and activity. Inhibition of Toll-like receptor 9 with a telomere-derived Toll-like receptor 9 inhibitory oligonucleotide or transient Toll-like receptor 9 knockdown with small interfering RNA attenuated human coronary artery endothelial cell responses to CpG DNA. In wild-type mice, CpG DNA shortened the bleeding time parallel with dramatic increases in plasma thrombin-antithrombin complex and tissue factor levels. Pretreatment with inhibitory oligonucleotide or anti-tissue factor antibody or genetic deletion of TLR9 prevented these changes, whereas depleting monocytes with clodronate resulted in a modest partial inhibition. CONCLUSIONS: Our findings demonstrate that bacterial DNA through Toll-like receptor 9 shifted the balance of tissue factor and tissue factor pathway inhibitor toward procoagulant phenotype in human coronary artery endothelial cells and activated blood coagulation in mice. Our study identifies Toll-like receptor 9 inhibitory oligonucleotides as potential therapeutic agents for the prevention of coagulation in pathologies where bacterial DNA may abundantly be present.