Sepsis is associated with a systemic activation of coagulation and an excessive inflammatory response. Anticoagulants have been shown to inhibit both coagulation and inflammation in sepsis. In this study, we used both genetic and pharmacologic approaches to analyze the role of tissue factor and protease-activated receptors in coagulation and inflammation in a mouse endotoxemia model. We used mice expressing low levels of the procoagulant molecule, tissue factor (TF), to analyze the effects of TF deficiency either in all tissues or selectively in hematopoietic cells. Low TF mice had reduced coagulation, inflammation, and mortality compared with control mice. Similarly, a deficiency of TF expression by hematopoietic cells reduced lipopolysaccharide (LPS)-induced coagulation, inflammation, and mortality. Inhibition of the downstream coagulation protease, thrombin, reduced fibrin deposition and prolonged survival without affecting inflammation. Deficiency of either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) alone did not affect inflammation or survival. However, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality. These data demonstrate that hematopoietic cells are the major pathologic site of TF expression during endotoxemia and suggest that multiple protease-activated receptors mediate crosstalk between coagulation and inflammation.

Fetal loss in patients with antiphospholipid (aPL) antibodies has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically activation of complement with generation of the anaphylotoxin C5a, is an essential trigger of fetal injury. In this study, we analyzed the role of the procoagulant molecule tissue factor (TF) in a mouse model of aPL antibody-induced pregnancy loss. We found that either blockade of TF with a monoclonal antibody in wild-type mice or a genetic reduction of TF prevented aPL antibodyinduced inflammation and pregnancy loss. In response to aPL antibody-generated C5a, neutrophils express TF potentiating inflammation in the deciduas and leading to miscarriages. Importantly, we showed that TF in myeloid cells but not fetal-derived cells (trophoblasts) was associated with fetal injury, suggesting that the site for pathologic TF expression is neutrophils. We found that TF expression in neutrophils contributes to respiratory burst and subsequent trophoblast injury IntroductionThrombosis and inflammation are linked in many clinical conditions. 1 Tissue factor (TF), the major cellular initiator of the coagulation protease cascade, plays important roles in both thrombosis and inflammation. 2 The coagulation cascade is initiated by the complex of TF and factor VIIa (FVIIa). The TF:FVIIa complex activates its substrates factor X and factor IX by limited proteolysis. Activated FX (FXa) then converts prothrombin to thrombin, which cleaves fibrinogen and activates platelets leading to the formation of a hemostatic plug. TF also contributes to inflammation. TF complexes (TF:FVIIa and TF:FVIIa:FXa) induce the expression of TNF-␣, interleukins, and adhesion molecules by cleaving protease activated receptors (PARs). [3][4][5] Monocytes from patients with antiphospholipid (aPL) antibodies express TF and in vitro experiments showed that monocytes incubated with aPL antibodies express TF. 6,7 A variety of inflammatory stimuli, including mitogens, bacterial cell products, components of the complement system, and cytokines, is known to induce the expression of TF on the surface of endothelial cells, monocytes, and neutrophils. 8,9 TF expression on these cells is a characteristic feature of acute and chronic inflammation in conditions such as sepsis, atherosclerosis, Crohn disease, systemic lupus erythematosus, and transplant rejection reactions. 10-14 TF on monocytes and synovial cells promotes leukocyte adhesion and transendothelial migration, potentiating inflammation in joints, 15 while decreased TF activity abrogates the systemic expression of inflammatory mediators in several animal models. 16,17 The antiphospholipid syndrome (APS) is considered a thrombophilic disorder. However, animal studies from our laboratory have shown the importance of inflammation in the pathogenesis of aPL-induced pregnancy loss, a common complication in APS. 18,19 Using a mouse model of APS, we demonstrated that complement activation, through the action of anaphylotoxin C5a, promotes neutrop...

LPS stimulation of monocytes/macrophages induces the expression of genes encoding proinflammatory cytokines and the procoagulant protein, tissue factor. Induction of these genes is mediated by various signaling pathways, including mitogen-activated protein kinases, and several transcription factors, including Egr-1, AP-1, ATF-2, and NF-κB. We used a genetic approach to determine the role of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) pathway in the regulation of LPS signaling and gene expression in isolated macrophages and in mice. The PI3K-Akt pathway is negatively regulated by the phosphatase and tensin homologue (PTEN). We used peritoneal exudate cells from Pik3r1-deficient mice, which lack the p85α regulatory subunit of PI3K and have reduced PI3K activity, and peritoneal macrophages from PTENflox/flox/LysMCre mice (PTEN−/−), which have increased Akt activity. Analysis of LPS signaling in Pik3r1−/− and PTEN−/− cells indicated that the PI3K-Akt pathway inhibited activation of the ERK1/2, JNK1/2, and p38 mitogen-activated protein kinases and reduced the levels of nuclear Egr-1 protein and phosphorylated ATF-2. Modulating the PI3K-Akt pathway did not affect LPS-induced degradation of IκBα or NF-κB nuclear translocation. LPS induction of TNF-α, IL-6, and tissue factor gene expression was increased in Pik3r1−/− peritoneal exudate cells and decreased in PTEN−/− peritoneal macrophages compared with wild-type (WT) cells. Furthermore, LPS-induced inflammation and coagulation were enhanced in WT mice containing Pik3r1−/− bone marrow compared with WT mice containing WT bone marrow and in mice lacking the p85α subunit in all cells. Taken together, our results indicate that the PI3K-Akt pathway negatively regulates LPS signaling and gene expression in monocytes/macrophages.

Objective-In endotoxemia, lipopolysaccharide (LPS) induces a systemic inflammatory response and intravascular coagulation. Monocytes orchestrate the innate immune response to LPS by expressing a variety of pro-inflammatory cytokines, such as tumor necrosis factor-␣ (TNF-␣), and the procoagulant molecule, tissue factor (TF). In this study, we analyzed the role of the phosphoinositide 3-kinase (PI3K)-Akt pathway in the activation of coagulation and the innate immune response in a mouse model of endotoxemia. Methods and Results-Wortmannin and LY294002 were used to inhibit the PI3K-Akt pathway. We found that wortmannin inhibited LPS-induced Akt phosphorylation in blood cells. Inhibition of the PI3K-Akt pathway significantly increased TF mRNA expression in blood cells, TF antigen, and thrombin-antithrombin III levels in the plasma, and fibrin deposition in the liver of endotoxemic mice. Inhibition of the PI3K-Akt pathway also strongly enhanced LPS-induced cytokine expression and the levels of soluble E-selectin in the plasma, suggesting enhanced activation of both monocytes and endothelial cells. Wortmannin treatment also increased the number of macrophages in the liver and kidney of endotoxemic mice. Finally, wortmannin and LY294002 dramatically reduced the survival time of endotoxemic mice. Conclusions-These data suggest that the PI3K-Akt pathway suppresses LPS-induced inflammation and coagulation in endotoxemic mice.

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