The coagulant and inflammatory exacerbation in sepsis is counterbalanced by the protective protein C (PC) pathway. Activated PC (APC) was shown to use the endothelial cell PC receptor (EPCR) as a coreceptor for cleavage of protease activated receptor 1 (PAR1) on endothelial cells. Gene profiling demonstrated that PAR1 signaling could account for all APC-induced protective genes, including the immunomodulatory monocyte chemoattractant protein-1 (MCP-1), which was selectively induced by activation of PAR1, but not PAR2. Thus, the prototypical thrombin receptor is the target for EPCR-dependent APC signaling, suggesting a role for this receptor cascade in protection from sepsis.
Cell-surface tissue factor (TF) binds the serine protease factor VIIa to activate coagulation or, alternatively, to trigger signaling through the G protein-coupled, protease-activated receptor 2 (PAR2) relevant to inflammation and angiogenesis. Here we demonstrate that TF⅐VIIa-mediated coagulation and cell signaling involve distinct cellular pools of TF. The surface-accessible, extracellular Cys 186 -Cys 209 disulfide bond of TF is critical for coagulation, and protein disulfide isomerase (PDI) disables coagulation by targeting this disulfide. A TF mutant (TF C209A) with an unpaired Cys 186 retains TF⅐VIIa signaling activity, and it has reduced affinity for VIIa, a characteristic of signaling TF on cells with constitutive TF expression. We further show that PDI suppresses TF coagulant activity in a nitric oxide-dependent pathway, linking the regulation of TF thrombogenicity to oxidative stress in the vasculature. Furthermore, a unique monoclonal antibody recognizes only the noncoagulant, cryptic conformation of TF. This antibody inhibits formation of the TF⅐PAR2 complex and TF⅐VIIa signaling, but it does not prevent coagulation activation. These experiments delineate an upstream regulatory mechanism that controls TF function, and they provide initial evidence that TF⅐VIIa signaling can be specifically inhibited with minimal effects on coagulation.allosteric disulfide ͉ protein disulfide ͉ isomerase ͉ S-nitrosylation ͉ G protein-coupled receptor
Coagulation activation by tissue factor (TF) is implicated in cancer progression, cancer-associated thrombosis and metastasis. The role of direct TF signaling pathways in cancer, however, remains incompletely understood. Here we address how TF contributes to primary tumor growth by using a unique pair of isotype-matched antibodies that inhibit either coagulation (monoclonal antibody [Mab]-5G9) or direct signaling (Mab-10H10). We demonstrate that the inhibitory antibody of direct TF-VIIa signaling not only blocks TF-VIIa mediated activation of PAR2, but also disrupts the interaction of TF with integrins. In epithelial and TF-expressing endothelial cells, association of TF with beta1 integrins is regulated by TF extracellular ligand binding and independent of PAR2 signaling or proteolytic activity of VIIa. In contrast, alpha3beta1 integrin association of TF is constitutive in breast cancer cells and blocked by Mab-10H10 but not by Mab-5G9. Mab-5G9 has antitumor activity in vivo, but we show here that Mab-10H10 is at least as effective in suppressing human xenograft tumors in 2 different models. Breast tumor growth was also attenuated by blocking PAR2 signaling. These results show that tumor cell TF-PAR2 signaling is crucial for tumor growth and suggest that anti-TF strategies can be applied in cancer therapy with minor impairment of TF-dependent hemostatic pathways.
The crucial role of cell signaling in hemostasis is clearly established by the action of the downstream coagulation protease thrombin that cleaves platelet-expressed G-protein-coupled protease activated receptors (PARs). Certain PARs are cleaved by the upstream coagulation proteases factor Xa (Xa) and the tissue factor (TF)-factor VIIa (VIIa) complex, but these enzymes are required at high nonphysiological concentrations and show limited recognition specificity for the scissile bond of target PARs. However, defining a physiological mechanism of PAR activation by upstream proteases is highly relevant because of the potent anti-inflammatory in vivo effects of inhibitors of the TF initiation complex. Activation of substrate factor X (X) by the TF-VIIa complex is here shown to produce enhanced cell signaling in comparison to the TF-VIIa complex alone, free Xa, or Xa that is generated in situ by the intrinsic activation complex. Macromolecular assembly of X into a ternary complex of TF-VIIa-X is required for proteolytic conversion to Xa, and product Xa remains transiently associated in a TFVIIa-Xa complex. By trapping this complex with a unique inhibitor that preserves Xa activity, we directly show that Xa in this ternary complex efficiently activates PAR-1 and -2. These experiments support the concept that proinflammatory upstream coagulation protease signaling is mechanistically coupled and thus an integrated part of the TF-VIIa-initiated coagulation pathway, rather than a late event during excessive activation of coagulation and systemic generation of proteolytic activity.
Metastasis Is a multistep process which requires highly adapted Interactions of tumor cells with host target organs. Compared with nonmetastatic cells, ietstic human melanoma cells express 1000-fold higher levels of tissue factor (TF), the major cellular initiator of the plasma coagu- (11,12). Studies with the highly specific thrombin inhibitor hirudin suggest that generation of the pleiotropic serine protease thrombin may trigger essential events during metastasis of melanoma cells (13). However, other seine proteases of the coagulation system, such as factor VII, have also been suggested as potentially important host factors for metastasis (14). Indeed, factor VII at the site of implantation may bind to tissue factor (TF), its receptor and catalytic cofactor. TF can be expressed on host endothelial cells in response to induction by a tumor-secreted cytokine, vascular permeability factor (15,16
Hemostasis initiates angiogenesis-dependent wound healing, and thrombosis is frequently associated with advanced cancer. Although activation of coagulation generates potent regulators of angiogenesis, little is known about how this pathway supports angiogenesis in vivo. Here we show that the tissue factor (TF)-VIIa protease complex, independent of triggering coagulation, can promote tumor and developmental angiogenesis through protease-activated receptor-2 (PAR-2) signaling. In this context, the TF cytoplasmic domain negatively regulates PAR-2 signaling. Mice from which the TF cytoplasmic domain has been deleted (TF Delta CT mice) show enhanced PAR-2-dependent angiogenesis, in synergy with platelet-derived growth factor BB (PDGF-BB). Ocular tissue from diabetic patients shows PAR-2 colocalization with phosphorylated TF specifically on neovasculature, suggesting that phosphorylation of the TF cytoplasmic domain releases its negative regulatory control of PAR-2 signaling in angiogenesis. Targeting the TF-VIIa signaling pathway may thus enhance the efficacy of angiostatic treatments for cancer and neovascular eye diseases.
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...
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