Alterations to Sphingomyelin Metabolism Affect Hemostasis and Thrombosis.

BACKGROUND: Our recent studies suggest that sphingomyelin levels in the plasma membrane influence TF (tissue factor) procoagulant activity. The current study was performed to investigate how alterations to sphingomyelin metabolic pathway would affect TF procoagulant activity and thereby affect hemostatic and thrombotic processes. METHODS: Macrophages and endothelial cells were transfected with specific siRNAs or infected with adenoviral vectors to alter sphingomyelin levels in the membrane. TF activity was measured in factor X activation assay. Saphenous vein incision-induced bleeding and the inferior vena cava ligation-induced flow restriction mouse models were used to evaluate hemostasis and thrombosis, respectively. RESULTS: Overexpression of SMS (sphingomyelin synthase) 1 or SMS2 in human monocyte-derived macrophages suppresses ATP-stimulated TF procoagulant activity, whereas silencing SMS1 or SMS2 increases the basal cell surface TF activity to the same level as of ATP-decrypted TF activity. Consistent with the concept that sphingomyelin metabolism influences TF procoagulant activity, silencing of acid sphingomyelinase or neutral sphingomyelinase 2 or 3 attenuates ATP-induced enhanced TF procoagulant activity in macrophages and endothelial cells. Niemann-Pick disease fibroblasts with a higher concentration of sphingomyelin exhibited lower TF activity compared with wild-type fibroblasts. In vivo studies revealed that LPS+ATP-induced TF activity and thrombin generation were attenuated in ASMase-/- mice, while their levels were increased in SMS2-/- mice. Further studies revealed that acid sphingomyelinase deficiency leads to impaired hemostasis, whereas SMS2 deficiency increases thrombotic risk. CONCLUSIONS: Overall, our data indicate that alterations in sphingomyelin metabolism would influence TF procoagulant activity and affect hemostatic and thrombotic processes.

The Gab2-MALT1 axis regulates thromboinflammation and deep vein thrombosis.

Deep vein thrombosis (DVT) is the third most common cause of cardiovascular mortality. Several studies suggest that DVT occurs at the intersection of dysregulated inflammation and coagulation upon activation of inflammasome and secretion of interleukin 1ß (IL-1ß) in restricted venous flow conditions. Our recent studies showed a signaling adapter protein, Gab2 (Grb2-associated binder 2), plays a crucial role in propagating inflammatory signaling triggered by IL-1ß and other inflammatory mediators in endothelial cells. The present study shows that Gab2 facilitates the assembly of the CBM (CARMA3 [CARD recruited membrane-associated guanylate kinase protein 3]-BCL-10 [B-cell lymphoma 10]-MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) signalosome, which mediates the activation of Rho and NF-κB in endothelial cells. Gene silencing of Gab2 or MALT1, the effector signaling molecule in the CBM signalosome, or pharmacological inhibition of MALT1 with a specific inhibitor, mepazine, significantly reduced IL-1ß-induced Rho-dependent exocytosis of P-selectin and von Willebrand factor (VWF) and the subsequent adhesion of neutrophils to endothelial cells. MALT1 inhibition also reduced IL-1ß-induced NF-κB-dependent expression of tissue factor and vascular cell adhesion molecule 1. Consistent with the in vitro data, Gab2 deficiency or pharmacological inhibition of MALT1 suppressed the accumulation of monocytes and neutrophils at the injury site and attenuated venous thrombosis induced by the inferior vena cava ligation-induced stenosis or stasis in mice. Overall, our data reveal a previously unrecognized role of the Gab2-MALT1 axis in thromboinflammation. Targeting the Gab2-MALT1 axis with MALT1 inhibitors may become an effective strategy to treat DVT by suppressing thromboinflammation without inducing bleeding complications.

Selective inhibition of activated protein C anticoagulant activity protects against hemophilic arthropathy in mice.

Recurrent spontaneous or trauma-related bleeding into joints in hemophilia leads to hemophilic arthropathy (HA), a debilitating joint disease. Treatment of HA consists of preventing joint bleeding by clotting factor replacement, and in extreme cases, orthopedic surgery. We recently showed that administration of endothelial cell protein C receptor (EPCR) blocking monoclonal antibodies (mAb) markedly reduced the severity of HA in factor VIII (FVIII)-/- mice. EPCR blocking inhibits activated protein C (APC) generation and EPCR-dependent APC signaling. The present study was aimed to define the role of inhibition of APC anticoagulant activity, APC signaling, or both in suppressing HA. FVIII-/- mice were treated with a single dose of isotype control mAb, MPC1609 mAb, that inhibits anticoagulant, and signaling properties of APC, or MAPC1591 mAb that only blocks the anticoagulant activity of APC. Joint bleeding was induced by needle puncture injury. HA was evaluated by monitoring joint bleeding, change in joint diameter, and histopathological analysis of joint tissue sections for synovial hypertrophy, macrophage infiltration, neoangiogenesis, cartilage degeneration, and chondrocyte apoptosis. No significant differences were observed between MPC1609 and MAPC1591 in inhibiting APC anticoagulant activity in vitro and equally effective in correcting acute bleeding induced by the saphenous vein incision in FVIII-/- mice. Administration of MAPC1591, and not MPC1609, markedly reduced the severity of HA. MAPC1591 inhibited joint bleed-induced inflammatory cytokine interleukin-6 expression and vascular leakage in joints, whereas MPC1609 had no significant effect. Our data show that an mAb that selectively inhibits APC's anticoagulant activity without compromising its cytoprotective signaling offers a therapeutic potential alternative to treat HA.

SARS-CoV-2 infection induces the activation of tissue factor-mediated coagulation via activation of acid sphingomyelinase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with the hypercoagulable state. Tissue factor (TF) is the primary cellular initiator of coagulation. Most of the TF expressed on cell surfaces remains cryptic. Sphingomyelin (SM) is responsible for maintaining TF in the encrypted state, and hydrolysis of SM by acid sphingomyelinase (ASMase) increases TF activity. ASMase was shown to play a role in virus infection biology. In the present study, we investigated the role of ASMase in SARS-CoV-2 infection-induced TF procoagulant activity. Infection of human monocyte-derived macrophages (MDMs) with SARS-CoV-2 spike protein pseudovirus (SARS-CoV-2-SP-PV) markedly increased TF procoagulant activity at the cell surface and released TF+ extracellular vesicles. The pseudovirus infection did not increase either TF protein expression or phosphatidylserine externalization. SARS-CoV-2-SP-PV infection induced the translocation of ASMase to the outer leaflet of the plasma membrane, which led to the hydrolysis of SM in the membrane. Pharmacologic inhibitors or genetic silencing of ASMase attenuated SARS-CoV-2-SP-PV-induced increased TF activity. Inhibition of the SARS-CoV-2 receptor, angiotensin-converting enzyme-2, attenuated SARS-CoV-2-SP-PV-induced increased TF activity. Overall, our data suggest that SARS-CoV-2 infection activates the coagulation by decrypting TF through activation of ASMase. Our data suggest that the US Food and Drug Administration-approved functional inhibitors of ASMase may help treat hypercoagulability in patients with COVID-19.

Endothelial Cell Protein C Receptor Deficiency Attenuates Streptococcus pneumoniae-induced Pleural Fibrosis.

Streptococcus pneumoniae is the leading cause of hospital community-acquired pneumonia. Patients with pneumococcal pneumonia may develop complicated parapneumonic effusions or empyema that can lead to pleural organization and subsequent fibrosis. The pathogenesis of pleural organization and scarification involves complex interactions between the components of the immune system, coagulation, and fibrinolysis. EPCR (endothelial protein C receptor) is a critical component of the protein C anticoagulant pathway. The present study was performed to evaluate the role of EPCR in the pathogenesis of S. pneumoniae infection-induced pleural thickening and fibrosis. Our studies show that the pleural mesothelium expresses EPCR. Intrapleural instillation of S. pneumoniae impairs lung compliance and lung volume in wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. Intrapleural S. pneumoniae infection induces pleural thickening in wild-type mice. Pleural thickening is more pronounced in EPCR-overexpressing mice, whereas it is reduced in EPCR-deficient mice. Markers of mesomesenchymal transition are increased in the visceral pleura of S. pneumoniae-infected wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. The lungs of wild-type and EPCR-overexpressing mice administered intrapleural S. pneumoniae showed increased infiltration of macrophages and neutrophils, which was significantly reduced in EPCR-deficient mice. An analysis of bacterial burden in the pleural lavage, the lungs, and blood revealed a significantly lower bacterial burden in EPCR-deficient mice compared with wild-type and EPCR-overexpressing mice. Overall, our data provide strong evidence that EPCR deficiency protects against S. pneumoniae infection-induced impairment of lung function and pleural remodeling.

Oxidative Stress Product, 4-Hydroxy-2-Nonenal, Induces the Release of Tissue Factor-Positive Microvesicles From Perivascular Cells Into Circulation.

OBJECTIVE: TF (Tissue factor) plays a key role in hemostasis, but an aberrant expression of TF leads to thrombosis. The objective of the present study is to investigate the effect of 4-hydroxy-2-nonenal (HNE), the most stable and major oxidant produced in various disease conditions, on the release of TF+ microvesicles into the circulation, identify the source of TF+ microvesicles origin, and assess their effect on intravascular coagulation and inflammation. Approach and Results: C57BL/6J mice were administered with HNE intraperitoneally, and the release of TF+ microvesicles into circulation was evaluated using coagulation assays and nanoparticle tracking analysis. Various cell-specific markers were used to identify the cellular source of TF+ microvesicles. Vascular permeability was analyzed by the extravasation of Evans blue dye or fluorescein dextran. HNE administration to mice markedly increased the levels of TF+ microvesicles and thrombin generation in the circulation. HNE administration also increased the number of neutrophils in the lungs and elevated the levels of inflammatory cytokines in plasma. Administration of an anti-TF antibody blocked not only HNE-induced thrombin generation but also HNE-induced inflammation. Confocal microscopy and immunoblotting studies showed that HNE does not induce TF expression either in vascular endothelium or circulating monocytes. Microvesicles harvested from HNE-administered mice stained positively with CD248 and α-smooth muscle actin, the markers that are specific to perivascular cells. HNE was found to destabilize endothelial cell barrier integrity. CONCLUSIONS: HNE promotes the release of TF+ microvesicles from perivascular cells into the circulation. HNE-induced increased TF activity contributes to intravascular coagulation and inflammation.

A critical role of endothelial cell protein C receptor in the intestinal homeostasis in experimental colitis.

Crohn's disease and ulcerative colitis are the two forms of disorders of the human inflammatory bowel disease with unknown etiologies. Endothelial cell protein C receptor (EPCR) is a multifunctional and multiligand receptor, which is expressed on the endothelium and other cell types, including epithelial cells. Here, we report that EPCR is expressed in the colon epithelial cells, CD11c+, and CD21+/CD35+ myeloid cells surrounding the crypts in the colon mucosa. EPCR expression was markedly decreased in the colon mucosa during colitis. The loss of EPCR appeared to associate with increased disease index of the experimental colitis in mice. EPCR-/- mice were more susceptible to dextran sulfate sodium (DSS)-induced colitis, manifested by increased weight loss, macrophage infiltration, and inflammatory cytokines in the colon tissue. DSS treatment of EPCR-/- mice resulted in increased bleeding, bodyweight loss, anemia, fibrin deposition, and loss of colon epithelial and goblet cells. Administration of coagulant factor VIIa significantly attenuated the DSS-induced colon length shortening, rectal bleeding, bodyweight loss, and disease activity index in the wild-type mice but not EPCR-/- mice. In summary, our data provide direct evidence that EPCR plays a crucial role in regulating the inflammation in the colon during colitis.

Acid sphingomyelinase plays a critical role in LPS- and cytokine-induced tissue factor procoagulant activity.

Tissue factor (TF) is a cofactor for factor VIIa and the primary cellular initiator of coagulation. Typically, most TF on cell surfaces exists in a cryptic coagulant-inactive state but are transformed to a procoagulant form (decryption) following cell activation. Our recent studies in cell model systems showed that sphingomyelin (SM) in the outer leaflet of the plasma membrane is responsible for maintaining TF in an encrypted state in resting cells, and the hydrolysis of SM leads to decryption of TF. The present study was carried out to investigate the relevance of this novel mechanism in the regulation of TF procoagulant activity in pathophysiology. As observed in cell systems, administration of adenosine triphosphate (ATP) to mice enhanced lipopolysaccharide (LPS)-induced TF procoagulant activity in monocytes. Treatment of mice with pharmacological inhibitors of acid sphingomyelinase (ASMase), desipramine and imipramine, attenuated ATP-induced TF decryption. Interestingly, ASMase inhibitors also blocked LPS-induced TF procoagulant activity without affecting the LPS-induced de novo synthesis of TF protein. Additional studies showed that LPS induced translocation of ASMase to the outer leaflet of the plasma membrane and reduced SM levels in monocytes. Studies using human monocyte-derived macrophages and endothelial cells further confirmed the role of ASMase in LPS- and cytokine-induced TF procoagulant activity. Overall, our data indicate that LPS- or cytokine-induced TF procoagulant activity requires the decryption of newly synthesized TF protein by ASMase-mediated hydrolysis of SM. The observation that ASMase inhibitors attenuate TF-induced coagulation raises the possibility of their therapeutic use in treating thrombotic disorders associated with aberrant expression of TF.

Factor VIIa induces anti-inflammatory signaling via EPCR and PAR1.

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. ß-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 ß-arrestin-1 dependent pathway. The present study suggests new therapeutic potentials for FVIIa, which is currently in clinical use for treating bleeding disorders.

Endothelial cell protein C receptor-dependent signaling.

PURPOSE OF REVIEW: Endothelial cell protein C receptor (EPCR), a transmembrane glycoprotein present on the surface of endothelial cells and other cell types, is an essential component of the protein C (PC) anticoagulant system. EPCR is also shown to play a critical role in mediating activated protein C (APC)-induced cytoprotective signaling. The purpose of this review is to outline the mechanisms of EPCR-dependent cell signaling and discuss recent findings made in this area. RECENT FINDINGS: Recent studies showed that the cleavage of protease-activated receptor (PAR)1 at a noncanonical site by APC-EPCR or the canonical site by thrombin when PC occupies EPCR induces ß-arrestin-2-mediated biased cytoprotective signaling. Factor VIIa binding to EPCR is also shown to induce the cytoprotective signaling. EPCR is found to be a reliable surface marker for identifying human hematopoietic stem cells in culture. EPCR, binding to diverse ligands, is thought to play a role in the pathogenesis of severe malaria, immune functions, and cancer by either blocking the APC-mediated signaling or by mechanisms that are yet to be elucidated. SUMMARY: Recent studies provide a mechanistic basis to how EPCR contributes to PAR1-mediated biased signaling. EPCR may play a role in influencing a wide array of biological functions by binding to diverse ligands.

Sphingomyelin encrypts tissue factor: ATP-induced activation of A-SMase leads to tissue factor decryption and microvesicle shedding.

A majority of tissue factor (TF) on cell surfaces exists in an encrypted state with minimal to no procoagulant activity. At present, it is unclear whether limited availability of phosphatidylserine (PS) and/or a specific membrane lipid in the outer leaflet of the plasma membrane contributes to TF encryption. Sphingomyelin (SM) is a major phospholipid in the outer leaflet, and SM metabolism is shown to be altered in many disease settings that cause thrombotic disorders. The present study is carried out to investigate the effect of SM metabolism on TF activity and TF+ microvesicles (MVs) release. In vitro studies using TF reconstituted into liposomes containing varying molar ratios of SM showed that a high molar ratio of SM in the proteoliposomes inhibits TF coagulant activity. Treatment of macrophages with sphingomyelinase (SMase) that hydrolyzes SM in the outer leaflet results in increased TF activity at the cell surface and TF+ MVs release without increasing PS externalization. Adenosine triphosphate (ATP) stimulation of macrophages that activates TF and induces MV shedding also leads to translocation of acid-sphingomyelinase (A-SMase) to the plasma membrane. ATP stimulation increases the hydrolysis of SM in the outer leaflet. Inhibition of A-SMase expression or activity not only attenuates ATP-induced SM hydrolysis, but also inhibits ATP-induced TF decryption and TF+ MVs release. Overall, our novel findings show that SM plays a role in maintaining TF in an encrypted state in resting cells and hydrolysis of SM following cell injury removes the inhibitory effect of SM on TF activity, thus leading to TF decryption.

Intrapleural Adenoviral-mediated Endothelial Cell Protein C Receptor Gene Transfer Suppresses the Progression of Malignant Pleural Mesothelioma in a Mouse Model.

Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer with a high mortality rate as it responds poorly to standard therapeutic interventions. Our recent studies showed that expression of endothelial cell protein C receptor (EPCR) in MPM cells suppresses tumorigenicity. The present study was aimed to investigate the mechanism by which EPCR suppresses MPM tumor growth and evaluate whether EPCR gene therapy could suppress the progression of MPM in a mouse model of MPM. Measurement of cytokines from the pleural lavage showed that mice implanted with MPM cells expressing EPCR had elevated levels of IFNγ and TNFα compared to mice implanted with MPM cells lacking EPCR. In vitro studies demonstrated that EPCR expression renders MPM cells highly susceptible to IFNγ + TNFα-induced apoptosis. Intrapleural injection of Ad.EPCR into mice with an established MPM originating from MPM cells lacking EPCR reduced the progression of tumor growth. Ad.EPCR treatment elicited recruitment of macrophages and NK cells into the tumor microenvironment and increased IFNγ and TNFα levels in the pleural space. Ad.EPCR treatment resulted in a marked increase in tumor cell apoptosis. In summary, our data show that EPCR expression in MPM cells promotes tumor cell apoptosis, and intrapleural EPCR gene therapy suppresses MPM progression.

Role of tissue factor in Mycobacterium tuberculosis-induced inflammation and disease pathogenesis.

Tuberculosis (TB) is a chronic lung infectious disease characterized by severe inflammation and lung granulomatous lesion formation. Clinical manifestations of TB include hypercoagulable states and thrombotic complications. We previously showed that Mycobacterium tuberculosis (M.tb) infection induces tissue factor (TF) expression in macrophages in vitro. TF plays a key role in coagulation and inflammation. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth in the lung and dissemination to other organs. Wild-type C57BL/6 and transgenic mice expressing human TF, either very low levels (low TF) or near to the level of wild-type (HTF), in place of murine TF were infected with M.tb via aerosol exposure. Levels of TF expression, proinflammatory cytokines and thrombin-antithrombin complexes were measured post M.tb infection and mycobacterial burden in the tissue homogenates were evaluated. Our results showed that M.tb infection did not increase the overall TF expression in lungs. However, macrophages in the granulomatous lung lesions in all M.tb-infected mice, including low TF mice, showed increased levels of TF expression. Conspicuous fibrin deposition in the granuloma was detected in wild-type and HTF mice but not in low TF mice. M.tb infection significantly increased expression levels of cytokines IFN-γ, TNF-α, IL-6 and IL-1ß in lung tissues. However, no significant differences were found in proinflammatory cytokines among the three experimental groups. Mycobacterial burden in lungs and dissemination into spleen and liver were essentially similar in all three genotypes. Our data indicate, in contrast to that observed in acute bacterial infections, that TF-mediated coagulation and/or signaling does not appear to contribute to the host-defense in experimental tuberculosis.

4-Hydroxy-2-nonenal enhances tissue factor activity in human monocytic cells via p38 mitogen-activated protein kinase activation-dependent phosphatidylserine exposure.

OBJECTIVE: 4-hydroxy-2-nonenal (HNE) is one of the major aldehydes formed during lipid peroxidation and is believed to play a role in the pathogenesis of atherosclerosis. The objective of the present study is to investigate the effect of HNE on tissue factor (TF) procoagulant activity expressed on cell surfaces. APPROACH AND RESULTS: TF activity and antigen levels on intact cells were measured using factor Xa generation and TF monoclonal antibody binding assays, respectively. Exposure of phosphatidylserine on the cell surface was analyzed using thrombin generation assay or by binding of a fluorescent dye-conjugated annexin V. 2',7'-dichlorodihydrofluorescein diacetate was used to detect the generation of reactive oxygen species. Our data showed that HNE increased the procoagulant activity of unperturbed THP-1 cells that express traces of TF antigen, but had no effect on unperturbed endothelial cells that express no measurable TF antigen. HNE increased TF procoagulant activity but not TF antigen of both activated monocytic and endothelial cells. HNE treatment generated reactive oxygen species, activated p38 mitogen-activated protein kinase, and increased the exposure of phosphatidylserine at the outer leaflet in THP-1 cells. Treatment of THP-1 cells with an antioxidant, N-acetyl cysteine, suppressed the above HNE-induced responses and negated the HNE-mediated increase in TF activity. Blockade of p38 mitogen-activated protein kinase activation inhibited HNE-induced phosphatidylserine exposure and increased TF activity. CONCLUSIONS: HNE increases TF coagulant activity in monocytic cells through a novel mechanism involving p38 mitogen-activated protein kinase activation that leads to enhanced phosphatidylserine exposure at the cell surface.

Rab GTPases regulate endothelial cell protein C receptor-mediated endocytosis and trafficking of factor VIIa.

Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa.

Endothelial cell protein C receptor opposes mesothelioma growth driven by tissue factor.

The procoagulant protein tissue factor (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 tissue factor-mediated effects on cancer progression. In this study, we investigated the influence of tissue factor, 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 tissue factor, EPCR or PAR1, and an orthotopic nude mouse model of MPM. Intrapleural administration of MPM cells expressing tissue factor and PAR1 but lacking EPCR and PAR2 (F2RL1) generated large tumors in the pleural cavity. Suppression of tissue factor or PAR1 expression in these cells markedly reduced tumor growth. In contrast, tissue factor overexpression in nonaggressive MPM cells that expressed EPCR and PAR1 with minimal levels of tissue factor did not increase their limited tumorigenicity. More importantly, ectopic expression of EPCR in aggressive MPM cells attenuated their growth potential, whereas EPCR silencing in nonaggressive MPM cells engineered to overexpress tissue factor 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 tissue factor promotes tumor growth through PAR1, and they show how EPCR can attenuate the growth of tissue factor-expressing tumor cells.

Mycobacterium tuberculosis infection and tissue factor expression in macrophages.

A number of earlier studies reported the occurrence of thrombotic complications, particularly disseminated intravascular coagulation and deep vein thrombosis, in tuberculosis (TB) patients. The aberrant expression of tissue factor (TF), the primary activator of coagulation cascade, is known to be responsible for thrombotic disorders in many diseases including bacterial infections. Further, expression of TF by cells of the monocyte/macrophage lineage is also shown to contribute to the development and progression of local and systemic inflammatory reactions. In the present study, we have investigated whether Mycobacterium tuberculosis (Mtb) infection induces TF expression in macrophages, and various host and pathogenic factors responsible for TF expression. We have tested the effect of live virulent Mtb H37Rv, gamma-irradiated Mtb H37Rv (γ-Mtb) and various components derived from Mtb H37Rv on TF expression in macrophages. The data presented in the manuscript show that both live virulent Mtb and γ-Mtb treatments markedly increased TF activity in macrophages, predominantly in the CD14(+) macrophages. Detailed studies using γ-Mtb showed that the increased TF activity in macrophages following Mtb treatment is the result of TF transcriptional activation. The signaling pathways of TF induction by Mtb appears to be distinct from that of LPS-induced TF expression. Mtb-mediated TF expression is dependent on cooperation of CD14/TLR2/TLR4 and probably yet another unknown receptor/cofactor. Mtb cell wall core components, mycolyl arabinogalactan peptidoglycan (mAGP), phosphatidylinositol mannoside-6 (PIM6) and lipomannan (LM) were identified as factors responsible for induction of TF in the order of mAGP>PIM6>LM. A direct contact between bacteria and macrophage and not Mtb-released soluble factors is critical for TF induction by Mtb. In summary, our data show that Mtb induces TF expression in macrophages and Mtb signaling pathways that elicit TF induction require cooperation of multiple receptors, co-receptors/co-factors including Toll-like receptors. The importance of TF in granuloma formation and containment of Mtb is discussed.

Tissue factor pathway inhibitor attenuates the progression of malignant pleural mesothelioma in nude mice.

Malignant pleural mesothelioma (MPM) is a rare cancer that is refractory to current treatments. It is characterized by a robust deposition of transitional fibrin that is in part promoted by tumor cells. MPM cells express tissue factor (TF) and the tissue factor pathway inhibitor (TFPI), but their contribution to the pathogenesis of MPM has been unclear. We found that REN MPM cells fail to express TFPI. Based on the tumor growth-promoting properties of TF, we hypothesized that the stable transfection of TFPI into REN MPM cells would decrease their aggressiveness. We tested our hypothesis using in vitro, in vivo, and ex vivo analyses. TFPI knock-in decreased the proliferation, invasion, and TF activity of REN cells in vitro. REN TFPI knock-in cells, empty vector, and naive control cells were next injected intrapleurally into nude mice. The expression of TFPI significantly decreased tissue invasion, inflammation, and the deposition of fibrin and collagen associated with tumor tissue, pleural effusions, and tumor burden. In ex vivo analyses, REN cells were cultured from harvested tumors. The overexpression of TFPI was maintained in cells propagated from TFPI knock-in tumors, and attenuated the activation of Factor X and the invasiveness of tumor cells. These analyses demonstrate that TFPI reduces the aggressiveness of MPM in vitro and in vivo, and that its effect involves the inhibition of TF procoagulant activity. These observations suggest that the interactions of TF and TFPI represent a novel therapeutic target in the treatment of MPM.

c-Maf-dependent growth of Mycobacterium tuberculosis in a CD14(hi) subpopulation of monocyte-derived macrophages.

Macrophages are a major component of the innate immune response, comprising the first line of defense against various intracellular pathogens, including Mycobacterium tuberculosis. In this report, we studied the factors that regulate growth of M. tuberculosis H37Rv in subpopulations of human monocyte-derived macrophages (MDMs). In healthy donors, M. tuberculosis H37Rv grew 5.6-fold more rapidly in CD14(hi) MDMs compared with that in CD14(lo)CD16(+) MDMs. Compared with CD14(lo)CD16(+) cells, M. tuberculosis H37Rv-stimulated CD14(hi) monocytes produced more IL-10 and had increased mRNA expression for c-Maf, a transcription factor that upregulates IL-10 gene expression. c-Maf small interfering RNA (siRNA) inhibited IL-10 production and growth of M. tuberculosis in CD14(hi) cells. Compared with CD14(lo)CD16(+) monocytes, M. tuberculosis H37Rv-stimulated CD14(hi) cells had increased expression of 22 genes whose promoters contained a c-Maf binding site, including hyaluronan synthase 1 (HAS1). c-Maf siRNA inhibited HAS1 expression in M. tuberculosis-stimulated CD14(hi) monocytes, and HAS1 siRNA inhibited growth of M. tuberculosis in CD14(hi) MDMs. M. tuberculosis H37Rv upregulated expression of HAS1 protein and its product, hyaluronan, in CD14(hi) MDMs. We conclude that M. tuberculosis grows more rapidly in CD14(hi) than in CD14(lo)CD16(+) MDMs because CD14(hi) cells have increased expression of c-Maf, which increases production of two key factors (hyaluronan and IL-10) that promote growth of M. tuberculosis.

Cystine 186-cystine 209 disulfide bond is not essential for the procoagulant activity of tissue factor or for its de-encryption.

Tissue factor (TF) on cell surfaces resides mostly in a cryptic state. It is not entirely clear how cryptic TF differs from procoagulantly active TF and how deencryption occurs. Here, we critically evaluated the importance of cystine 186-cystine 209 (Cys186-Cys209) bond formation for TF procoagulant activity and its de-encryption. Chinese hamster ovary cells transfected with TF(C186S), TF(C209S), or TF(C186S/C209S) expressed little procoagulant activity at the cell surface. TF monoclonal antibody and activated factor VII (FVIIa) binding studies showed that little TF protein was present at the cell surface in cells expressing mutant TF. Similar data were obtained in human umbilical vein endothelial cells (HUVECs) transduced to express TF(C186S), TF(C209S), or TF(C186S/C209S). Analysis of TF activity in HUVECs expressing similar levels of wild-type TF and TF(C186S/C209S) showed that TF mutant in the presence of saturating concentrations of FVIIa exhibited similar coagulant activity as that of wild-type TF. More importantly, treatment of HUVECs expressing TF(C186S/C209S) with HgCl(2) or ionomycin increased the cell-surface TF activity to the same extent as that of the wild-type TF. Our data provide clear evidence that TF lacking the Cys186-Cys209 bond is coagulantly active once it is complexed with FVIIa, and TF de-encryption does not require Cys186-Cys209 disulfide bond formation.