Genetic duplication of tissue factor reveals subfunctionalization in venous and arterial hemostasis.

Tissue factor (TF) is an evolutionarily conserved protein necessary for initiation of hemostasis. Zebrafish have two copies of the tissue factor gene (f3a and f3b) as the result of an ancestral teleost fish duplication event (so called ohnologs). In vivo physiologic studies of TF function have been difficult given early lethality of TF knockout in the mouse. We used genome editing to produce knockouts of both f3a and f3b in zebrafish. Since ohnologs arose through sub- or neofunctionalization, they can unmask unknown functions of non-teleost genes and could reveal whether mammalian TF has developmental functions distinct from coagulation. Here we show that a single copy of either f3a or f3b is necessary and sufficient for normal lifespan. Complete loss of TF results in lethal hemorrhage by 2-4 months despite normal embryonic and vascular development. Larval vascular endothelial injury reveals predominant roles for TFa in venous circulation and TFb in arterial circulation. Finally, we demonstrate that loss of TF predisposes to a stress-induced cardiac tamponade independent of its role in fibrin formation. Overall, our data suggest partial subfunctionalization of TFa and TFb. This multigenic zebrafish model has the potential to facilitate study of the role of TF in different vascular beds.

Tissue factor cytoplasmic domain exacerbates post-infarct left ventricular remodeling via orchestrating cardiac inflammation and angiogenesis.

The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and is implicated in cardiac pathology, the contribution of its cytoplasmic domain to post-infarct myocardial injury and adverse left ventricular (LV) remodeling remains unknown. Methods: Myocardial infarction was induced in wild-type mice or mice lacking the TF cytoplasmic domain (TF∆CT) by occlusion of the left anterior descending coronary artery. Heart function was monitored with echocardiography. Heart tissue was collected at different time-points for histological, molecular and flow cytometry analysis. Results: Compared with wild-type mice, TF∆CT had a higher survival rate during a 28-day follow-up after myocardial infarction. Among surviving mice, TF∆CT mice had better cardiac function and less LV remodeling than wild-type mice. The overall improvement of post-infarct cardiac performance in TF∆CT mice, as revealed by speckle-tracking strain analysis, was attributed to reduced myocardial deformation in the peri-infarct region. Histological analysis demonstrated that TF∆CT hearts had in the infarct area greater proliferation of myofibroblasts and better scar formation. Compared with wild-type hearts, infarcted TF∆CT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1) - Rac1 axis. In particular, infarcted TF∆CT hearts displayed markedly lower ratios of inflammatory M1 macrophages and reparative M2 macrophages (M1/M2). In vitro experiment with primary macrophages demonstrated that deletion of the TF cytoplasmic domain inhibited macrophage polarization toward the M1 phenotype. Furthermore, infarcted TF∆CT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Finally, the overall cardioprotective effects observed in TF∆CT mice could be abolished by subcutaneously infusing a cocktail of PAR1-activating peptide and PAR2-inhibiting peptide via osmotic minipumps. Conclusions: Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation.

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.

Myeloid cell-derived coagulation tissue factor is associated with renal tubular damage in mice fed an adenine diet.

Patients with chronic kidney disease (CKD) commonly exhibit hypercoagulability. Increased levels of uremic toxins cause thrombogenicity by increasing tissue factor (TF) expression and activating the extrinsic coagulation cascade. TF is induced in monocytes and macrophages under pathological conditions, such as inflammatory diseases. However, the role of monocyte myeloid cell TF in CKD progression remains unclear. We aimed to clarify this issue, and the present study found that patients with CKD had elevated levels of D-dimer, a marker of fibrin degradation, which was associated with decreased estimated glomerular filtration rate and increased serum levels of uremic toxins, such as indoxyl sulfate. In vitro studies showed that several uremic toxins increased cellular TF levels in monocytic THP-1 cells. Mice with TF specifically deleted in myeloid cells were fed an adenine diet to cause uremic kidney injury. Myeloid TF deletion reduced tubular injury and pro-inflammatory gene expression in the kidneys of adenine-induced CKD but did not improve renal function as measured by plasma creatinine or blood urea nitrogen. Collectively, our findings suggest a novel concept of pathogenesis of coagulation-mediated kidney injury, in which elevated TF levels in monocytes under uremic conditions is partly involved in the development of CKD.

Structure-based design and discovery of novel anti-tissue factor antibodies with cooperative double-point mutations, using interaction analysis.

The generation of a wide range of candidate antibodies is important for the successful development of drugs that simultaneously satisfy multiple requirements. To find cooperative mutations and increase the diversity of mutants, an in silico double-point mutation approach, in which 3D models of all possible double-point mutant/antigen complexes are constructed and evaluated using interaction analysis, was developed. Starting from an antibody with very high affinity, four double-point mutants were designed in silico. Two of the double-point mutants exhibited improved affinity or affinity comparable to that of the starting antibody. The successful identification of two active double-point mutants showed that a cooperative mutation could be found by utilizing information regarding the interactions. The individual single-point mutants of the two active double-point mutants showed decreased affinity or no expression. These results suggested that the two active double-point mutants cannot be obtained through the usual approach i.e. a combination of improved single-point mutants. In addition, a triple-point mutant, which combines the distantly located active double-point mutation and an active single-point mutation collaterally obtained in the process of the double-point mutation strategy, was designed. The triple-point mutant showed improved affinity. This finding suggested that the effects of distantly located mutations are independent and additive. The double-point mutation approach using the interaction analysis of 3D structures expands the design repertoire for mutants, and hopefully paves a way for the identification of cooperative multiple-point mutations.

Beyond thrombosis: the impact of tissue factor signaling in cancer.

Tissue factor (TF) is the primary initiator of the coagulation cascade, though its effects extend well beyond hemostasis. When TF binds to Factor VII, the resulting TF:FVIIa complex can proteolytically cleave transmembrane G protein-coupled protease-activated receptors (PARs). In addition to activating PARs, TF:FVIIa complex can also activate receptor tyrosine kinases (RTKs) and integrins. These signaling pathways are utilized by tumors to increase cell proliferation, angiogenesis, metastasis, and cancer stem-like cell maintenance. Herein, we review in detail the regulation of TF expression, mechanisms of TF signaling, their pathological consequences, and how it is being targeted in experimental cancer therapeutics.

Comparison of native and citrated whole blood samples for rapid thromboelastography in Beagles.

OBJECTIVE: To examine the extent to which rapid thromboelastography (r-TEG) could decrease the testing time in comparison with that required for kaolin-activated thromboelastography (TEG), and to compare 2 types of blood samples (ie, native and citrated whole blood [WB]), for determining r-TEG values in healthy dogs. DESIGN: Prospective observational study. SETTING: University teaching hospital. ANIMALS: Sixteen healthy Beagles. INTERVENTIONS: Kaolin-activated TEG test using citrated WB samples and r-TEG test using native and citrated WB samples were performed in 16 dogs. At 60 minutes after the initial blood sampling, further samples were collected from a subset of 6 dogs in the same manner to evaluate intraindividual repeatability of r-TEG. MEASUREMENTS AND MAIN RESULTS: The mean time to maximum amplitude (MA) for r-TEG with native and citrated WB samples was recorded as 1313.9 ± 250.9 seconds and 1351.3 ± 264.6 seconds (mean ± SD), respectively, and 1779.9 ± 197.0 seconds for kaolin-activated TEG. Coefficients of variation with native and citrated WB samples for r-TEG values, TEG-activated clotting time, clot formation time, α angle, and MA, were determined to be 13.4% versus 18.8%, 11.1% versus 16.6%, 4.2% versus 5.1%, and 10.0% versus 10.0%, respectively. Intraindividual variations were lower for native WB samples than for citrated WB samples. CONCLUSIONS: The r-TEG test significantly decreased the mean time to MA compared with the kaolin-activated TEG test. In addition, native WB samples showed lower coefficients of variation and intraindividual variation than citrated WB samples in r-TEG analysis; this suggests that native WB samples can provide more consistent results. Therefore, the r-TEG method using native WB samples is recommended for assessment of dogs' hemostatic status when an early diagnosis is required.

Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.

Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.

Tumor microenvironment mediated by suppression of autophagic flux drives liver malignancy.

The physiological role of autophagy in the catabolic process of the body involves protein synthesis and degradation in homeostasis under normal and stressed conditions. In hepatocellular carcinoma (HCC), the role of tumor microenvironment (TME) has been concerned as the main issue in fighting against this deadly malignancy. During the last decade, the crosstalk between tumor cells and their TME in HCC extensively accumulated. However, a deeper knowledge for the actual function of autophagy in this interconnection which involved in supporting tumor development, progression and chemoresistance in HCC is needed but still largely unknown. Recent studies have shown that coagulants tissue factor (TF) and factor VII (FVII) has a pathological role in promoting tumor growth by activating protease-activated receptor 2 (PAR2). Autophagy-associated LC3A/B-II formation was selectively suppressed by FVII/PAR2 signaling which mediated by mTOR activation through Atg7 but not Atg5/Atg12 axis. The coagulant-derived autophagic suppression seemed potentiate a vicious circle of malignancy in producing more FVII and PAR2 which facilitate in vivo and in vitro tumor progression of HCC and the investigations are consistent with the clinical observations. In this review, we briefly summarize the current understanding of autophagy and discuss recent evidence for its role in HCC malignancy.

Effect of Tissue Factor on Colorectal Cancer Stem Cells.

BACKGROUND/AIM: Tissue factor (TF) expression increases cancer stem cell (CSC) activity in breast and lung cancer. There are ongoing studies focused on targeting CSCs via anti-TF treatment, for breast and lung cancer therapy. Herein, the aim was to determine whether targeting TF could have an anti-CSC therapeutic role in colorectal cancer (CRC). MATERIALS AND METHODS: Evaluation of colonosphere-forming efficiency (CFE) and aldehyde dehydrogenase (ALDH) expression level was used to quantify CSC activity in two CRC cell lines, after TF knockdown (TFKD) or TF over-expression (TFOE). RESULTS: TFKD resulted in increased levels of ALDH in SW620 (1.31±0.04-fold, p<0.001) and DLD-1 (1.63±0.14-fold, p=0.04) cells. CFE was increased in SW620 (1.21±0.23% vs. 2.03±0.29%, p=0.01) and DLD-1 (0.41±0.12% vs. 0.68±0.9%, p=0.01) cells. Conversely, TFOE decreased ALDH expression (0.72±0.04-fold, p=0.001) and CFE (0.33±0.05% vs. 0.66±0.14%, p=0.006) in DLD-1, but had no impact on SW620 cells. CONCLUSION: In the examined CRC cell lines, TF expression was inversely related to CSC activity suggesting that anti-TF therapies may not have a role in CRC treatment.

Chitinase 3-like-1 promotes intrahepatic activation of coagulation through induction of tissue factor in mice.

Coagulation is a critical component in the progression of liver disease. Identification of key molecules involved in the intrahepatic activation of coagulation (IAOC) will be instrumental in the development of effective therapies against liver disease. Using a mouse model of concanavalin A (ConA)-induced hepatitis, in which IAOC plays an essential role in causing liver injury, we uncovered a procoagulant function of chitinase 3-like 1 (Chi3l1). Chi3l1 expression is dramatically elevated after ConA challenge, which is dependent on ConA-induced T cell activation and the resulting interferon γ and tumor necrosis factor α productions. Compared with wild-type mice, Chi3l1-/- mice show less IAOC, reduced tissue factor (TF) expression, and attenuated liver injury. Reconstituting Chi3l1-/- mice with recombinant TF triggers IAOC and augments liver injury. CONCLUSION: Our data demonstrate that Chi3l1, through induction of TF via mitogen-activated protein kinase activation, promotes IAOC and tissue injury. (Hepatology 2018;67:2384-2396).

Mechanisms and risk factors of thrombosis in cancer.

The close relationship between cancer and thrombosis is known since more than a century. Venous thromboembolism (VTE) may be the first manifestation of an occult malignancy in an otherwise healthy individual. Cancer patients commonly present with abnormalities of laboratory coagulation tests, indicating an ongoing subclinical hypercoagulable condition. The results of laboratory tests demonstrate that a process of fibrin formation and removal parallels the development of malignancy, which is of particular interest since fibrin and other clotting products are important for both thrombogenesis and tumor progression. Besides general clinical risk factors (i.e. age, previous VTE, immobility, etc.), other factors typical of cancer can increase the thrombotic risk in these patients, including the type of cancer, advanced disease stage, and cancer therapies. In addition, biological factors, including tumor cell-specific prothrombotic properties and the host cell inflammatory response to the tumor, play a central role in the pathogenesis of cancer-associated thrombosis. Cancer cells produce and release procoagulant and fibrinolytic proteins, as well as inflammatory cytokines. In addition, they are capable of directly adhering to host cells (i.e. endothelial cells, monocytes, platelets, and neutrophils), thereby stimulating additional prothrombotic properties of the host effector cells. Tumor-shed procoagulant microparticles also contribute to the patient hypercoagulable state. Finally, the changes of stromal cells of the tumor 'niche' induced by tissue factor (TF) highlight new interactions between hemostasis and cancer. Of interest, most of these mechanisms, besides activating the hemostatic system, also promote tumor growth and metastasis, and are regulated by oncogenic events. Indeed, molecular studies demonstrate that oncogenes responsible for the cellular neoplastic transformation drive the programs of hemostatic protein expression and microparticle liberation by cancer tissues. Human and animal experimental models demonstrate that activation of cancer-associated prothrombotic mechanisms parallels the development of overt thrombotic syndromes in vivo.

IL-33 stimulates the release of procoagulant microvesicles from human monocytes and differentially increases tissue factor in human monocyte subsets.

Monocytes and monocyte-derived microvesicles (MVs) are the main source of circulating tissue factor (TF). Increased monocyte TF expression and increased circulating levels of procoagulant MVs contribute to the formation of a prothrombotic state in patients with cardiovascular disease. Interleukin (IL)-33 is a pro-inflammatory cytokine involved in atherosclerosis and other inflammatory diseases, but its role in regulating thrombosis is still unclear. The aim of the present study was to investigate the effects of IL-33 on the procoagulant properties of human monocytes and monocyte-derived MVs. IL-33 induced a time- and concentration-dependent increase of monocyte TF mRNA and protein levels via binding to the ST2-receptor and activation of the NF-κB-pathway. The IL-33 treated monocytes also released CD14+TF+ MVs and IL-33 was found to increase the TF activity of both the isolated monocytes and monocyte-derived MVs. The monocytes were classified into subsets according to their CD14 and CD16 expression. Intermediate monocytes (IM) showed the highest ST2 receptor expression, followed by non-classical monocytes (NCM), and classical monocytes (CM). IL-33 induced a significant increase of TF only in the IM (p<0.01), with a tendency in NCM (p=0.06), but no increase was observed in CM. Finally, plasma levels of IL-33 were positively correlated with CD14+TF+ MVs in patients undergoing carotid endarterectomy (r=0.480; p=0.032; n=20). We hereby provide novel evidence that the proinflammatory cytokine IL-33 induces differential TF expression and activity in monocyte subsets, as well as the release of procoagulant MVs. In this manner, IL-33 may contribute to the formation of a prothrombotic state characteristic for cardiovascular disease.

Tissue factor: A potent stimulator of Von Willebrand factor synthesis by human umbilical vein endothelial cells.

Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of Von Willebrand factor. The aim of this study was to examine the effects of the inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) and the coagulation factors, tissue factor and thrombin on the release and cleavage potential of ultra-large von Willebrand factor (ULVWF) and its cleavage protease by cultured human umbilical vein endothelial cells (HUVEC). HUVEC were treated with IL-6, IL-8, and TNF-α, tissue factor (TF) and thrombin, and combinations thereof for 24 hours under static conditions. The cells were then exposed to shear stress after which the VWF-propeptide levels and the VWF cleavage protease, ADAMTS13 content were measured. All treatments and their combinations, excluding IL-6, significantly stimulated the secretion of VWF from HUVEC. The VWF secretion from the HUVEC was stimulated most by the combination of TF with TNF-α. Slightly lower levels of ADAMTS13 secretion were found with all treatments. This may explain the thrombogenicity of patients with inflammation where extremely high VWF levels and slightly lower ADAMTS13 levels are present.

Tissue factor induces VEGF expression via activation of the Wnt/ß-catenin signaling pathway in ARPE-19 cells.

PURPOSE: The purpose of the present study was to investigate the potential signal mechanism of tissue factor (TF) in the regulation of the expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (ARPE-19) cells. METHODS: An in vitro RPE cell chemical hypoxia model was established by adding cobalt chloride (CoCl2) in the culture medium. The irritative concentration of CoCl2 was determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit. VEGF production in ARPE-19 cells was measured with enzyme-linked immunosorbent assay (ELISA) and western blotting. The Wnt signaling pathway-associated molecules, including phospho-glycogen synthase kinase 3ß (p-GSK3ß), GSK3ß, p-ß-catenin and ß-catenin, were detected with western blotting. pEGFP-N3-hTF was constructed and verified with digestion of the restriction enzyme and sequencing analysis. Human TF overexpression and silencing plasmids were transfected into the ARPE-19 cells to clarify the causal relationship between TF and VEGF expression. The Transwell coculture system of ARPE-19 cells and RF/6A rhesus macaque choroid-retinal endothelial cells was performed to evaluate cell invasion and tube formation ability. RESULTS: Our anoxic model of ARPE-19 cells showed that TF expression was upregulated in accordance with variations in hypoxia-inducible factor 1-alpha (HIF-1α) and VEGF levels. Silencing and overexpression of TF decreased and increased VEGF expression, respectively. The Wnt/ß-catenin signaling pathway played an important role in this effect. Results from the ARPE-19 cell and RF/6A cell coculture system showed that the enhancement of TF expression in the ARPE-19 cells led to significantly faster invasion and stronger tube-forming ability of the RF/6A cells, while siRNA-mediated TF silencing caused the opposite effects. Pharmacological disruption of Wnt signaling IWR-1-endo inhibited the effects compared to the TF-overexpressing group, indicating the importance of the Wnt/ß-catenin signaling pathway in the process of TF-induced VEGF expression and angiogenesis. CONCLUSIONS: Involvement of the activation of the Wnt/ß-catenin signaling pathway is an important mediator for TF-induced VEGF production during the process of angiogenesis. Thus, our findings may ascertain the molecular regulation of TF in neovessel formation and show significant therapeutic implications.

Simulated thrombin responses in venous valves.

OBJECTIVE: Venous thromboembolism frequently results in thrombi formation near or within the pocket of a venous valve due to recirculating hemodynamics, which has been largely attributed to hypoxia-induced tissue factor (TF) expression. Numerical models are now capable of assessing the spatiotemporal behavior of the TF-initiated coagulation cascade under nonuniform hemodynamics. The aim of this study was to use such a numerical simulation to analyze the degree and location of thrombin formation with respect to TF position in the presence of disturbed flow induced by an open venous valve. METHODS: Thrombin formation was simulated using a computational model that captures the hemodynamics, kinetics, and chemical transport of 22 biochemical species. Disturbed flow is described by the presence of a valve in the equilibrium phase of the valve cycle with leaflets in a fully open position. Three different positions of TF downstream of the valve opening were investigated. RESULTS: The critical amount of TF required to initiate a thrombotic response is reduced by up to 80% when it is positioned underneath the recirculating regions near the valve opening. In addition, because of the increased surface area of the open valve cusp in conjunction with recirculating hemodynamics, it was observed that thrombin is generated inside the valve pocket even when the exposed region of TF is downstream of the valve. CONCLUSIONS: The presence of prothrombotic surface reactions in conjunction with recirculating hemodynamics provides an additional mechanism for thrombus formation in venous valves that does not require direct damage or dysfunction to the valve itself.

Relationship between trauma-induced coagulopathy and progressive hemorrhagic injury in patients with traumatic brain injury.

Progressive hemorrhagic injury (PHI) can be divided into coagulopathy-related PHI and normal coagu- lation PHI. Coagulation disorders after traumatic brain injuries can be included in trauma-induced coagulopathy (TIC). Some studies showed that TIC is associated with PHI and increases the rates of disability and mortality. In this review, we discussed some mechanisms in TIC, which is of great importance in the development of PHI, including tissue factor (TF) hypothesis, protein C pathway and thrombocytopenia. The main mechanism in the relation of TIC to PHI is hypocoagulability. We also reviewed some coagulopathy parameters and proposed some possible risk factors, predictors and therapies.

Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling.

The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.

Pregnancy-associated plasma protein-A promotes TF procoagulant activity in human endothelial cells by Akt-NF-κB axis.

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase with a controversial role in pathophysiology of cardiovascular disease. It seems involved in progression of atherosclerosis and is widely represented in atherosclerotic plaque. PAPP-A plasma levels are elevated in patients with acute coronary syndromes (ACS), thus it has been suggested that it might be a prognostic marker for developing major cardiovascular events. However, the pathophysiological link(s) between PAPP-A and ACS are still unknown. Several studies have indicated that tissue factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates whether PAPP-A, at concentrations measurable in ACS patients, might induce TF expression in human endothelial cells in culture (HUVEC). In HUVEC, PAPP-A induced TF-mRNA transcription as demonstrated by real time PCR and expression of functionally active TF as demonstrated by FACS analysis and pro-coagulant activity assay. PAPP-A induced TF expression through the activation of Akt/NF-κB axis, as demonstrated by luciferase assay and by suppression of TF-mRNA transcription as well as of TF expression/activity by Akt and NF-κB inhibitors. These data indicate that PAPP-A promotes TF expression in human endothelial cells and support the hypothesis that this proteinase, besides being involved in progression of atherosclerosis, does not represent an independent risk factor for adverse cardiovascular events, but it rather might play an "active" role in the pathophysiology of ACS as an effector molecule able to induce a pro-thrombotic phenotype in endothelial cells.