Inflammatory cytokines alter the hemostatic balance of endothelial cells (ECs). Alternatively spliced human tissue factor (asHTF), a soluble isoform of tissue factor (TF), has recently been detected in ECs, possibly contributing to procoagulability. Agonists regulating asHTF expression and release are yet unknown. This study examines the effect of TNF-α and IL-6 on the endothelial expression of both TF variants and delineates the impact of asHTF on the procoagulability of extracellular fluids. asHTF and TF mRNA were assessed by real-time PCR, and asHTF, TF, and tissue factor pathway inhibitor (TFPI) proteins by Western blot and fluorescence microscopy before and after stimulation with TNF-α (10 ng/mL) or IL-6 (10 ng/L). The procoagulability of cell supernatant was analyzed by a chromogenic assay with or without phospholipid vesicles. We found asHTF mRNA to be maximally increased 10 minutes after TNF-α and 40 minutes after IL-6 treatment (asHTF/GAPDH ratio 0.0223±0.0069 versus 0.0012±0.0006 for control, P <0.001 and 0.0022±0.0004 versus 0.0012±0.0007, P <0.05, respectively). Not only was asHTF increased, but also TFPI decreased after cytokine treatment. asHTF was found in the supernatant as early as 5 hours after TNF-α stimulation, supporting factor Xa generation after relipidation (6.55±1.13 U versus 2.99±0.59 U in control supernatant, P <0.00001). Removal of asHTF from supernatants by immunoprecipitation diminished its procoagulability to baseline. The soluble TF isoform expressed and released from ECs in response to inflammatory cytokines becomes procoagulant in the presence of phospholipids. Thus, asHTF released from ECs is a marker for and a contributor to imbalanced hemostasis.

The coagulation cascade is activated during viral infections. This response may be part of the host defense system to limit spread of the pathogen. However, excessive activation of the coagulation cascade can be deleterious. In fact, inhibition of the tissue factor/factor VIIa complex reduced mortality in a monkey model of Ebola hemorrhagic fever. Other studies showed that incorporation of tissue factor into the envelope of herpes simplex virus increases infection of endothelial cells and mice. Furthermore, binding of factor X to adenovirus serotype 5 enhances infection of hepatocytes but also increases the activation of the innate immune response to the virus. Coagulation proteases activate protease-activated receptors (PARs). Interestingly, we and others found that PAR1 and PAR2 modulate the immune response to viral infection. For instance, PAR1 positively regulates TLR3-dependent expression of the antiviral protein interferon β, whereas PAR2 negatively regulates expression during coxsackievirus group B infection. These studies indicate that the coagulation cascade plays multiple roles during viral infections.

The blood coagulation system and immune system of higher organisms are thought to have a common ancestral origin. During infections, the blood coagulation system is activated and components of the hemostatic system are directly involved in the immune response and immune system modulations. The current view is that the activation of coagulation is beneficial for infections with bacteria and viruses. It limits pathogen dissemination and supports pathogen killing and tissue repair. On the other hand, over‐activation can lead to thrombosis with subsequent depletion of hemostatic factors and secondary bleeding. This review will summarize the current knowledge on blood coagulation and pathogen infection with focus on most recent studies of the role of the different parts of the blood coagulation system in selected bacterial and viral infections.

Tissue factor (TF) is the primary activator of the coagulation cascade. During endotoxemia, TF expression leads to disseminated intravascular coagulation. However, the relative contribution of TF expression by different cell types to the activation of coagulation has not been defined. In this study, we investigated the effect of either a selective inhibition of TF expression or cell type-specific deletion of the TF gene (F3) on activation of coagulation in a mouse model of endotoxemia. We found that inhibition of TF on either hematopoietic or nonhematopoietic cells reduced plasma thrombin-antithrombin (TAT) levels 8 hours after administration of bacterial lipopolysaccharide (LPS). In addition, plasma TAT levels were significantly reduced in endotoxemic mice lacking the TF gene in either myeloid cells (TFflox/flox,LysMCre mice) or in both endothelial cells (ECs) and hematopoietic cells (TFflox/flox,Tie-2Cre mice). However, deletion of the TF gene in ECs alone had no effect on LPS-induced plasma TAT levels. Similar results were observed in mice lacking TF in vascular smooth muscle cells. Finally, we found that mouse platelets do not express TF pre-mRNA or mRNA. Our data demonstrate that in a mouse model of endotoxemia activation of the coagulation cascade is initiated by TF expressed by myeloid cells and an unidentified nonhematopoietic cell type(s).

Hypercholesterolemia is a major risk factor for atherosclerosis. It also is associated with platelet hyperactivity, which increases morbidity and mortality from cardiovascular disease. However, the mechanisms by which hypercholesterolemia produces a procoagulant state remain undefined. Atherosclerosis is associated with accumulation of oxidized lipoproteins within atherosclerotic lesions. Small quantities of oxidized lipoproteins are also present in the circulation of patients with coronary artery disease. We therefore hypothesized that hypercholesterolemia leads to elevated levels of oxidized LDL (oxLDL) in plasma and that this induces expression of the procoagulant protein tissue factor (TF) in monocytes. In support of this hypothesis, we report here that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma microparticle (MP) TF activity. Furthermore, a high-fat diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor–deficient mice and African green monkeys. Genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte-derived TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity. Simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hypercholesterolemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6 complex.

Coagulation is a host defense system that limits the spread of pathogens. Coagulation proteases, such as thrombin, also activate cells by cleaving PARs. In this study, we analyzed the role of PAR-1 in coxsackievirus B3–induced (CVB3-induced) myocarditis and influenza A infection. CVB3-infected Par1^–/– mice expressed reduced levels of IFN-β and CXCL10 during the early phase of infection compared with Par1^+/+ mice that resulted in higher viral loads and cardiac injury at day 8 after infection. Inhibition of either tissue factor or thrombin in WT mice also significantly increased CVB3 levels in the heart and cardiac injury compared with controls. BM transplantation experiments demonstrated that PAR-1 in nonhematopoietic cells protected mice from CVB3 infection. Transgenic mice overexpressing PAR-1 in cardiomyocytes had reduced CVB3-induced myocarditis. We found that cooperative signaling between PAR-1 and TLR3 in mouse cardiac fibroblasts enhanced activation of p38 and induction of IFN-β and CXCL10 expression. Par1^–/– mice also had decreased CXCL10 expression and increased viral levels in the lung after influenza A infection compared with Par1^+/+ mice. Our results indicate that the tissue factor/thrombin/PAR-1 pathway enhances IFN-β expression and contributes to the innate immune response during single-stranded RNA viral infection.