IntroductionRecent studies from our laboratory 1,2 and others 3,4 have shown that factor VIIa (FVIIa), a clotting protease that binds to tissue factor (TF) and initiates the activation of the coagulation cascade, also binds to the endothelial cell protein C receptor (EPCR), a receptor for anticoagulant protein C/activated protein C (APC). EPCR controls coagulation by promoting the activation of protein C by thrombin-thrombomodulin complexes. 5 In addition to controlling coagulation, EPCR has been shown to modulate several nonhemostatic functions by supporting APC-induced protease activated receptor-1 (PAR1)-mediated cell signaling. [6][7][8][9][10][11][12][13] Although direct evidence for an association of FVIIa with EPCR in vivo is yet to come, several recent observations are a strong indication that FVIIa does in fact interact with EPCR in vivo. Both human and murine FVIIa administered to mice were shown to associate with endothelium, and blockade of EPCR with EPCR-specific antibodies was shown to prolong the human FVIIa circulatory-half life in mice. 2,14 Analysis of FVII, FVIIa, and soluble EPCR levels in a large group of healthy individuals revealed that those with the EPCR Gly variants, whose circulating levels of soluble EPCR were higher, had higher levels of circulating FVII and FVIIa, suggesting that EPCR in vivo serves as a reservoir for FVII. 15,16 At present, the physiologic importance of FVIIa's interaction with EPCR is not entirely clear. Our recent studies suggest that EPCR may play a role in the clearance and/or transport of FVIIa. 2 Although we are unable to find evidence for the modulation of FVIIa's coagulant activity by EPCR, 1 others have shown that FVIIa binding to EPCR on endothelial cells downregulates FVIIa's coagulant activity. 4 Similarly, EPCR was shown to down-regulate FVIIa generation on endothelial cells by reducing FVII accessibility to phospholipids at the cell surface. 17 Despite divergent views on the potential mechanisms by which APC binding to EPCR provides cytoprotective activity through PAR1-mediated cell signaling, it is generally believed that complex formation of APC with EPCR potentiates APC cleavage of PAR1, and that PAR1 activation is responsible for eliciting protective signaling responses. 6,13,[18][19][20] In agreement with this notion, APC was shown to cleave PAR1 on endothelial cells, and EPCRblocking antibodies that prevent APC binding to EPCR inhibited APC cleavage of PAR1. 18 In studies performed in a heterologous cell model system expressing transfected EPCR and PAR1 or PAR2 reporter constructs, we found no evidence that the FVIIa bound to EPCR was capable of cleaving either PAR1 or PAR2 or of inducing cell signaling. 1 In earlier studies, APC was shown to cleave PAR1 reporter constructs expressed in endothelial cells (EA.hy926 cells), but this cleavage required high concentrations of APC (75nM or higher) and was EPCR independent. 10,21 In the same studies, an APC-mediated protective effect was seen with much lower concentrations of APC, and this effect was E...

Efferocytosis by alveolar phagocytes (APs) is pivotal in maintenance of lung homeostasis. Increased efferocytosis by APs results in protection against lethal acute lung injury due to pulmonary infections whereas defective efferocytosis by APs results in chronic lung inflammation. In this report, we show that pulmonary delivery of Bacillus Calmette-Guerin (BCG) significantly enhances efferocytosis by APs. Increased efferocytosis by APs maintains lung homeostasis and protects mice against lethal influenza pneumonia. Intranasally treated wild type C57Bl/6 (WT) mice with BCG showed significant increase in APs efferocytosis in vivo compared to their PBS-treated counterparts. All BCG-treated WT mice survived lethal influenza A virus (IAV) infection whereas all PBS-treated mice succumbed. BCG-induced resistance was abrogated by depleting AP prior to IAV infection. BCG treatment increased uptake, and digestion/removal of apoptotic cells by APs. BCG significantly increased the expression of TIM4 on APs and increased expression of Rab5 and Rab7. We demonstrated that increased efferocytosis by APs through pulmonary delivery of BCG initiated rapid clearance of apoptotic cells from the alveolar space, maintained lung homeostasis, reduced inflammation and protected host against lethal IAV pneumonia.

The pro-coagulant protein Tissue Factor (TF, F3) is a powerful growth promoter in many tumors but its mechanism of action is not well understood. More generally, it is unknown whether hemostatic factors expressed on tumor cells influence TF-mediated effects on cancer progression. In this study, we investigated the influence of TF, endothelial cell protein C receptor (EPCR, PROCR) and protease activated receptor-1 (PAR1, F2R) on the growth of malignant pleural mesothelioma (MPM), using human MPM cells that lack or express TF, EPCR or PAR1 and an orthotopic nude mouse model of MPM. Intrapleural administration of MPM cells expressing TF and PAR1 but lacking EPCR and PAR2 (F2RL1) generated large tumors in the pleural cavity. Suppression of TF or PAR1 expression in these cells markedly reduced tumor growth. In contrast, TF overexpression in non-aggressive MPM cells that expressed EPCR and PAR1 with minimal levels of TF did not increase their limited tumorigenicity. More importantly, ectopic expression of EPCR in aggressive MPM cells attenuated their growth potential, whereas EPCR silencing in non-aggressive MPM cells engineered to overexpress TF increased their tumorigenicity. Immunohistochemical analyses revealed that EPCR expression in tumor cells reduced tumor cell proliferation and enhanced apoptosis. Overall, our results enlighten the mechanism by which TF promotes tumor growth through PAR1, and they show how EPCR can attenuate the growth of TF-expressing tumor cells.

Recent studies show that endothelial cell protein C receptor (EPCR) interacts with diverse ligands, in addition to its known ligands protein C and activated protein C (APC). We showed in earlier studies that procoagulant clotting factor VIIa (FVIIa) binds EPCR and downregulates EPCR-mediated anticoagulation and induces an endothelial barrier protective effect. Here, we investigated the effect of FVIIa's interaction with EPCR on endothelial cell inflammation and lipopolysaccharide (LPS)-induced inflammatory responses in vivo. Treatment of endothelial cells with FVIIa suppressed tumor necrosis factor α (TNF-α)- and LPS-induced expression of cellular adhesion molecules and adherence of monocytes to endothelial cells. Inhibition of EPCR or protease-activated receptor 1 (PAR1) by either specific antibodies or small interfering RNA abolished the FVIIa-induced suppression of TNF-α- and LPS-induced expression of cellular adhesion molecules and interleukin-6. β-Arrestin-1 silencing blocked the FVIIa-induced anti-inflammatory effect in endothelial cells. In vivo studies showed that FVIIa treatment markedly suppressed LPS-induced inflammatory cytokines and infiltration of innate immune cells into the lung in wild-type and EPCR-overexpressing mice, but not in EPCR-deficient mice. Mechanistic studies revealed that FVIIa treatment inhibited TNF-α-induced ERK1/2, p38 MAPK, JNK, NF-κB, and C-Jun activation indicating that FVIIa-mediated signaling blocks an upstream signaling event in TNFα-induced signaling cascade. FVIIa treatment impaired the recruitment of TNF-receptor-associated factor 2 into the TNF receptor 1 signaling complex. Overall, our present data provide convincing evidence that FVIIa binding to EPCR elicits anti-inflammatory signaling via a PAR1- and β-arrestin-1 dependent pathway. The present study suggests new therapeutic potentials for FVIIa, which is currently in clinical use for treating bleeding disorders.

Recent studies established that clotting factor VIIa (FVIIa) binds endothelial cell protein C receptor (EPCR). It has been speculated that FVIIa interaction with EPCR might augment the hemostatic effect of rFVIIa in therapeutic conditions. The present study is carried out to investigate the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of rFVIIa in hemophilia therapy. Active-site inhibited FVIIa, which is capable of binding to EPCR but has no ability to activate factor X, reduced the concentration of rFVIIa required to correct the bleeding following the saphenous vein injury in mouse hemophilia model systems. Higher doses of rFVIIa were required to restore hemostasis in EPCR overexpressing hemophilia mice compared to hemophilia mice expressing normal levels of EPCR. Administration of FVIII antibody induced only mild hemophilic bleeding in EPCR-deficient mice, which was corrected completely with a low dose of rFVIIa. Administration of therapeutic concentrations of rFVIIa increased plasma protein C levels in EPCR overexpressing mice, indicating the displacement of protein C from EPCR by rFVIIa. EPCR levels did not significantly alter the bioavailability of rFVIIa in plasma. Overall, our data indicate that EPCR levels influence the hemostatic effect of rFVIIa in treating hemophilia. Our present findings suggest that FVIIa displacement of anticoagulant protein C from EPCR that results in down-regulation of activated protein C generation and not the direct effect of EPCR-FVIIa on FX activation is the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of rFVIIa in hemophilia therapy.

Summary Background Recent studies have shown that factor VIIa binds to endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C. At present, the physiologic significance of FVIIa interaction with EPCR in vivo remains unclear. Objective: To investigate whether exogenously administered FVIIa, by binding to EPCR, induces a barrier protective effect in vivo. Methods Lipopolysaccharide (LPS)-induced vascular leakage in the lung and kidney, and vascular endothelial growth factor (VEGF)-induced vascular leakage in the skin, were used to evaluate the FVIIa-induced barrier protective effect. Wild-type, EPCR-deficient, EPCR-overexpressing and hemophilia A mice were used in the studies. Results Administration of FVIIa reduced LPS-induced vascular leakage in the lung and kidney; the FVIIa-induced barrier protective effect was attenuated in EPCR-deficient mice. The extent of VEGF-induced vascular leakage in the skin was highly dependent on EPCR expression levels. Therapeutic concentrations of FVIIa attenuated VEGF-induced vascular leakage in control mice but not in EPCR-deficient mice. Blockade of FVIIa binding to EPCR with a blocking mAb completely attenuated the FVIIa-induced barrier protective effect. Similarly, administration of protease-activated receptor 1 antagonist blocked the FVIIa-induced barrier protective effect. Hemophilic mice showed increased vascular permeability, and administration of therapeutic concentrations of FVIIa improved barrier integrity in these mice. Conclusions This is the first study to demonstrate that FVIIa binding to EPCR leads to a barrier protective effect in vivo. This finding may have clinical relevance, as it indicates additional advantages of using FVIIa in treating hemophilic patients.

Summary Background Recombinant factor VIIa (rFVIIa) has been used widely for treating hemophilia patients with inhibitory autoantibodies against factor VIII or IX. Its mechanism of action is not entirely known. A majority of in vitro studies suggested that pharmacological concentrations of rFVIIa restore hemostasis in hemophilia in a phospholipid-dependent mechanism, independent of tissue factor (TF). However, a few studies suggested that a TF-dependent mechanism plays a primary role in rFVIIa correction of bleeding in hemophilia patients. Here, we investigated the potential contribution of TF in rFVIIa-induced hemostasis in hemophilia employing a model system of FVIII antibody-induced hemophilia in TF transgenic mice. Methods Mice expressing low levels of human TF (LTF mice), relatively high levels of human TF (HTF mice) or wild-type mice (WT mice) were administered with neutralizing anti-FVIII antibodies to induce hemophilia in these mice. The mice were then treated with varying concentrations of rFVIIa. rFVIIa-induced hemostasis was evaluated with the saphenous vein bleeding model. Results Administration of FVIII inhibitory antibodies induced the hemophilic bleeding phenotype in all three genotypes. rFVIIa administration rescued the bleeding phenotype in all three genotypes. No significant differences were observed in rFVIIa-induced correction in the bleeding of LTF and HTF mice administered with FVIII antibodies. Conclusions Our results provide strong evidence supporting that the hemostatic effect of pharmacological doses of rFVIIa stems from a TF-independent mechanism.

Cigarette smoke has been associated with susceptibility to different pulmonary and airway diseases. Impaired alveolar macrophages (AMs) that are major phagocytes in the lung have been associated with patients with airway diseases and active smokers. In the current report, we show that exposure to second-hand cigarette smoke (SHS) significantly reduced efferocytosis in vivo. More importantly, delivery of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) to the alveolar space restored and refurbished the efferocytosis capability of AMs. Exposure to SHS significantly reduced expression of CD16/32 on AMs, and treatment with GM-CSF not only restored but also significantly increased the expression of CD16/32 on AMs. GM-CSF treatment increased uptake and digestion/removal of apoptotic cells by AMs. The latter was attributed to increased expression of Rab5 and Rab7. Increased efferocytosis of AMs was also tested in a disease condition. AMs from GM-CSF-treated, influenza-infected, SHS-exposed mice showed significantly better efferocytosis activity, and mice had significantly less morbidity compared with phosphate-buffered saline-treated group. GM-CSF-treated mice had increased amphiregulin levels in the lungs, which in addition to efferocytosis of AMs may have attributed to their protection against influenza. These results will have great implications for developing therapeutic approaches by harnessing mucosal innate immunity to treat lung and airway diseases and protect against pneumonia.