Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers 1,2 , and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation 3 . The precise mechanism coupling coagulation and inflammation remains unresolved 4-7 . Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1b to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.Disseminated intravascular coagulation and systemic inflammation are signs of excessive activation of the innate immune system. Both are attenuated by genetic reduction of tissue factor and its protease ligand coagulation factor VIIa, leading to improved survival in endotoxaemia 6,8 . In a model of severe, but not completely lethal lipopolysaccharide (LPS) challenge 9 , we show that PAR1 deficiency protects mice from lethality (Fig. 1a). PAR1 2/2 mice initially developed elevated inflammation and coagulation markers indistinguishable from the wild type (Fig. 1b, c). Unlike the wild type, PAR1 2/2 mice progressively resolved systemic inflammation beginning at 12 h. To address whether coagulation amplifies inflammation signalling. a, Survival advantage of PAR1 2/2 mice in 90% lethal LPS challenge induced by intraperitoneal injection of 8 mg kg -1 LPS (summary of three independent experiments, n $ 28 per genotype, PAR1 2/2 survival advantage for each individual experiment, P , 0.05). b, Reduced late-stage inflammation in PAR1 2/2 mice documented by IL-6 and IL-1b levels (mean 6 s.d., n 5 18 per group, asterisks indicate groups that are different from the wild type (WT), P , 0.05). c, TAT levels in wild-type and PAR1 2/2 mice, or wild-type mice treated at 10 h with PAR1 antagonist RWJ58259 (P1ant) or the thrombin inhibitor hirudin (Hir). d, Intervention with PAR1 antagonist or hirudin improves survival, similarly to PAR1 deficiency (n 5 8 per group, P , 0.02 relative to wild-type control). e, Intervention with P...

Tissue factor (TF), the initiator of the coagulation cascade, mediates coagulation factor VIIa-dependent activation of protease activated receptor-2 (PAR2). Here we delineate an unexpected role for coagulation signaling in obesity and its complications. Mice lacking PAR2 (F2rl1) or the cytoplasmic domain of TF (F3) are protected from high fat diet (HFD) induced weight gain and insulin resistance. In hematopoietic cells, genetic deletion of TF-PAR2 signaling reduces adipose tissue macrophage inflammation and specific pharmacological inhibition of macrophage TF signaling rapidly ameliorates insulin resistance. In contrast, non-hematopoietic cell TF-VIIa-PAR2 signaling specifically promotes obesity. Mechanistically, adipocyte TF cytoplasmic domain dependent VIIa signaling suppresses Akt phosphorylation with concordant adverse transcriptional changes of key regulators of obesity and metabolism. Pharmacological blockade of adipocyte TF in vivo reverses these effects of TF-VIIa signaling and rapidly improves energy expenditure. Thus, TF signaling is a potential therapeutic target to improve impaired metabolism and insulin resistance in obesity.

Thrombosis is initiated by tissue factor (TF), a coagulation cofactor/receptor expressed in the vessel wall, on myeloid cells, and on microparticles (MPs) with variable procoagulant activity. However, the molecular pathways that generate prothrombotic TF in vivo are poorly defined. The oxidoreductase protein disulfide isomerase (PDI) is thought to be involved in the activation of TF. Here, we found that in mouse myeloid cells, ATPtriggered signaling through purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7 receptor; encoded by P2rx7) induced activation (decryption) of TF procoagulant activity and promoted release of TF + MPs from macrophages and SMCs. The generation of prothrombotic MPs required P2X7 receptor-dependent production of ROS leading to increased availability of solvent-accessible extracellular thiols. An antibody to PDI with antithrombotic activity in vivo attenuated the release of procoagulant MPs. In addition, P2rx7 -/-mice were protected from TF-dependent FeCl 3 -induced carotid artery thrombosis. BM chimeras revealed that P2X7 receptor prothrombotic function was present in both hematopoietic and vessel wall compartments. In contrast, an alternative anti-PDI antibody showed activities consistent with cellular activation typically induced by P2X7 receptor signaling. This anti-PDI antibody restored TF-dependent thrombosis in P2rx7 -/-mice. These data suggest that PDI regulates a critical P2X7 receptor-dependent signaling pathway that generates prothrombotic TF, defining a link between inflammation and thrombosis with potential implications for antithrombotic therapy.

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Protease activated receptor 1 (PAR1) signaling can play opposing roles in sepsis, either promoting dendritic cell (DC)-dependent coagulation and inflammation or reducing sepsis lethality due to activated protein C (aPC) therapy. To further define this PAR1 paradox, we focused on the vascular effects of PAR1 signaling. Pharmacological perturbations of the intravascular coagulant balance were combined with genetic mouse models to dissect the roles of endogenously generated thrombin and aPC during escalating systemic inflammation. Acute blockade of the aPC pathway with a potent inhibitory antibody revealed that thrombin-PAR1 signaling increases inflammation-induced vascular hyperpermeability. Conversely, aPC-PAR1 signaling and the endothelial cell PC receptor (EPCR) prevented vascular leakage, and pharmacologic or genetic blockade of this pathway sensitized mice to LPSinduced lethality. Signaling-selective aPC variants rescued mice with defective PC activation from vascular leakage and lethality. Defects in the aPC pathway were fully compensated by sphingosine 1 phosphate receptor 3 (S1P3) deficiency or by selective agonists of the S1P receptor 1 (S1P1), indicating that PAR1 signaling contributes to setting the tone for the vascular S1P1/S1P3 balance. Thus, the activating proteases and selectivity in coupling to S1P receptor subtypes determine vascular PAR1 signaling specificity in systemic inflammatory response syndromes in vivo. (Blood. 2009; 113:2859-2866)