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.

IntroductionMicroparticles (MPs) are small (100-1000 nm) membrane-bound bodies that are released from cells during activation or cell death. 1 A crucial step in the MP formation is the loss of plasma membrane asymmetry leading to the exposure of phosphatidylserine (PS). 1 PS on the MPs allows their detection by flow cytometry using annexin V. In addition, flow cytometry can be used to determine the cell type that released the MPs because MPs possess cell surface markers of their cell origin. In addition to membrane-bound cell surface receptors, MPs can also contain mRNA, microRNA, cytokines, and growth factors. 2 Indeed, it was shown that endothelial cells incubated with MPs derived from cells expressing mRNA encoding green fluorescent protein subsequently expressed green fluorescent protein. 3 Thus, MPs can act as mediators of cell to cell communication, either locally or at a distance via the circulation.Although platelets are the primary source of MPs in the circulation under normal conditions, MPs released by monocytes are increased during experimental human and mouse endotoxemia 4,5 and systemic bacterial infections, 6 as well as other diseases. [7][8][9][10][11] It is thought that these monocyte MPs may contribute to disseminated intravascular coagulation, which often occurs during sepsis. The highly procoagulant nature of MPs is probably the result of the exposure of PS on the MP surface and the expression of tissue factor, the primary activator of the extrinsic coagulation cascade. Interestingly, elevated numbers of CD14-positive, tissue factor-positive MPs were found in a septic patient with disseminated intravascular coagulation. 6 Elevated proinflammatory cytokine production also occurs during endotoxemia and sepsis. One important proinflammatory cytokine up-regulated in response to bacterial infection and lipopolysaccharide (LPS) stimulation is IL-1␤. IL-1␤ is unusual because it does not contain an N-terminal signal sequence for secretion and therefore must be released from the cell via an alternative mechanism. In addition, it is synthesized as a larger precursor protein that must be cleaved into the active cytokine. Cleavage is mediated by an active inflammasome. The LPS-activated inflammasome contains the nucleotide-binding domain, leucine rich repeat containing protein (NLR) NLRP3, an adaptor molecule known as apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1. 12 The current model suggests that LPS induces a conformational change in NLRP3 that allows interaction with ASC, via homotypic pyrin domain interactions. 13 Importantly, it has previously been shown that IL-1␤ can be packaged and released in MPs and that this process requires adenosine triphosphate activation of P2 ϫ 7, a receptor required for inflammasome activation and IL-1␤ release. 14 Proinflammatory cytokines, such as IL-1␤ and TNF-␣, induce the expression of cell adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, on the endot...

IntroductionThe link between cancer and venous thromboembolism (VTE) is referred to as Trousseau syndrome. Interestingly, different cancer types are associated with different rates of VTE, with pancreatic cancer having one of the highest rates. 1,2 A VTE risk-scoring model has been developed that stratifies ambulatory cancer patients undergoing chemotherapy into 3 VTE risk categories based on 5 parameters: (1) the site of the primary tumor, (2) prechemotherapy leukocyte count, (3) platelet count, (4) hemoglobin level, and (5) body mass index. 3 Recently, this model was expanded to include the biomarkers D-dimer and soluble P-selectin. 4 Another potential circulating biomarker of VTE risk in pancreatic cancer patients is microparticle (MP) tissue factor (TF). [5][6][7][8][9] Full-length TF (flTF) is a transmembrane protein that activates the coagulation cascade. 10 In addition, an alternatively spliced form of TF (asTF) has been identified that lacks a membrane anchor and therefore can be released as a soluble protein. 11 Increased TF expression is correlated with poor prognosis in pancreatic cancer. [12][13][14] Cultured human pancreatic tumor lines express variable levels of both flTF and asTF and release TF-positive MPs containing flTF into the culture medium. [14][15][16][17][18][19] In some patients with pancreatic cancer, high levels of TF-positive MPs are found in the circulation and, in a small pilot study, were predictive of VTE. [5][6][7]9,20 In a mouse model of human colorectal tumors, human TF protein is released into the circulation. 21 In nude mice bearing orthotopic human pancreatic tumors (L3.6pl) plasma levels of human TF protein were correlated with the levels of thrombin-antithrombin (TAT) complex, a marker of the activation of coagulation. 22 Further, plasma from these tumor-bearing mice was found to enhance thrombin generation in vitro in a human TF-dependent manner. 22 Another study found that human (SOJ-4) and mouse (PANC02) pancreatic cell lines expressed TF, and the investigators observed an accumulation of tumor-derived MPs at the site of thrombosis and increased thrombosis in a microvascular model. 18 The objective of the present study was to determine the role of tumor-derived TF in the activation of coagulation and thrombosis in a xenograft mouse model of human pancreatic tumors. We found that only TF-positive tumors activated coagulation and that this activation was abolished by inhibition of human TF. Two TF-positive pancreatic tumor cell lines activated coagulation, but only one had detectable levels of circulating TF-positive MPs, which suggested that activation of coagulation was due to TF expression by the tumor itself rather than to TF on the MPs. Mice with elevated levels of TF-positive MPs exhibited increased thrombosis in a saphenous vein model, but not in an inferior vena cava (IVC) stenosis model. Methods Cell linesHuman pancreatic (MIAPaCa-2 [CRL-1420] Abs and proteinsPE-labeled mouse IgG control (#555574), mouse anti-human TF (#550312) and FITC-conjugated anti-human MUC...

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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.