thromboplastin and Glioma

The purpose of this paper is to review the rationale for the development of coagulation-reactive drugs for the experimental therapy of gliomas. Numerous reactants familiar to students of blood coagulation have been shown to contribute to neoplastic proliferation, invasion and metastasis. Recently, considerable progress has been made in demonstrating the ability of drugs capable of inhibiting these reactants to alter cancer progression. Biological features of gliomas within the realm of blood coagulation suggest that clinical trials of such drugs warrant consideration. This approach offers the prospect of a novel treatment for this devastating tumour type that does not share the toxicities of conventional cancer therapies.

Topics: Anticoagulants; Aprotinin; Blood Coagulation; Brain Neoplasms; Factor Xa; Glioma; Heparin; Heparin, Low-Molecular-Weight; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lipoproteins; Thrombin; Thrombomodulin; Thrombophilia; Thromboplastin; Urokinase-Type Plasminogen Activator

Coagulation activation in human gliomas may have two consequences: (1) activation of systemic coagulation reactions leading to the development of venous thromboembolic disease, and (2) stimulation of tumor growth and invasion. Anticoagulation in patients with gliomas, therefore, may not only prevent thrombosis but also have anticancer activity. Tissue factor and thrombin are appropriate targets for intervention, and several drugs are suitable for testing. Low-molecular-weight heparin and direct thrombin inhibitors are useful for reducing thrombin production and activity, and recombinant tissue factor pathway inhibitor and statins are examples of drugs that target tissue factor directly. This article reviews the implications of coagulation activation in human gliomas and provides a rationale for clinical testing of anticoagulants as part of a treatment strategy for this devastating human cancer.

Topics: Anticoagulants; Blood Coagulation; Glioma; Humans; Neoplasm Proteins; Thromboplastin

Glioma is the most common brain tumor and the main cause of death from primary brain tumors. Due to the limitations of current diagnostic and treatment methods, the prognosis of high-grade glioma is not optimistic and is prone to venous thrombosis. Epithelial-mesenchymal transition (EMT) is a vital step for glioma cells to obtain a highly migratory and invasive cell phenotype. Tissue factor (TF) is the downstream target of several carcinogenic pathways. According to reports, the TF gene is highly methylated and down-regulated in IDH1 mutant gliomas with good prognosis. We aimed to investigate the impact of EMT on the expression of TF in glioma cells, as well as the corresponding mechanism. Our data indicated that the expression level of TF in glioma tissues increased, and was positively correlated with the WHO classification of glioma. After inducing EMT in glioma cells in vitro, TF expression increased significantly, indicating that EMT in glioma cells can promote TF expression. Further studies have shown that the expression level of ZEB1 is positively correlated with the WHO classification of glioma tissues and the expression level of TF in glioma tissues. This study proved that EMT promotes the expression of TF in glioma cells through the miR-200a/ZEB1 axis. In summary, these results indicated that EMT can promote the expression of TF in glioma cells via the miR-200a/ZEB1 axis. For gliomas, TF is related to EMT and has the potential to become an effective target against EMT and thrombotic events.

Topics: Brain Neoplasms; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glioma; Humans; MicroRNAs; Thromboplastin; Zinc Finger E-box-Binding Homeobox 1

Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type-based diagnosis and antithrombotic intervention.

Topics: Animals; Extracellular Vesicles; Glioblastoma; Glioma; Humans; Mice; Thromboplastin; Thrombosis

Tissue Factor (TF) has been well established in angiogenesis, invasion, metastasis, and prognosis in glioma. A noninvasive assessment of TF expression status in glioma is therefore of obvious clinical relevance. Dynamic contrast-enhanced (DCE) MRI parameters have been used to evaluate microvascular characteristics and predict molecular expression status in tumors. Our aim is to investigate whether quantitative DCE-MRI parameters could assess TF expression in glioma.. Thirty-two patients with histopathologically diagnosed supratentorial glioma who underwent DCE-MRI were retrospectively recruited. Extended Tofts linear model was used for DCE-MRI post-processing. Hot-spot, whole tumor cross-sectional approaches, and histogram were used for analysis of model based parameters. Four serial paraffin sections of each case were stained with TF, CD105, CD34 and α-Sooth Muscle Actin, respectively for evaluating the association of TF and microvascular properties. Pearson correlation was performed between percentage of TF expression area and DCE-MRI parameters, multiple microvascular indexes.. Volume transfer constant (K. Our results indicate that K

Topics: Adult; Aged; Contrast Media; Cross-Sectional Studies; Female; Glioma; Humans; Magnetic Resonance Imaging; Male; Middle Aged; Neovascularization, Pathologic; Prognosis; Retrospective Studies; Supratentorial Neoplasms; Thromboplastin

Nuclear medicine examinations for imaging gliomas have been introduced into clinical practice to evaluate the grade of malignancy and determine sampling locations for biopsies. However, these modalities have some limitations. Tissue factor (TF) is overexpressed in various types of cancers, including gliomas. We thus generated an anti-human TF monoclonal antibody (mAb) clone 1849. In the present study, immunohistochemistry performed on glioma specimens using anti-TF 1849 mAb showed that TF expression in gliomas increased in proportion to the grade of malignancy based on the World Health Organization (WHO) classification, and TF was remarkably expressed in necrosis and pseudopalisading cells, the histopathological hallmarks of glioblastoma multiforme (GBM). Furthermore, in both fluorescence and single-photon emission computed tomography/computed tomography (SPECT/CT) imaging studies, anti-TF 1849 IgG efficiently accumulated in TF-overexpressing intracranial tumours in mice. Although further investigation is required for a future clinical use of immuno-SPECT with

Topics: Animals; Antibodies, Monoclonal; Brain; Brain Neoplasms; Cell Line, Tumor; Female; Glioma; Humans; Immunoconjugates; Indium Radioisotopes; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Imaging; Single Photon Emission Computed Tomography Computed Tomography; Thromboplastin; Xenograft Model Antitumor Assays

Mutant isocitrate dehydrogenase 1 (IDH1) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II-IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26-30 % of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0 %). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85-90 % in wild-type versus 2-6 % in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE.

Topics: Adult; Aged; Aged, 80 and over; Alcohol Oxidoreductases; Animals; Antineoplastic Agents; Blood Platelets; Brain Neoplasms; Calcimycin; Calcium Ionophores; Cohort Studies; Female; Glioma; Humans; Isocitrate Dehydrogenase; Male; Mice; Middle Aged; Mutation; Thrombin; Thromboplastin; Thrombosis

The coagulation system links immediate (hemostatic) and late (inflammatory, angiogenic) tissue responses to injury, a continuum that often is subverted in cancer. Here we provide evidence that tumor dormancy is influenced by tissue factor (TF), the cancer cell-associated initiator of the coagulation system and a signaling receptor. Thus, indolent human glioma cells deficient for TF remain viable but permanently dormant at the injection site for nearly a year, whereas the expression of TF leads to a step-wise transition to latent and overt tumor growth phases, a process that is preceded by recruitment of vascular (CD105(+)) and myeloid (CD11b(+) and F4/80(+)) cells. Importantly, the microenvironment orchestrated by TF expression drives permanent changes in the phenotype, gene-expression profile, DNA copy number, and DNA methylation state of the tumor cells that escape from dormancy. We postulate that procoagulant events in the tissue microenvironment (niche) may affect the fate of occult tumor cells, including their biological and genetic progression to initiate a full-blown malignancy.

Topics: Animals; Cell Line, Tumor; DNA Copy Number Variations; DNA Methylation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioma; Humans; Mice; Mutation; Neoplastic Processes; Statistics, Nonparametric; Thromboplastin; Tumor Microenvironment

In human gliomas, tissue factor (TF) is overexpressed, associated with the grade of malignancy and influences tumour biology. Intra-tumoural fibrin/fibrinogen deposition and activation of the fibrinolytic system also play a role in tumour cell proliferation and angiogenesis. The first aim of the present study was to investigate TF expression and the presence of fibrin/fibrinogen and D-dimers in canine glioma biopsies, graded according to the World Health Organization (WHO) classification of tumours of the central nervous system. The second aim was to investigate the occurrence of intravascular thrombosis (IVT) in canine gliomas, as a potential histological marker of glioma type or grade of malignancy. An immunohistochemical study using antibodies against TF, fibrin/fibrinogen and D-dimers was performed with 24 glioma samples, including 15 oligodendrogliomas, 6 astrocytomas and 3 mixed gliomas. Immunohistochemical data were statistically analysed to determine whether there was any relationship between glioma type and grade of malignancy. All gliomas were moderate to strongly positive for TF and the staining score was significantly higher (P = 0.04) in high-grade (III or IV) than in low-grade (II) gliomas. Intra-tumoural fibrin/fibrinogen deposition was detected in all tumour biopsies assessed, and D-dimers were detected in 17/24 gliomas. IVT was a frequent finding, but was not linked to a specific glioma type or malignancy grade. TF expression, fibrin/fibrinogen deposition, extravascular fibrinolytic system activation and IVT occur in canine gliomas. Canine glioma might be a suitable model for studying coagulation and fibrinolysis as potential therapeutic targets for human gliomas.

Topics: Animals; Astrocytoma; Biomarkers; Dog Diseases; Dogs; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Gene Expression; Glioma; Immunohistochemistry; Oligodendroglioma; Spain; Thromboplastin; Thrombosis

Glioblastoma multiforme (GBM) is an aggressive form of glial brain tumors, associated with angiogenesis, thrombosis, and upregulation of tissue factor (TF), the key cellular trigger of coagulation and signaling. Since TF is upregulated by oncogenic mutations occurring in different subsets of human brain tumors we investigated whether TF contributes to tumourigenesis driven by oncogenic activation of EGFR (EGFRvIII) and RAS pathways in the brain. Here we show that TF expression correlates with poor prognosis in glioma, but not in GBM. In situ, the TF protein expression is heterogeneously expressed in adult and pediatric gliomas. GBM cells harboring EGFRvIII (U373vIII) grow aggressively as xenografts in SCID mice and their progression is delayed by administration of monoclonal antibodies blocking coagulant (CNTO 859) and signaling (10H10) effects of TF in vivo. Mice in which TF gene is disrupted in the neuroectodermal lineage exhibit delayed progression of spontaneous brain tumors driven by oncogenic N-ras and SV40 large T antigen (SV40LT) expressed under the control of sleeping beauty transposase. Reduced host TF levels in low-TF/SCID hypomorphic mice mitigated growth of glioma subcutaneously but not in the brain. Thus, we suggest that tumor-associated TF may serve as therapeutic target in the context of oncogene-driven disease progression in a subset of glioma.

Topics: Adolescent; Adult; Animals; Brain; Brain Neoplasms; Cell Line, Tumor; ErbB Receptors; Gene Deletion; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Mice; Mice, SCID; Oncogenes; Prognosis; Thromboplastin

Brain tumor patients have an increased risk of venous thromboembolism (VTE). An important role in cancer-related VTE has been suggested for tissue factor (TF), the main initiator of the coagulation cascade. We conducted a prospective cohort study to determine whether expression levels of TF in brain tumors are associated with future VTE.. We immunohistochemically determined TF-expression in brain tumor specimens of 96 adult patients (8 low-grade and 82 high-grade gliomas, 6 embryonal tumors) that were included in the Vienna Cancer and Thrombosis Study (CATS). Each patient was prospectively followed until the occurrence of VTE and/or death within a period of two years or loss of follow-up.. Fifteen brain tumor patients (15.6%) developed VTE during follow-up. Seventy-seven brain tumors (80.2%) stained positive for TF. Staining was strong in 13 (13.5%), moderate in 64 (66.7%) and negative in 19 (19.8%) tumors. No statistically significant association between TF-expression (negative, focal, widespread) and the occurrence of VTE was found. The hazard ratio (HR) for VTE was 1.30 (95% confidence interval [CI]: 054 - 3.14, p=0.567) when patients with negative-, focal- and widespread TF expression were compared and not statistically significant. Also when tumors were categorized into two groups (focal/widespread versus negative TF-expression), the HR for future VTE was not statistically significant (HR: 1.45, 95% CI: 0.44 - 7.37; p=0.578). An association can still not be definitely excluded, as this study was underpowered.. Our data indicate that TF-expression levels in brain tumors are not strongly associated with future VTE.

Topics: Brain Neoplasms; Female; Glioma; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasms, Germ Cell and Embryonal; Risk Factors; Thromboplastin; Venous Thromboembolism

Tissue factor (TF) is upregulated in several malignant diseases, including gliomas. Here, we demonstrate pronounced differences in the expression of TF and its interactors factor VII and protease-activated receptor 2 (PAR-2) in nine human glioma cell lines (U87, U251, U343, U373, MZ-18, MZ-54, MZ-256, MZ-304, Hs 683) as detected by RT-PCR and Western blot analysis. Inhibition of TF signaling by a neutralizing monoclonal antibody (mAb TF9-10H10) led to significantly reduced proliferation in high-grade astroglial (MZ-18 and MZ-304) and oligodendroglial (Hs 683) cell lines abundantly expressing TF, but not in U373 cells expressing low amounts of TF. Scratch migration assays and Boyden chamber assays indicated that mAb TF9-10H10 and lentiviral knockdown of TF significantly reduced cell migration and invasion of MZ-18, MZ-304 and Hs 683 cells, both under normoxic and hypoxic conditions. Of note, all three cell lines displayed increased cell migration and invasion under hypoxic conditions (1% O(2)), which was associated with enhanced expression of TF and increased phosphorylation of p44/42 mitogen-activated protein kinase (ERK1/2). Silencing of TF blocked activation of the ERK pathway, induction of TF expression and the potentiating effect of hypoxia on cell migration and invasion. RNA interference against PAR-2 abrogated the autocrine effects of TF on cell proliferation, migration and invasion, indicating that TF signals via PAR-2 in glioma cells. Our results suggest an important role for the TF/FVIIa/PAR-2/ERK axis in tumor growth and invasion of glioma and suggest that TF may be a suitable target for the development of novel therapies against high-grade glioma.

Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Knockdown Techniques; Genetic Vectors; Glioma; Humans; Lentivirus; MAP Kinase Signaling System; Neoplasm Invasiveness; Receptor, PAR-2; RNA Interference; Thromboplastin

This article characterizes the vascular effects following vascular-targeted photodynamic therapy with a photosensitizer which actively targets endothelial cells.. This strategy was considered by coupling a chlorin to a heptapeptide targeting neuropilin-1 in human malignant glioma-bearing nude mice. A laser Doppler microvascular perfusion monitor was used to monitor microvascular blood perfusion in tumor tissue. Endothelial cells' ultra structural integrity was observed by transmission electron microscopy. The consequences of photosensitization on tumor vessels, tissue factor expression, fibrinogen consumption, and thrombogenic effects were studied by immunohistochemical staining.. Treatment of glioma-bearing mice with the conjugate showed a statistically significant tumor growth delay. Vascular effect was characterized by a decrease in tumor tissue blood flow at about 50% baseline during treatment not related to variations in temperature. This vascular shutdown was mediated by tumor blood vessels' congestion. A pro-thrombotic behavior of targeted endothelial cells in the absence of ultra structural changes led to the induction of tissue factor expression from the earliest times post-treatment. Expression of tissue factor-initiated thrombi formation was also related to an increase in fibrinogen consumption.. Using a peptide-conjugated photosensitizer targeting neuropilin-1, induction of tissue factor expression immediately post-treatment, led to the establishment of thrombogenic effects within the vessel lumen.

Topics: Animals; Blood Vessels; Endothelial Cells; Female; Glioma; Humans; Mice; Mice, Nude; Neuropilin-1; Oligopeptides; Photochemotherapy; Photosensitizing Agents; Porphyrins; Thromboplastin; Thrombosis

Epidermal growth factor receptor variant III (EGFRvIII), the most common EGFR mutation, is associated with cell migration of glioblastoma multiforme (GBM) cases; however, the mechanism has not been elucidated. In this study, we found that the EGFRvIII-promoted glioma cell migration was closely linked to high levels of tyrosine phosphorylation in focal adhesion kinase (FAK) Y397. We also demonstrated that EGFRvIII formed a complex with FAK, resulting in enhanced tyrosine phosphorylation levels of FAK Y397 and EGFR Y1068. After knockdown of FAK expression via anti-FAK shRNA, the U87ΔEGFR cell migration was significantly inhibited, accompanying with the reduced phosphorylation levels of extracellular signal-regulated kinase (ERK1/2). Furthermore, the role of ERK1/2 in FAK-regulated cell migration was confirmed. Taken together, our results suggest that FAK and its downstream molecule ERK were involved in EGFRvIII-promoted glioma cell migration in U87ΔEGFR cells.

Topics: Cell Movement; Epidermis; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Glioblastoma; Glioma; Humans; Phosphorylation; Signal Transduction; Thromboplastin

ErbB oncogenes drive the progression of several human cancers. Our study shows that in human carcinoma (A431) and glioma (U373) cells, the oncogenic forms of epidermal growth factor receptor (EGFR; including EGFRvIII) trigger the up-regulation of tissue factor (TF), the transmembrane protein responsible for initiating blood coagulation and signaling through interaction with coagulation factor VIIa. We show that A431 cancer cells in culture exhibit a uniform TF expression profile; however, these same cells in vivo exhibit a heterogeneous TF expression and show signs of E-cadherin inactivation, which is coupled with multilineage (epithelial and mesenchymal) differentiation. Blockade of E-cadherin in vitro, leads to the acquisition of spindle morphology and de novo expression of vimentin, features consistent with epithelial-to-mesenchymal transition. These changes were associated with an increase in EGFR-dependent TF expression, and with enhanced stimulation of vascular endothelial growth factor production, particularly following cancer cell treatment with coagulation factor VIIa. In vivo, cells undergoing epithelial-to-mesenchymal transition exhibited an increased metastatic potential. Furthermore, injections of the TF-blocking antibody (CNTO 859) delayed the initiation of A431 tumors in immunodeficient mice, and reduced tumor growth, vascularization, and vascular endothelial growth factor expression. Collectively, our data suggest that TF is regulated by both oncogenic and differentiation pathways, and that it functions in tumor initiation, tumor growth, angiogenesis, and metastasis. Thus, TF could serve as a therapeutic target in EGFR-dependent malignancies.

Topics: Animals; Cadherins; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Epithelial Cells; ErbB Receptors; Flow Cytometry; Glioma; Humans; Mesoderm; Mice; Mice, SCID; Neoplasm Metastasis; Neovascularization, Pathologic; Thromboplastin; Up-Regulation; Vascular Endothelial Growth Factor A; Vimentin

That there is a correlation between cancer and procoagulant states is well-known. C6 glioma cell line was originally induced in random-bred Wistar-Furth rats and is morphologically similar to glioblastoma multiforme, the most common aggressive glioma resistant to therapeutic interventions.. In this study we analyzed the molecular mechanisms responsible for the highly procoagulant properties of C6 glioma cells.. The presence of tissue factor (TF) and phosphatidylserine (PS) in C6 cells was investigated by flow-cytometric and functional analyses. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins.. TF was identified by flow-cytometric and functional [factor (F) Xa formation in the presence of cells and FVIIa] assays. Alternatively, conversion of FX into FXa was also observed in the presence of C6 cells, FIXa and FVIIIa. This effect was both cell- and FVIIIa-dependent, being consistent with formation of the intrinsic tenase complex. C6 cells were also able to activate prothrombin in the presence of FXa and FVa, thus supporting formation of the prothrombinase complex. This ability was similar to positive controls performed with PS-containing vesicles. Accordingly, exposure of PS on C6 cells was demonstrated by flow cytometry employing specific anti-PS antibodies. In addition, annexin V, which blocks PS binding sites, inhibited FX and prothrombin conversion by their respective C6-assembled activating complexes.. C6 glioma cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant TF exposure and from the presence of the anionic lipid PS at the outer leaflet of cell membrane. Therefore, this animal cell line may be used to explore new aspects concerning the role of blood coagulation proteins in tumor biology, especially those affecting the central nervous system.

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Cell Line, Tumor; Cysteine Endopeptidases; Flow Cytometry; Glioma; Humans; Neoplasm Proteins; Phosphatidylserines; Rats; Thrombin; Thromboplastin

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Cell Line, Tumor; Cysteine Endopeptidases; Data Interpretation, Statistical; Glioma; Humans; Neoplasm Proteins; Phosphatidylserines; Rats; Thrombin; Thromboplastin

Tissue factor (TF), a cell surface receptor of factor VII/VIIa, was initially recognized as an initiator of the extrinsic coagulation pathway. TF has recently been found to be expressed highly in certain types of malignant tumors. In addition, TF expression appears to be a marker for malignant angiogenesis in human solid tumors. However TF expression and its relationship to angiogenesis and tumor progression in human glioma are still unclear.. Quantitative reverse transcription PCR and immunofluorescence were performed on the U251 glioma cell line, 5 normal brain specimens, and 34 glioma surgical specimens. Of the gliomas, 10 were grade IV, 12 grade III, 7 grade II, and 5 grade I. Microvessels were highlighted using a monoclonal antibody specific to human von Willebrand factor.. TF was strongly positive in 90% of the grade IV cases, 58% of grade III, 43% of grade II, and 20% of grade I. TF staining was not present in any normal brain specimens. The mean level of TF mRNA in normal brain tissue was 5.0 x 10(3) copies/microg RNA. Among the gliomas, TF mRNA ranged from 1.7 x 10(5) to 6.8 x 10(7) copies/microg, with a mean of 4.6 x 10(6). TF expression was highest in glioblastomas that showed greatest vascularity.. These findings support a role for TF in angiogenesis in glioma. TF is expressed in glioma and the level of expression correlates with the histologic grade of malignancy and vascularity.

Topics: Antigens; Brain Neoplasms; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Glioma; Humans; Immunohistochemistry; Neoplasm Staging; Neovascularization, Pathologic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin; Tumor Cells, Cultured

Initiation of the coagulation serine protease cascade in mammalian cells is mediated by tissue factor (TF), which is a cell surface receptor and cofactor for coagulation factor VII (FVII) and its activated form FVII (FVIIa). Increasing evidence suggests that TF is expressed in a wide range of cancer cells and plays important roles in cancer progression and metastasis. In this study, we investigated the association between the expression level of TF transcript and histologic features of glioma.. RNA was extracted from normal brain tissues and glioma tissues. We developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify TF gene expression and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at the mRNA level in human glioma.. The dynamic range of the assay was 10(3)-10(8) copy/microg RNA. The relationship between Ct and log starting concentration was linear (r2 > or = 0.99). The mean expression of TF in healthy brain tissue was 6.2 x 10(3) copy/microg RNA. Overexpression of TF was found in 42 brain glioma samples, mean value is 2.9 x 10(6) copy/microg RNA.. TF mRNA transcript is expressed in glioma and the level of expression correlates with histologic grade of malignancy. This new simple, rapid, semiautomated assay is a major alternative to Northern blot and competitive quantitative PCR for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and support future TF-based clinical applications.

Topics: Biomarkers, Tumor; Brain Neoplasms; Calibration; Glioma; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Neoplasm Staging; Polymerase Chain Reaction; Quality Control; Reproducibility of Results; RNA, Messenger; RNA, Neoplasm; Sensitivity and Specificity; Thromboplastin; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor in human gliomas. VEGF-induced proteins in endothelial cells, tissue factor (TF), osteopontin (OPN) and alphavbeta3 integrin have been implicated as important molecules by which VEGF promotes angiogenesis in vivo. Sixty-eight gliomas were immunohistochemically stained with TF, VEGF, OPN and alphavbeta3 integrin antibody. Twenty-three tumours, six normal brains and nine glioma cell lines were evaluated for their mRNA expression of VEGF and TF by reverse transcription polymerase chain reaction analysis. The data indicated that TF as well as VEGF was a strong regulator of human glioma angiogenesis. First, TF expression in endothelial cells which was observed in 74% of glioblastomas, 54% of anaplastic astrocytomas and none of low-grade astrocytomas, correlated with the microvascular density of the tumours. Double staining for VEGF and TF demonstrated co-localization of these two proteins in the glioblastoma tissues. Second, there was a correlation between TF and VEGF mRNA expression in the glioma tissues. Third, glioma cell conditioned medium containing a large amount of VEGF up-regulated the TF mRNA expression in human umbilical vein endothelial cells. OPN and alphavbeta3 integrin, were also predominantly observed in the microvasculature of glioblastomas associated with VEGF expression. Microvascular expression of these molecules could be an effective antiangiogenesis target for human gliomas.

Topics: Brain Neoplasms; Endothelial Growth Factors; Glioblastoma; Glioma; Humans; Lymphokines; Microcirculation; Neovascularization, Pathologic; Osteopontin; Receptors, Vitronectin; Sialoglycoproteins; Thromboplastin; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The relationship between coagulation cascade activation and glioma cell proliferation was examined. The human glioma cell lines T98G, TM-1 and normal human astrocyte cell strain (NHA) were examined. Using anti-tissue factor (TF) antibody, immunocytochemical detection of TF antigen was obtained in both cell lines and cell strain. TF antigen in cell lysates was also measured by enzyme linked immunosorbent assay (ELISA). In a one-stage clotting assay, T98G, TM-1 and NHA revealed procoagulant activity (PCA) in normal human plasma and factor VII deficient plasma. PCA in normal human plasma was significantly inhibited by both inhibitory anti-TF antibody and cysteine protease inhibitor HgCl2. This result indicates that T98G, TM-1 and NHA cells express not only TF but also cancer procoagulant (CP) at the same time. In a cell proliferation assay, thrombin induced proliferation in T98G and TM-1 cells in a dose-dependent fashion and in NHA cell in a bell-shaped fashion. This mitogenic stimulant was inhibited by the specific thrombin inhibitor hirudin. The combinations of coagulation factors II, V, and X with or without factor VII induced proliferation in T98G, TM-1, and NHA cells. The maximal mitogenic stimulatory effects were larger in glioma cells than in NHA. These mitogenic stimulatory effects were also inhibited by hirudin. Each coagulation factor on its own or in any other combination of coagulation factors had no proliferative effect. Thus, these mitogenic stimulatory effects were considered to be the effect of thrombin. In conclusion, T98G and TM-1 human glioma cells express two different types of procoagulants TF and CP. In the presence of coagulation factors, these glioma cells can generate thrombin and this thrombin generation is capable of inducing glioma cell proliferation in vitro.

Topics: Antibodies; Blood Physiological Phenomena; Cell Division; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme-Linked Immunosorbent Assay; Factor VII Deficiency; Glioma; Humans; Immunohistochemistry; Magnesium Chloride; Microscopy, Confocal; Neoplasm Proteins; Reference Values; Thrombin; Thromboplastin; Tumor Cells, Cultured

To clarify factors that may contribute to the development of intratumoral hemorrhage, we analyzed the expression of tissue factor (TF), an initiator of the extrinsic coagulation pathway, and of tissue factor pathway inhibitor (TFPI) in glioblastomas with or without massive intratumoral hematoma. Among 196 glioma cases reviewed, there were 13 with macroscopic intratumoral hemorrhage. We focused on the glioblastomas and used immunoblot- and immunohistochemical methods to compare the expression of TF and TFPI in 9 glioblastomas with macroscopic hematoma and 30 glioblastomas without macroscopic hemorrhage. Although TF was expressed in most glioblastomas irrespective of the presence or absence of macroscopic hemorrhage, the staining patterns differed significantly: TF-positive glioma cells were diffusely present in the non-hemorrhage group; in the group with hemorrhage, positive cells, primarily macrophages, were scattered throughout the tissue examined. The expression of TFPI was significantly higher in the group with than in the group without hemorrhage. Our results suggest that local suppression of the TF-dependent coagulation cascade is a contributing factor that permits the occurrence of intratumoral hemorrhage.

Topics: Adult; Aged; Brain Neoplasms; Child, Preschool; Female; Glioblastoma; Glioma; Hematoma; Hemorrhage; Humans; Immunoblotting; Immunohistochemistry; Lipoproteins; Macrophages; Magnetic Resonance Imaging; Male; Middle Aged; Thromboplastin; Tomography, X-Ray Computed

Tissue factor (TF), a cell surface receptor of factor VII/VIIa, was initially recognized as an initiator of the extrinsic coagulation pathway. TF has recently been found to be expressed highly in certain types of malignant tumors. In addition, TF belongs to the interferon receptor family and is one of the immediate early genes, suggesting that TF may participate in the regulation of cell growth. However, the correlation between the expression of TF and cell growth is still unclear.. Expression of TF in 6 glioma cell lines and 44 glioma surgical specimens was studied by Northern blot analysis, Western blot analysis, immunohistochemistry, and in situ hybridization.. Northern blot analysis showed that glioma cells expressed minor novel transcripts of 3.3 kb and 1.6 kb, in addition to the transcripts of 2.2 kb and 3.1 kb that were previously reported. Western blot analysis revealed that the level of TF protein did not correlate with that of TF transcripts. Although immunohistochemical analysis of surgical specimens showed that all gliomas were positive for TF, it was interesting that 1 of 10 benign gliomas (10%) was positive for TF (malignancy Grade I-II), 13 of 14 anaplastic astrocytomas (86%) (malignancy grade III) and 19 of 20 glioblastomas (95%) (malignancy grade IV) were moderately or strongly positive for TF. In situ hybridization showed the expression of TF mRNA in glioma cells.. TF is expressed in glioma and the level of expression correlates with the histologic grade of malignancy.

Topics: Astrocytoma; Blotting, Northern; Blotting, Western; Cell Division; Gene Expression Regulation, Neoplastic; Genes, Immediate-Early; Glioblastoma; Glioma; Humans; Immunohistochemistry; In Situ Hybridization; Receptors, Cell Surface; Receptors, Interferon; RNA, Messenger; Thromboplastin; Transcription, Genetic; Tumor Cells, Cultured

Tissue factor (TF) is a cell surface glycoprotein that initiates the extrinsic coagulation protease cascade and it is expressed in some tumor cells. TF belongs to the interferon receptor family, and it is one of the early immediate genes, suggesting that TF has a biological function other than hemostasis. We investigated the expression of TF in gliomas. Immunocytochemistry showed the expression of TF in 3 glioma cell lines. Immunohistochemical analysis of 44 surgical specimens revealed that all gliomas were positive for TF, and 19 (95%) of 20 glioblastomas, 12 (86%) of 14 anaplastic astrocytomas and 1 (10%) of 10 benign gliomas were moderately or strongly positive for TF. Our study showed that TF is expressed in gliomas, and that the level of TF expression is correlated with the grade of malignancy of the glioma, suggesting that TF may participate in cell growth.

Topics: Brain Neoplasms; Cell Division; Glioblastoma; Glioma; Humans; Immunohistochemistry; Neoplasm Staging; Thromboplastin; Tumor Cells, Cultured

Reported is the immunoreactivity of tumoral and peritumoral vessels of 7 spinal cord gliomas induced in rats by means of ethylnitrosourea. Immunostaining for Factor VIII/RAg has been more strongly pronounced in endothelial cells of newly formed vessels at the tumor periphery, in comparison with tumoral vessels, and this is likely to suggest a differentiation process during extensive neovascularization of the tumor periphery. Endothelial distribution of vimentin in the peripheral vessels is more intensive than within the tumors, suggesting its relation to rapid new blood vessel formation. Immunostaining for GFAP and vimentin overlaps in astrocytes trapped within neoplasms, in astrocytes at the tumor periphery, and in the peritumoral zone.

Topics: Animals; Capillaries; Cell Transformation, Neoplastic; Endothelium, Vascular; Female; Glial Fibrillary Acidic Protein; Glioma; Immunohistochemistry; Pregnancy; Rats; Rats, Inbred Strains; Spinal Cord Neoplasms; Thromboplastin; Vimentin

Microvesicles (diameter ca 200 nm) from the cell-free supernatant of U87MG human glioblastoma cell caused platelet aggregation and coagulation in a manner identical with that previously shown for the intact cells. Both activities were inhibited by dansylarginine -N-(3-ethyl-1,5-pentanediyl) amide (DAPA), confirming the thrombin-dependent nature of both activities. The specific activities per microgram of protein were 2-10 times greater in the microvesicles than in the plasma membrane fraction, suggesting localization in specific membrane domains. Sucrose density centrifugation gave a single protein peak (density 1.14) with congruent procoagulant and platelet aggregating activities. Both activities required the extrinsic pathway, as shown by studies with factor-deficient plasmas, and both were inhibited by heating (60 min/100 degrees C), by reduction and alkylation, and by incubation of the microvesicles with rabbit anti-bovine brain tissue factor antibody. These observations were confirmed using microvesicles from the HL-60 human promyelocytic leukemia cells, which are known to contain tissue factor activity. The results suggest that both procoagulant and proaggregating activities are causally related through the presence of tissue factor in the microvesicles. Studies with the Baumgartner perfusion apparatus showed that U87MG microvesicles increased the size of adherent thrombi nearly tenfold and that these thrombi were associated with nucleated cells from the blood. The increase in adherent thrombi did not occur if perfusion was carried out in the presence of DAPA, confirming the role of thrombin in their formation.

Topics: Antibodies; Arginine; Blood Coagulation; Cell Line; Centrifugation, Density Gradient; Dansyl Compounds; Glioma; Humans; Neoplastic Stem Cells; Perfusion; Platelet Aggregation; Thromboplastin; Thrombosis

The thromboplastic activity and the fibrinolytic activity were examined in 7 human meningiomas and 6 gliomas obtained at neurosurgery. Two different haemostatic patterns emerged, meningiomas having lower thromboplastic and higher fibrinolytic activity than that of gliomas. This difference might help to explain the better haemostatic capacity of gliomas during and after operation than that of meningiomas.

Topics: Adult; Aged; Brain Neoplasms; Female; Fibrinolysis; Glioma; Hemostasis; Humans; Male; Meningioma; Middle Aged; Thromboplastin