thromboplastin and Ovarian-Neoplasms

Tissue factor (TF) is a cell surface receptor for coagulation factor VII (fVII). The TF-activated fVII (fVIIa) complex is an essential initiator of the extrinsic blood coagulation process. Interactions between cancer cells and immune cells via coagulation factors and adhesion molecules can promote progression of cancer, including epithelial ovarian cancer (EOC). This process is not necessarily advantageous, as tumor tissues generally undergo hypoxia due to aberrant vasculature, followed by reduced access to plasma components such as coagulation factors. However, hypoxia can activate TF expression. Expression of fVII, intercellular adhesion molecule-1 (ICAM-1), and multiple pro-inflammatory cytokines can be synergistically induced in EOC cells in response to hypoxia along with serum deprivation. Thus, pro-inflammatory responses associated with the TF-fVIIa-ICAM-1 interaction are expected within hypoxic tissues. Tumor tissue consists of multiple components such as stromal cells, interstitial fluid, albumin, and other micro-factors such as proton and metal ions. These factors, together with metabolism reprogramming in response to hypoxia and followed by functional modification of TF, may contribute to coagulation factor-driven inflammatory responses in EOC tissues. The aim of this review was to describe potential coagulation factor-driven inflammatory responses in hypoxic EOC tissues. Arguments were extended to clinical issues targeting this characteristic tumor environment.

Topics: Animals; Blood Coagulation; Blood Coagulation Factors; Carcinoma, Ovarian Epithelial; Female; Humans; Hypoxia; Inflammation; Inflammation Mediators; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Signal Transduction; Thromboplastin

The paper reviews the current studies on mechanism of thrombo-embolic complications in patients with malignancy. The neoplastic cells can express procoagulants like tissue factor (TF), cancer procoagulant (CP) and others, which induce thrombin formation in blood plasma and cause thrombo-embolic events, disseminated intravascular coagulation and other complications. Both procoagulants are acting via extrinsic pathway--TF makes complexes with factor VII and calcium ions, while CP acts directly on factor X transforming it into active form (Xa). Gynecological tumor-related complications occur in 20-30% patients with ovarian or uterine cancer.

Topics: Antineoplastic Agents; Female; Humans; Neoplasms; Ovarian Neoplasms; Thromboembolism; Thromboplastin; Uterine Neoplasms

Ovarian cancer (OC) is the deadliest gynecologic malignancy, with an overall 5-year survival rate of less than 30%. The existing paradigm for OC detection involves a serum marker, CA125, and ultrasound examination, neither of which is sufficiently specific for OC. This study addresses this deficiency through the use of a targeted ultrasound microbubble directed against tissue factor (TF).. TF expression was examined in both OC cell lines and patient-derived tumor samples via western blotting and IHC. In vivo microbubble ultrasound imaging was analyzed using high grade serous ovarian carcinoma orthotopic mouse models.. While TF expression has previously been described on angiogenic, tumor-associated vascular endothelial cells (VECs) of several tumor types, this is first study to show TF expression on both murine and patient-derived ovarian tumor-associated VECs. Biotinylated anti-TF antibody was conjugated to streptavidin-coated microbubbles and in vitro binding assays were performed to assess the binding efficacy of these agents. TF-targeted microbubbles successfully bound to TF-expressing OC cells, as well as an in vitro model of angiogenic endothelium. In vivo, these microbubbles bound to the tumor-associated VECs of a clinically relevant orthotopic OC mouse model.. Development of a TF-targeted microbubble capable of successfully detecting ovarian tumor neovasculature could have significant implications towards increasing the number of early-stage OC diagnoses. This preclinical study shows potential for translation to clinical use, which could ultimately help increase the number of early OC detections and decrease the mortality associated with this disease.

Topics: Animals; Early Detection of Cancer; Endothelial Cells; Female; Humans; Mice; Microbubbles; Ovarian Neoplasms; Thromboplastin; Ultrasonography

The fallopian tube fimbrial epithelium, which is exposed to the follicular fluid (FF) contents of ovulation, is regarded as the main origin of ovarian high-grade serous carcinoma. Previously, we found that growth factors in FF, such as IGF2, are responsible for the malignant transformation of fallopian tube epithelium. However, ovulation is a monthly transient event, whereas carcinogenesis requires continuous, long-term exposure. Here, we found the transformation activity of FF sustained for more than 30 days after drainage into the peritoneal fluid (PF). Hepatocyte growth factor (HGF), activated through the ovulation injury-tissue factor-thrombin-HGF activator (HGFA)-HGF cleavage cascade confers a sustained transformation activity to fallopian tube epithelium, high-grade serous carcinoma. Physiologically, the high reserve of the coagulation-HGF cascade sources a sustained level of HGF in PF, then to the blood circulation. This HGF axis promotes the growth of the corpus luteum and repair of tissue injury after ovulation.

Topics: Adult; Animals; Apoptosis; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Corpus Luteum; Cystadenocarcinoma, Serous; Fallopian Tube Neoplasms; Female; Follicular Fluid; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor II; Mice; Mice, Inbred NOD; Mice, SCID; Ovarian Neoplasms; Ovulation; Peptide Hydrolases; Prognosis; Serine Endopeptidases; Thrombin; Thromboplastin; Xenograft Model Antitumor Assays

Ovarian cancer is a known risk factor of venous thromboembolism (VTE). Thrombogenic factor expression and lymphocytic infiltrate have been reported in endometriosis and ovarian cancers. We reviewed 30 cases of ovarian carcinomas (high grade serous carcinoma, 10; endometrioid carcinoma, 10; clear cell carcinoma (CCC), 10) and 16 endometriotic lesions. We immunohistochemically investigated the expressions of tissue factor (TF), podoplanin, P-selectin, and number of CD4 and CD8 positive lymphocytes in cancer tissue and endometriotic lesions, along with their relationship with VTE. The expression of TF was higher in CCC. The TF expression and the number of CD8 positive cells were higher in cancer tissues with VTE than in those without VTE. The podoplanin or P-selectin expression did not differ among histological types or between cases with and without VTE. Our results demonstrated a high TF expression and intraepithelial CD8 cells in CCC, which were associated with VTE. The results suggest that infiltrating lymphocytes may affect TF expression that, in turn, influences VTE.

Topics: Adenocarcinoma, Clear Cell; Aged; Carcinoma, Endometrioid; CD8-Positive T-Lymphocytes; Female; Humans; Lymphocytes, Tumor-Infiltrating; Membrane Glycoproteins; Middle Aged; Ovarian Neoplasms; P-Selectin; Thromboplastin; Thrombosis; Venous Thromboembolism

Ovarian clear cell carcinoma (OCCC) and high grade serous ovarian cancer (HGSOC) are associated with the highest risk of VTE among patients with epithelial ovarian cancer (EOC). Tissue factor (TF) is a transmembrane glycoprotein which can trigger thrombosis. We sought to evaluate if there is an association between VTE and tumor expression of tissue factor (TF), plasma TF, and microvesicle TF (MV TF) activity in this high-risk population.. We performed a case-control study of OCCC and HGSOC patients with and without VTE. 105 patients who underwent surgery at a tertiary care center between January 1995 and October 2013 were included. Plasma TF was measured with an enzyme-linked immunosorbent assay. A TF-dependent Factor Xa generation assay was used to measure MV TF activity. Immunohistochemical (IHC) analysis was performed to evaluate tumor expression of TF.. 35 women with OCCC or HGSOC diagnosed with VTE within 9months of surgery were included in the case group. Those with VTE had a worse OS, p<0.0001, with a greater than three-fold increase in risk of death, HR 3.33 (CI 1.75-6.35). There was no significant difference in median plasma TF level or MV TF activity level between patients with and without VTE. OCCC patients had greater expression of TF in their tumors than patients with HGSOC, p<0.0001.. TFMV activity and plasma TF level were not predictive of VTE in this patient population. Given the extensive expression of TF in OCCC tumors, it is unlikely IHC expression will be useful in risk stratification for VTE in this population.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Ovarian Epithelial; Case-Control Studies; Enzyme Multiplied Immunoassay Technique; Female; Humans; Immunohistochemistry; Middle Aged; Neoplasm Staging; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Survival Analysis; Thromboplastin; Venous Thrombosis

Most cancer cells trigger thrombin generation (TG) to various extent. In the present study, we dissected the mechanisms responsible for the procoagulant activity of pancreatic adenocarcinoma cells (BXPC3), a highly thrombogenic cancer type, and breast cancer cells (MCF7), a less thombogenic tumor type. TG of normal plasma was assessed by the Thrombinoscope (CAT®) in the presence or absence of cancer cells. TG was also assessed in plasma depleted of clotting factors, in plasma spiked with tissue factor (TF) and/or procoagulant phospholipids, in plasma spiked with an anti-TF monoclonal antibody or with corn trypsin inhibitor (CTI). The presence of alternatively spliced TF (asTF), TF activity (TFa) and cancer procoagulant (CP) levels were determined. TFa and asTF were highly expressed by BXPC3 cells, compared to MCF7 cells, while CP levels were higher in MCF7 cells. BXPC3 cells had a stronger effect on TG than MCF7 cells. Accordingly, anti-TF had more inhibitory activity on TG triggered by BXPC3 cells while CTI had more pronounced inhibitory effect on TG triggered by MCF7 cells. TG enhancement by both BXPC3 and MCF7 cells was mediated by FVII and intrinsic tenase while FXII and FXI were also important for MCF7 cells. The induction of TG by BXPC3 cells was mainly driven by the TF pathway while TG generation triggered by MCF7 cells was also driven by FXII activation. Therefore, hypercoagulability results from a combination of the inherent procoagulant properties of cancer cell-associated TF as well as of procoagulant phospholipids in the plasma microenvironment.

Topics: Antibodies, Monoclonal; Breast Neoplasms; Cell Line, Tumor; Colonic Neoplasms; Cysteine Endopeptidases; Factor XII; Female; HCT116 Cells; HT29 Cells; Human Umbilical Vein Endothelial Cells; Humans; MCF-7 Cells; Neoplasm Proteins; Ovarian Neoplasms; Pancreatic Neoplasms; Platelet-Rich Plasma; Thrombin; Thromboplastin

A documented relationship between ovarian cancer and thrombosis does exist. Low-molecular-weight heparins (LMWHs) are cornerstone drugs in the primary prevention and treatment of venous thromboembolic events in patients with cancer. However, cancer cells may alter the efficiency of these antithrombotic agents.. We aimed to characterize the procoagulant phenotype of human epithelial ovarian adenocarcinoma cells, IGROV1, and to compare the capacity of tinzaparin and enoxaparin to inhibit thrombin generation triggered by these cells.. Thrombin generation induced by different concentrations of IGROV1 cells on platelet poor plasma (PPP) was assessed by the calibrated automated thrombogram assay. Tissue factor (TF) expression was studied using Western blot analysis. Then, the experimental model of thrombin generation was used to compare the inhibitory effect of clinically relevant concentrations of both tinzaparin and enoxaparin. The inhibitory concentration 50 (IC50) of the mean rate index and the endogenous thrombin potential and the 2-fold increase in lag time were analyzed on the basis of the anti-Xa and anti-IIa activities of the LMWHs.. IGROV1 cells suspended into PPP resulted in a significant increase in thrombin generation in the absence of any exogenous source of TF and phospholipids. Tissue factor was expressed by IGROV1 cells. Tinzaparin was a more potent inhibitor of thrombin generation than enoxaparin. The inhibition of thrombin generation induced by IGROV1 cancer cells depended mainly on the anti-Xa activity of the LMWHs.. This experimental study in ovarian cancer cells demonstrates that the antithrombotic activity of LMWHs is not completely predicted by the anti-Xa or anti-IIa activities measured in PPP.

Topics: Cell Line, Tumor; Dose-Response Relationship, Drug; Enoxaparin; Factor Xa Inhibitors; Female; Fibrinolytic Agents; Heparin, Low-Molecular-Weight; Humans; Ovarian Neoplasms; Plasma; Prothrombin; Thrombin; Thromboplastin; Tinzaparin

Our 2007 study of 32 patients with ovarian cancer reported the possible involvement of tissue factor (TF) in the development of venous thromboembolism (VTE) before treatment, especially in clear cell carcinoma (CCC). This follow-up study further investigated this possibility in a larger cohort.. We investigated the intensity of TF expression (ITFE) and other variables for associations with VTE using univariate and multivariate analyses in 128 patients with epithelial ovarian cancer initially treated between November 2004 and December 2010, none of whom had received neoadjuvant chemotherapy. Before starting treatment, all patients were ultrasonographically screened for VTE. The ITFE was graded based on immunostaining of surgical specimens.. Histological types were serous carcinoma (n = 42), CCC (n = 12), endometrioid carcinoma (n = 15), mucinous carcinoma (n = 53), and undifferentiated carcinoma (n = 6). The prevalence of VTE was significantly higher in CCC (34%) than in non-CCC (17%, P = 0.03). As ITFE increased, the frequencies of CCC and VTE increased significantly (P < 0.001 and P = 0.014, respectively). Multivariate analysis identified TF expression and pretreatment dimerized plasmin fragment D level as significant independent risk factors for VTE development. These factors showed particularly strong impacts on advanced-stage disease (P = 0.021).. The 2007 cohort was small, preventing multivariate analysis. This study of a larger cohort yielded stronger evidence that the development of VTE in epithelial ovarian cancer may involve TF expression in cancer tissues.

Topics: Adult; Aged; Aged, 80 and over; Analysis of Variance; Biomarkers, Tumor; Carcinoma, Ovarian Epithelial; Cohort Studies; Female; Humans; Middle Aged; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Thromboplastin; Venous Thromboembolism

Targeting cancer stem cell (CSC) represents a promising therapeutic approach as it can potentially fight cancer at its root. The challenge is to identify a surface therapeutic oncotarget on CSC. Tissue factor (TF) is known as a common yet specific surface target for cancer cells and tumor neovasculature in several solid cancers. However, it is unknown if TF is expressed by CSCs. Here we demonstrate that TF is constitutively expressed on CD133 positive (CD133+) or CD24-CD44+ CSCs isolated from human cancer cell lines, tumor xenografts from mice and breast tumor tissues from patients. TF-targeted agents, i.e., a factor VII (fVII)-conjugated photosensitizer (fVII-PS for targeted photodynamic therapy) and fVII-IgG1Fc (Immunoconjugate or ICON for immunotherapy), can eradicate CSC via the induction of apoptosis and necrosis and via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, respectively. In conclusion, these results demonstrate that TF is a novel surface therapeutic oncotarget for CSC, in addition to cancer cell TF and tumor angiogenic vascular endothelial TF. Moreover, this research highlights that TF-targeting therapeutics can effectively eradicate CSCs, without drug resistance, isolated from breast, lung and ovarian cancer with potential to translate into other most commonly diagnosed solid cancer, in which TF is also highly expressed.

Topics: A549 Cells; Animals; Antibody-Dependent Cell Cytotoxicity; Cell Line, Tumor; Female; Humans; Lung Neoplasms; Male; Metalloporphyrins; Mice; Mice, SCID; Molecular Targeted Therapy; Neoplastic Stem Cells; Ovarian Neoplasms; Photochemotherapy; Thromboplastin; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Enhanced tissue factor (TF) expression in epithelial ovarian cancer (EOC) is associated with aggressive disease. Our objective was to evaluate the role of the TF-factor VIIa-protease-activated receptor-2 (PAR-2) pathway in human EOC.. TCGA RNAseq data from EOC databases were analyzed for PAR expression. Cell and microparticle (MP) associated TF protein expression (Western blot) and MP-associated coagulant activity were determined in human EOC (SKOV-3, OVCAR-3 and CaOV-3) and control cell lines. PAR-1 and PAR-2 protein expressions were similarly examined. The PAR dependence of VEGF-A release (ELISA) and chemotactic migration in response to FVIIa and cellular proliferation in response to thrombin was evaluated with small molecule antagonists.. Relative mRNA expression consistently demonstrated PAR-2>PAR-1≫PAR-3/4 in multiple EOC datasets. Human EOC cell line lysates confirmed expression of TF, PAR-1 and PAR-2 proteins. MPs isolated from EOC cell lines demonstrated markedly enhanced (4-10 fold) TF coagulant activity relative to control cell lines. FVIIa induced a dose-dependent increase in VEGF-A release (2.5-3 fold) from EOC cell lines that was abrogated by the PAR-2 antagonist ENMD-1068. FVIIa treatment of CaOV-3 and OVCAR-3 cells resulted in increased chemotactic migration that was abolished by ENMD-1068. Thrombin induced dose-dependent EOC cell line proliferation was completely reversed by the PAR-1 antagonist vorapaxar. Small molecule antagonists had no effect on these phenotypes without protease present.. Enhanced activity of the TF-FVIIa-PAR-2 axis may contribute to the EOC progression via PAR-2 dependent signaling that supports an angiogenic and invasive phenotype and local thrombin generation supporting PAR-1 dependent proliferation.

Topics: Blotting, Western; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemotaxis; Factor VIIa; Female; Humans; Neoplasm Invasiveness; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Receptor, PAR-1; Receptor, PAR-2; Receptors, Proteinase-Activated; RNA, Messenger; Signal Transduction; Thrombin; Thromboplastin; Vascular Endothelial Growth Factor A

Thromboembolic events occur frequently in ovarian cancer patients. Tissue factor (TF) is often overexpressed in tumours, including ovarian clear-cell carcinoma (CCC), a subtype with a generally poor prognosis. TF-coagulation factor VII (fVII) complexes on the cell surface activate downstream coagulation mechanisms. Moreover, cancer cells secrete extracellular vesicles (EVs), which act as vehicles for TF. We therefore examined the characteristics of EVs produced by ovarian cancer cells of various histological subtypes. CCC cells secreted high levels of TF within EVs, while the high-TF expressing breast cancer cell line MDA-MB-231 shed fewer TF-positive EVs. We also found that CCC tumours with hypoxic tissue areas synthesised TF and fVII in vivo, rendering the blood of xenograft mice bearing these tumours hypercoagulable compared with mice bearing MDA-MB-231 tumours. Incorporation of TF into EVs and secretion of EVs from CCC cells exposed to hypoxia were both dependent on the actin-binding protein, filamin-A (filA). Furthermore, production of these EVs was dependent on different protease-activated receptors (PARs) on the cell surface. These results show that CCC cells could produce large numbers of TF-positive EVs dependent upon filA and PARs. This phenomenon may be the mechanism underlying the increased incidence of venous thromboembolism in ovarian cancer patients.

Topics: Animals; Blood Coagulation; Cell Line, Tumor; Extracellular Vesicles; Factor VII; Factor Xa; Female; Filamins; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Mice; Mice, SCID; Neoplasm Transplantation; Ovarian Neoplasms; Receptors, Proteinase-Activated; Thromboplastin; Thrombosis; Venous Thromboembolism

Tissue factor (TF) is involved in tumor growth and metastasis and contributes to venous thromboembolism (VTE) in cancer, including gynecological malignancies. The diagnostic value of microvesicle-associated TF procoagulant activity (MV TF PCA) in women with suspected ovarian cancer, however, has not been studied.. To evaluate MV TF PCA as a diagnostic tool in women with an ovarian mass of unknown etiology and as a predictive biomarker for perioperative VTE.. Plasma MVs were isolated by high-speed centrifugation and analyzed for TF-specific PCA by single-stage clotting assay. In addition, plasma TF antigen and soluble P-selectin (sCD62P) were measured by ELISA.. D-Dimer, MV TF PCA, and sCD62P, but not the tumor marker, CA-125, significantly differentiated patients with malignant (n=40) from those with benign tumors (n=15) and healthy controls (n=34). In cancer patients, only D-Dimer and CA-125 correlated with the FIGO stage. An abnormal D-dimer had the highest sensitivity for the diagnosis of cancer, while MV TF PCA above the ROC curve-derived cut-off value of 182U/mL had the highest specificity. By multivariate logistic regression analysis, addition of MV TF PCA conferred diagnostic benefit to the single variables, CA-125 (p=0.052) and D-dimer (p=0.019). Perioperative VTE occurred in 16% of cancer patients and was associated with an advanced FIGO stage, but not MV TF PCA. There was no difference in plasma TF antigen levels between study groups.. MV TF PCA, but not plasma TF antigen, may provide valuable additional information for the diagnostic work-up of women with suspected ovarian cancer.

Topics: Adult; Aged; Biomarkers, Tumor; CA-125 Antigen; Cell-Derived Microparticles; Female; Fibrin Fibrinogen Degradation Products; Hemostasis; Humans; Middle Aged; Ovarian Neoplasms; Ovary; P-Selectin; Perioperative Period; Preoperative Period; Thromboplastin; Venous Thromboembolism

An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and "Metastasis Initiating Cell (MIC)" marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients.. With informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay.. The co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity.. We present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci.

Topics: Biomarkers; Blood Platelets; Cell Communication; Cell Movement; Chemotactic Factors; Female; Humans; Neoplasm Staging; Ovarian Neoplasms; Phenotype; Platelet Glycoprotein GPIb-IX Complex; Thromboplastin; Tumor Cells, Cultured

Preoperative evaluation of patients presenting with ovarian masses is challenging, partly due to shortcomings with the commonly used marker, CA-125. Ovarian cancer is associated with systemic coagulation activation. Measurement of D-dimer, serum tissue factor (TF), and the coagulation process as a whole are considered candidates for improving discrimination between benign and malignant ovarian masses. We therefore sought to identify possible benefits by analyzing preoperative coagulation status in conjunction with CA-125 in patients with ovarian masses. Preoperative blood from 95 patients with ovarian masses (75 benign, 20 malignant) and 30 controls was analyzed, prospectively. Thromboelastography served for global hemostatic assessment. Plasma TF antigen and D-dimer were measured by ELISA and microparticle-associated TF activity by thrombin generation assay. TF microparticles were enumerated by flow cytometry. Time to clot formation by thromboelastography was similar between patients having either benign or malignant ovarian tumors. Clot formation rate, clot strength, and coagulation index were significantly increased in patients having malignant versus benign tumors, indicating that thromboelastography differentiated malignant from benign tumors. D-dimer alone differentiated malignant from benign ovarian tumors and also improved differentiation when combined with CA-125. Circulating TF antigen, activity, and TF microparticle numbers, however, failed to differentiate benign from malignant tumors. Significant coagulation activation occurs in women with ovarian malignancies. Plasma D-dimer may help discriminate between patients with benign and malignant tumors. Thromboelastography may also contribute meaningfully when combined with CA-125 in the preoperative evaluation of ovarian masses. Larger studies are needed to assess these possibilities.

Topics: Adult; Aged; Biomarkers, Tumor; Blood Coagulation; CA-125 Antigen; Diagnosis, Differential; Female; Fibrin Fibrinogen Degradation Products; Fibrinogen; Hemostasis; Humans; Middle Aged; Ovarian Neoplasms; Preoperative Period; Sensitivity and Specificity; Thrombelastography; Thromboplastin

Ovarian cancer is known to display a particular association with venous thromboembolism (VTE) with reports up to 42% of patients developing thromboembolic complications. Tissue Factor (TF) and its inhibitor Tissue Factor Pathway Inhibitor (TFPI) have been implicated in VTE risk in cancer. The aim of this study was to measure tumour derived TF and TFPI and to investigate their potential role in VTE in ovarian cancer patients.. TF and TFPI mRNA expression was measured using TaqMan real time PCR in 99 ovarian tumour samples. Nineteen cases complicated by VTE were matched to 19 cases without VTE. TF and TFPI protein levels were measured using ELISA and immunohistochemistry was used to localize TF expression. The role of TF expression on overall survival was also determined.. TF mRNA and protein expression was increased in tumours from patients with clear cell carcinoma (p<0.001). TF protein expression was also increased in endometroid carcinoma (P<0.01) compared with benign tumours. TFPI mRNA expression was increased in clear cell carcinoma (P<0.01). TF mRNA and antigen level was increased in malignant tumours of patients who developed VTE compared with matched malignant õtumours of patients who remained thrombosis free (P<0.01). There was no difference in TFPI expression between the two groups.. TF expression in ovarian cancer is significantly higher in patients who develop VTE. TF expression was increased in clear cell ovarian cancer and endometroid cancer and this may explain the higher risk of VTE in these subgroups. TF derived from these tumours may be the trigger for VTE in ovarian cancer.

Topics: Aged; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lipoproteins; Middle Aged; Ovarian Neoplasms; Ovary; RNA, Messenger; Thromboplastin; Up-Regulation; Venous Thrombosis

Tissue factor (TF), an initiator of blood coagulation, participates in cancer progression and metastasis. We recently found that inhibition of MAPK/ERK upregulated both full length TF (flTF) and soluble isoform TF (asTF) gene expression and cell-associated TF activity in breast cancer MDA-MB-231 cells. We explored the possible mechanisms, especially the possible interaction with EGFR and PI3K/Akt pathways.. A plasmid containing TF promoter -2174 ~ +128 plus luciferase reporter gene was introduced into MDA-MB-231 cells to evaluate TF promoter activity. In order to study the interaction of these pathways, ERK inhibitor (PD98059), PI3K inhibitors (LY294002, wortmannin), Akt inhibitor (A6730), and EGFR inhibitor (erlotinib) as well as the corresponding siRNAs were used to treat MDA-MB-231 cells, and ovarian cancer OVCAR-3 and SKOV-3 cells. Quantitative PCR and western blot were used to determine TF expression. One stage clotting assays were used to measure pro-coagulation activity of the MDA-MB-231 cells.. We show that PI3K inhibitors LY294002, wortmannin and A6730 significantly inhibited TF promoter activity, and reduced TF mRNA and protein levels due to the inhibition of Akt phosphorylation. In contrast, ERK inhibitor PD98059 and ERK siRNA enhanced TF promoter activity by 2.5 fold and induced an increase in TF mRNA and protein levels in a dose dependent manner in these cells. The PI3K/Akt pathway was shown to be involved in PD98059-induced TF expression because the induction was inhibited by PI3K/Akt inhibitors. Most interestingly, the EGFR inhibitor erlotinib and EGFR siRNA also significantly suppressed PD98059- or ERK siRNA-induced TF promoter activity and TF protein expression. Similar results were found with ovarian cancer cells SKOV-3 and OVCAR-3. Furthermore, in MDA-MB-231, mRNA levels of asTF were regulated in a similar way to that of TF in response to the cell treatment.. This study showed a regulatory mechanism in which MAPK/ERK signals inhibit EGFR/PI3K/Akt-mediated TF expression in breast cancer MDA-MB-231 cells. The same regulation was observed in ovarian cancer OVCAR-3 and SKOV-3 cells. Interestingly, we observed that both flTF and asTF could be regulated in a parallel manner in MDA-MB-231. As the PI3K/Akt pathway and EGFR regulate TF expression in cancer cells, targeting these signaling components is expected to potentially inhibit TF expression-associated tumor progression.

Topics: Blood Coagulation; Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; Collagen; Drug Combinations; Enzyme Inhibitors; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Laminin; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Promoter Regions, Genetic; Proteoglycans; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Thromboplastin; Transcription, Genetic; Tumor Cells, Cultured

Microparticles are known to be increased in various malignancies. In this prospective study, microparticle levels were evaluated in patients with benign and malignant ovarian lesions.. Microparticles from platelets/megakaryocytes, activated platelets and endothelial cells, tissue factor exposing microparticles and D-dimer values were examined in patients with newly diagnosed ovarian lesions before surgery, and were correlated with tumor histology.. Higher counts of CD63-positive microparticles were detected in patients with ovarian cancer [mean=276×10(6) (range: 64-948)/l; n=12] as compared to patients with benign ovarian tumors [146×10(6) (45-390)/l; n=21; p=0.014]. D-dimer values were also increased in patients with cancer [860 (180-4500) ng/l versus 280 (170-2720) ng/l; p=0.001].. Elevated levels of CD63-positive microparticles and D-dimer reflect the procoagulant phenotype of these patients. However, for the discrimination between benign and malignant ovarian tumors, measuring preoperative levels of microparticles does not seem to be helpful.

Topics: Adult; Aged; Aged, 80 and over; Annexin A5; Case-Control Studies; Cell-Derived Microparticles; Female; Fibrin Fibrinogen Degradation Products; Humans; Integrin beta3; Middle Aged; Ovarian Neoplasms; Prospective Studies; Tetraspanin 30; Thromboplastin

Tumor-associated macrophage infiltration and up-regulation of tissue factor-factor VII (TF-FVIIa) complex have been observed in the peritoneum and stroma of epithelial ovarian cancer (EOC). However, it is not clear how tumor-associated macrophage and TF-FVIIa complex promotes EOC invasion. In the present study, we aimed to determine the mechanism by which interaction of TF-FVIIa and monocytes (MOs) promotes EOC metastasis.. Matrigel invasion assay was used to analyze the potential of EOC metastasis. Enzyme-linked immunosorbent assay and real-time polymerase chain reaction were used to detect expressions of cytokines and chemokines. Fluorescence-activated cell sorting was used to count the percentage of CD14, CD68, and CD163 of MOs.. We found that the TF-FVIIa complex caused dynamic changes in MOs cytokine and chemokine expression. CD14 and CD163 were also upregulated on MOs by TF-FVIIa. Epithelial ovarian cancer cells were cocultured with TF-FVIIa-stimulated MOs, demonstrating increased invasion potential. Interleukin 8 (IL-8) was proposed as the major chemoattractant mediating EOC invasion based on MOs messenger RNA and protein expression profiles. Anti-IL-8 monoclonal neutralizing antibody attenuated EOC cell invasion in a concentration-dependent manner, and tumor necrosis factor α from TF-FVIIa-stimulated MOs was observed to amplify IL-8 production. The following transcription factors in MOs were activated by TF-FVIIa and inhibited by the tissue factor pathway inhibitor: oncogenes HIF-1α, HIF-1β, Oct I, Oct II, and Egr-1; inflammatory mediators c-Fos and c-Rel; and STAT family members STAT5A and STAT5B.. Our study suggested that the interaction between the TF-FVIIa complex might play a role in mediating EOC invasion and metastasis depending on MOs mechanism.

Topics: Carcinoma, Ovarian Epithelial; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Factor VIIa; Female; Gene Expression Regulation, Neoplastic; Humans; Monocytes; Multiprotein Complexes; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Signal Transduction; Thromboplastin; Up-Regulation

We evaluated the expression of tissue factor (TF) in ovarian cancer (EOC) and the potential of hI-con1, an antibody-like molecule targeting TF, as a novel form of therapy against chemotherapy-resistant ovarian disease. We studied the expression of TF in 88 EOC by immunohistochemistry (IHC) and real-time-PCR (qRT-PCR) and the levels of membrane-bound-complement-regulatory-proteins CD46, CD55 and CD59 in primary EOC cell lines by flow-cytometry. Sensitivity to hI-con1-dependent-cell-mediated-cytotoxicity (IDCC), complement-dependent-cell-cytotoxicity and inhibition of IDCC by γ-immunoglobulin were evaluated in 5-h (51)chromium-release-assays. Cytoplasmic and/or membrane TF expression was observed in 24 out of 25 (96%) of the EOC samples tested by IHC, but not in normal ovarian-tissue. EOC with clear cell histology significantly overexpress TF when compared to serous, endometrioid, or undifferentiated tumors by qRT-PCR. With a single exception, all primary EOC that overexpressed TF demonstrated high levels of CD46, CD55 and CD59 and regardless of their histology or resistance to chemotherapy, were highly sensitive to IDCC. The effect of complement and physiologic doses of γ-immunoglobulin on IDCC in ovarian cancer cell lines overexpressing TF was tumor specific and related to the overexpression of CD59 on tumor cells. Small-interfering-RNA-mediated knockdown of CD59 expression in ovarian tumors significantly increased hI-con1-mediated cytotoxic activity in vitro. Finally, low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (P < 0.01). hI-con1 molecule induces strong cytotoxicity against primary chemotherapy-resistant ovarian cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of ovarian tumors refractory to standard treatment modalities.

Topics: Cell Line, Tumor; Factor VII; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoconjugates; Immunoglobulin Fc Fragments; Immunotherapy; Ovarian Neoplasms; Thromboplastin

Pulmonary tumor thrombotic microangiopathy (PTTM) is a rare clinicopathologic entity causing severe pulmonary hypertension, right-side heart failure, and sudden death. Its histologic features include widespread tumor emboli of the small arteries and arterioles of the lung, associated with thrombus formation and fibrocellular and fibromuscular intimal proliferation. The most frequent causative neoplasm for PTTM is gastric cancer, but lesions in other organs, including the ovary, have been occasionally identified as primary causes. Detailed molecular mechanisms underlying PTTM remain unclear, but some studies have suggested that tissue factor (TF) and vascular endothelial growth factor (VEGF) expressed by tumor cells may be involved in the pathogenesis for cases of gastric cancer. However, little is known about these molecules in PTTM caused by neoplasms of non-gastric origin. Here, we report the autopsy findings of a 42-year-old woman with ovarian cancer showing positive immunoreactivity for both TF and VEGF who died suddenly of PTTM. The present case provides support for the conclusion that these factors may be involved in the pathogenesis of PTTM, independent of the causal neoplasm.

Topics: Adenocarcinoma, Clear Cell; Adult; Antineoplastic Combined Chemotherapy Protocols; Autopsy; Chemotherapy, Adjuvant; Fatal Outcome; Female; Gynecologic Surgical Procedures; Heart Arrest; Humans; Immunohistochemistry; Lung Neoplasms; Neoplastic Cells, Circulating; Ovarian Neoplasms; Pulmonary Embolism; Thromboplastin; Thrombosis; Vascular Endothelial Growth Factor A

Thromboembolic events are a major complication in ovarian cancer patients. Tissue factor (TF) is frequently overexpressed in ovarian cancer tissue and correlates with intravascular thrombosis. TF binds to coagulation factor VII (fVII), changing it to its active form, fVIIa. This leads to activation of the extrinsic coagulation cascade. fVII is produced by the liver and believed to be supplied from blood plasma at the site of coagulation. However, we recently showed that ovarian cancer cells express fVII transcripts under normoxia and that this transcription is inducible under hypoxia. These findings led us to hypothesise that ovarian cancer cells are intrinsically associated with TF-fVIIa coagulation activity, which could result in thrombosis.. In this study, we examined whether ectopically expressed fVII could cause thrombosis by means of immunohistochemistry, RT-PCR, western blotting and flow cytometry.. Ectopic fVII expression occurs frequently in ovarian cancers, particularly in clear cell carcinoma. We further showed that ovarian cancer cells express TF-fVIIa on the cell surface under normoxia and that this procoagulant activity is enhanced by hypoxic stimuli. Moreover, we showed that ovarian cancer cells secrete microparticles (MPs) with TF-fVIIa activity. Production of this procoagulant secretion is enhanced under hypoxia.. These results raise the possibility that cancer cell-derived TF-fVIIa could cause thrombotic events in ovarian cancer patients.

Topics: Cell Hypoxia; Cell Line, Tumor; Cell-Derived Microparticles; Factor VII; Female; Humans; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Thromboplastin; Venous Thromboembolism

As a micro-wound and target-aimed technology without special limitation, Electric Pulses have been widely researched in tumor treatment and the effects have been demonstrated by a series of experiments, yet the mechanism has not been explained clearly. In this experiment, energy controllable steep pulse (ECSP) was used to treat nude mice bearing human ovarian tumor, and the result was compared with that of the control group. The expression of an important coagulant factor-tissue factor (TF) was analyzed, as TF was also a tumor indicator of invasion and metastasis, the result may indicate the relationship among ECSP, thrombosis and tumor invasion. In this study, to shed light on the mechanism of tumor treatment in electrical fields, nude mice bearing ovarian tumors were randomly divided into the treated group and the untreated group. We treated the former group and took out the tumor instantly. The thrombosis and necrosis of ovarian tumor were observed under microscope. The expression of TF was analyzed by SP immunohistochemistry and RT-PCR. Lower level of TF expression was noticed in the tumor tissue treated by ECSP, and more apparent thrombosis was also seen in this group. The results make it clear that ECSP can accelerate thrombosis and consume coagulant factors such as TF, and that low expression of TF in tumor tissue can cut out the signal paths of tumor invasion. So it is suggested that ECSP may restrain tumor invasion and metastasis by modulating thrombosis.

Topics: Animals; Electric Stimulation Therapy; Electromagnetic Fields; Electroporation; Female; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Ovarian Neoplasms; Random Allocation; RNA, Messenger; Thromboplastin

Ovarian cancer, and clear cell carcinoma in particular, reportedly increases the risk of venous thromboembolism (VTE). However, the mechanisms remain unclear. Tissue factor (TF) supposedly represents a major factor in the procoagulant activities of cancer cells. The present study examined the involvement of TF expression in VTE for patients with ovarian cancer. Subjects comprised 32 consecutive patients (mean age 49.8 years) with histologically confirmed ovarian cancer. Presence of VTE was examined using a combination of clinical features, D-dimer levels and venous ultrasonography. Immunohistochemical analysis was used to evaluate TF expression into 4 degrees. Venous thromboembolism was identified in 10 of the 32 patients (31%), including five of the 11 patients with clear cell carcinoma. Tissue factor expression was detected in cancer tissues from 24 patients and displayed significant correlations with VTE development (P=0.0003), D-dimer concentration (P=0.003) and clear cell carcinoma (P<0.05). Multivariate analysis identified TF expression as an independent predictive factor of VTE development (P<0.05). Tissue factor (TF) expression is a possible determinant of VTE development in ovarian cancer. In particular, clear cell carcinoma may produce excessive levels of TF and is more likely to develop VTE.

Topics: Adolescent; Adult; Aged; Female; Humans; Immunohistochemistry; Middle Aged; Ovarian Neoplasms; Thromboembolism; Thromboplastin

Tissue factor (TF) is a procoagulant that plays an important part in tumor angiogenesis. We sought to determine the role of preoperative serum TF levels in predicting clinical outcome in patients with ovarian cancer.. TF expression was determined by reverse transcriptase polymerase chain reaction in ovarian cell lines. Using enzyme-linked immunosorbent assay, we assessed preoperative serum TF levels in 98 women with invasive epithelial ovarian carcinoma, 30 with low malignant potential (LMP) tumors, 16 with benign tumors, and a separate validation group of 39 women with adnexal masses. Clinical information was gathered from chart review.. TF was expressed in four of the five ovarian cancer cell lines, but absent in the nontransformed cells. Ovarian cancer patients had a median preoperative serum TF level of 85.2 pg/mL, which was significantly higher than in those with LMP tumors (12.8 pg/mL; P < .01) and benign adnexal disease (30.7 pg/mL; P < .01). TF >or= 190 pg/mL was significantly associated with decreased patient survival (P < .01). After adjusting for other clinical variables in a multivariate Cox regression model, TF >or= 190 pg/mL was an independent prognostic factor (P < .01). Analysis of serum TF levels from the validation set confirmed that high TF (>or=190 pg/mL) was associated with a 3.4-fold increase in risk of death from disease (P = .02) and shorter survival (P = .01).. Preoperative serum TF levels are significantly elevated in patients with ovarian carcinoma. Elevated preoperative TF level is an independent prognostic factor for death from disease.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Profiling; Humans; Middle Aged; Neovascularization, Pathologic; Ovarian Neoplasms; Predictive Value of Tests; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Survival Analysis; Thromboplastin

Blood coagulation factor VII (fVII) is physiologically synthesized in the liver and released into the blood. Binding of fVII to tissue factor (TF) at sites of vascular injury triggers coagulation and hemostasis. TF/fVIIa complex formation on the surface of cancer cells plays important roles in cancer biology. Although fVII is synthesized by hepatocellular carcinoma, it remained unclear how TF/fVIIa complex formation and promigratory signaling can occur for most other cancers in extravascular locations. Here, we show by reverse transcription-PCR analysis that nonhepatic cancer cell lines constitutively express fVII mRNA and that endogenously synthesized fVIIa triggers coagulation activation on these cells. fVIIa expression in cancer cells is inducible under hypoxic conditions and hypoxia-inducible factor-2 alpha bound the promoter region of the FVII gene in chromatin immunoprecipitation analyses. Constitutive fVII expression in an ovarian cancer cell line enhanced both migration and invasion. Enhanced motility was blocked by anti-TF antibodies, factor Xa inhibition, and anti-protease-activated receptor-1 antibody treatment, confirming that TF/fVIIa stimulated migration by triggering cell signaling. This study shows that ectopic synthesis of fVII by cancer cells is sufficient to support proinvasive factor Xa-mediated protease-activated receptor-1 signaling and that this pathway is inducible under hypoxia.

Topics: Basic Helix-Loop-Helix Transcription Factors; Blood Coagulation; Carbon-Carbon Ligases; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Factor VII; Factor Xa; Female; Humans; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasms; Ovarian Neoplasms; Receptor, PAR-1; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Thromboplastin; Transfection

To construct the expression vector of human tissue factor (TF), and investigate the influence of TF/coagulant factor VIIa (FVIIa) complex on the transcriptional expression of urokinase plasminogen activator (u-PA) and u-PA receptor (u-PAR) in human ovarian cancer.. The human TF cDNA was obtained from placenta by RT-PCR and then inserted into eukaryotic expression vector pcDNA3 to obtain the TF-pcDNA3 combinant. This combinant was transfected into human ovarian cancer cell line A2780 by lipofectamine. Stably-transfected cells A2780/TF were screened. A2780 and A2780/TF cell lines were stimulated by FVIIa respectively, and the transcriptional levels of u-PA and u-PAR were examined by RT-PCR.. (1) The constructed product was identified as TF-pcDNA3 combinant by sequencing. (2) TF was highly expressed not only at transcriptional level in the stable-transfected A2780/TF cell (transfected cell 3.91 +/- 0.28, untransfected cell 0.97 +/- 0.23, P < 0.01), but also on the membrane of the cell surface [transfected cell (48.56 +/- 9.53)%, untransfected cell (2.73 +/- 1.15)%, P < 0.01]. (3) The u-PA and u-PAR mRNA levels in A2780 cell line did not change significantly after stimulated by FVIIa; (4) While stimulated by FVIIa, the u-PAR mRNA levels in A2780/TF cells increased significantly in both dose-dependent and time-dependent manner, while the u-PA mRNA levels did not change significantly; (5) In the A2780/TF cell line the enhanced expression of u-PAR mRNA by FVIIa was significantly inhibited by coincubated with anti-TF antibody.. TF/FVIIa complex could up-regulate the transcription of u-PAR in human ovarian cancer cells so as to enhance tumor invasion and metastasis.

Topics: Cell Line, Tumor; Factor VIIa; Female; Gene Expression Regulation, Neoplastic; Humans; Ovarian Neoplasms; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; Thromboplastin; Up-Regulation; Urokinase-Type Plasminogen Activator

To explore the role of tissue factor/activated factor VII (TF/FVIIa) complex in human ovarian cancer invasion and metastasis.. (1) Constructed an expression vector of TF, pcDNA3-TF and established a human ovarian cell line A2780/TF expressing high level TF by using molecular cloning and gene transfection techniques. (2) By Boyden chamber assay to count the numbers of A2780 and A2780/TF cells that penetrated the matrigel to the back of PVPF membrane after FVIIa stimulation. (3) BALB/c nude mice were used to establish experimental model of metastasis with A2780 or A2780/TF and the lung tissue sections were examined by microscopy for cancer metastasis.. (1) Compared with their parental A2780 cells, A2780/TF cells expressed high level of TF mRNA (3.99 +/- 0.15 vs 0.97 +/- 0.23, P < 0.01) and TF antigen on cell surface \\[(48.56 +/- 9.53)% vs (2.73 +/- 1.15)%, P < 0.01\\]. (2) After stimulation, the A2780/TF cell number on the back of PVPF membrane increased from basal level 157.3 +/- 19.2 to 447.7 +/- 39.4 (P < 0.01), which could decreased to basal level when coincubated with anti-TF antibody. (3) Cancer metastasis was found in 22.2% of nude mice transplanted with A2780 cells, while in 88.9% of those transplanted with A2780/TF cells.. TF could promote the invasion and metastasis of human ovarian cancer cells through TF/FVIIa pathway.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cloning, Molecular; Factor VIIa; Female; Genetic Vectors; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Ovarian Neoplasms; Thromboplastin; Transfection; Transplantation, Heterologous

The aim was to construct the expressive vector of human tissue factor (TF), and determine its expressive level in stable-transfected human ovarian cancer cell line. The human TFcDNA was obtained from human placenta by RT-PCR and then inserted into eukaryotic expressive vector pcDNA3 to obtain the TF-pcDNA3 recombinant. This recombinant gene was introduced into human ovarian cell line A2780 through transfection mediated by lipofectamine. Stable-transfected cells were screened by G418. The TF expressive levels were detected by RT-PCR and flow cytometry. The results showed that: (1) the constructed product was identified as TF-pcDNA3 recombinant by sequencing. (2) TF was highly expressed not only at transcriptional level in the stable-transfected A2780 cell (transfected cell 3.99 +/- 0.15, untransfected cell 0.97 +/- 0.23, P < 0.01), but also on the membrane of the cell surface [transfected cell (48.56 +/- 9.53)%, untransfected cell (2.73 +/- 1.15)%, P < 0.01]. It was concluded that TF gene was successfully cloned, and was introduced into human ovarian cancer cell, and the subline A2780/TF which stably expresses TF at high level was obtained. It will provide good experimental basis for investigating new mechanisms of tumor growth, invasion, metastasis, hypercoagulability, and for exploring a new strategy of gene therapy.

Topics: Animals; Cell Line, Tumor; Cloning, Molecular; Female; Humans; Mice; Ovarian Neoplasms; Recombinant Proteins; Thromboplastin; Transfection

Recent studies have shown that antithrombin III (AT III)/heparin is capable of inhibiting the catalytic activity of factor VIIa bound either to relipidated tissue factor (TF) in suspension or to TF expressed on cell surfaces. We report studies of the mechanism of which by AT III inhibits factor VIIa bound to cell surface TF and compare this inhibitory mechanism with that of tissue factor pathway inhibitor (TFPI)-induced inhibition of factor VIIa/TF. AT III alone and AT III/heparin to a greater extent reduced factor VIIa bound to cell surface TF. Our data show that the decrease in the amount of factor VIIa associated with cell surface TF in the presence of AT III was the result of (1) accelerated dissociation of factor VIIa from cell surface TF after the binding of AT III to factor VIIa/TF complexes and (2) the inability of the resultant free factor VIIa-AT III complexes to bind effectively to a new cell surface TF site. Binding of TFPI/factor Xa to cell surface factor VIIa/TF complexes markedly decreased the dissociation of factor VIIa from the resultant quaternary complex of factor VIIa/TF/TFPI/factor Xa. Addition of high concentrations of factor VIIa could reverse the AT III-induced inhibition of cell surface factor VIIa/TF activity but not TFPI/factor Xa-induced inhibition of factor VIIa/TF activity.

Topics: Antithrombin III; Cell Line; Factor VIIa; Factor X; Factor Xa Inhibitors; Female; Fibroblasts; Heparin; Humans; Lipoproteins; Ovarian Neoplasms; Thromboplastin; Tumor Cells, Cultured

The ability of anionic phospholipids (especially phosphatidylserine, PS) on the outer membrane leaflet of four tumour cell lines to support different stages of the extrinsic pathway of coagulation was probed using annexin V as an inhibitor. The procoagulant activity of two tumorigenic (MKN-28, human gastric carcinoma, Hep3B, human hepatoblastoma) and two non-tumorigenic (HepG2, human hepatocellular, HOC-1, human ovarian carcinoma) cell lines were observed to be inhibited by annexin V, although significant differences (observed as IC50 with respect to annexin V) were noted for each stage of coagulation and between different cell types. This was considered to suggest a restricted accessibility of PS in the vicinity of coagulation factors on the surface of the cell. PS levels, as estimated by binding of 125I-annexin V, were high on two of the cell lines tested, equivalent to 24 x 10(6) sites per cell for HepG2 (Kd 128 nM) and 6.5 x 10(6) sites per cell for MKN-28 (Kd 50 nM). During 9 days' culturing of HepG2 and MKN-28, the number of sites per cell remained constant. However, perhaps supporting a proposal of reduced availability, there was an observed fall in PS-dependent procoagulant activity of HepG2 and MKN-28 cells, subsequent to a peak on reaching confluency at 3 days. Both prothrombinase activity and total procoagulant activity fell, even though the number of 125I-annexin V binding sites remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Anions; Annexin A5; Blood Coagulation; Carcinoma, Hepatocellular; Female; Hepatoblastoma; Humans; Iodine Radioisotopes; Liver Neoplasms; Ovarian Neoplasms; Phosphatidylserines; Phospholipids; Stomach Neoplasms; Thromboplastin; Tumor Cells, Cultured

Because free factor VIIa is inactivated only very slowly by a plasma concentration of antithrombin III (AT III) even in the presence of heparin, it has been assumed that AT III plays no significant role in regulating the initiation of tissue factor-dependent blood coagulation. However, in the present study, we present evidence that factor VIIa bound to tissue factor, unlike free factor VIIa, is readily inactivated by AT III in the presence of heparin. In a reaction mixture containing calcium ions and approximately equimolar concentrations of relipidated tissue factor (8.9 nmol/L) and factor VIIa (10 nmol/L), AT III (100 micrograms/mL) plus heparin (1 U/mL) inhibited 50% of the factor VIIa coagulant activity of the reaction mixture within 5 minutes. AT III/heparin was also shown to inhibit the catalytic activity towards factor X of factor VIIa/tissue factor complexes formed on monolayers of an ovarian carcinoma cell line (OC-2008) that constitutively expresses surface membrane tissue factor. AT III, even in the absence of exogenously added heparin, substantially inhibited the functional activity of factor VIIa/cell surface tissue factor complexes on intact monolayers. AT III alone and AT III/heparin, to a greater extent, also inhibited factor VIIa on "nonfunctional" factor VIIa/tissue factor complexes on intact monolayers, with resultant inhibition of their expression of factor VIIa/tissue factor catalytic activity toward factor X after cell lysis. The potential physiologic significance of these findings is discussed.

Topics: Antithrombin III; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Factor VIIa; Female; Heparin; Humans; Kinetics; Molecular Weight; Ovarian Neoplasms; Protein Binding; Thromboplastin; Time Factors; Tumor Cells, Cultured

Proteins of the annexin/lipocortin family bind tightly to anionic phospholipids and platelets and act as in vitro anticoagulants. Annexins may be useful as tools to study the availability of anionic phospholipids on cell surfaces and their role in the regulation of blood coagulation. In the present study, we investigated the binding of annexin V (placental anticoagulant protein I) to a human ovarian carcinoma cell line, OC-2008, that constitutively expresses surface membrane tissue factor activity. Binding of annexin V to cell monolayers was calcium-dependent, specific, saturable and reversible; Scatchard analysis indicated a single class of binding sites with an apparent Kd of 9.4 +/- 3.1 nM and 5.2 +/- 1 x 10(6) sites per cell. Binding was completely inhibited by phospholipid vesicles containing phosphatidylserine, but was not inhibited by vesicles containing phosphatidylcholine. Annexin V inhibited the cell surface-dependent activity of prothrombinase complex, but did not inhibit the activity of the factor VIIa/tissue factor complex. In conclusion, these results suggest that anionic phospholipid is present on the extracellular face of OC-2008 cells; this anionic phospholipid is functionally important for the activity of the prothrombinase complex, but the importance of anionic phospholipid for the cell surface factor VIIa/tissue factor functional activity is unclear.

Topics: Annexin A5; Binding Sites; Blood Coagulation; Factor VIIa; Female; Humans; Multienzyme Complexes; Ovarian Neoplasms; Phosphatidylserines; Thromboplastin; Tumor Cells, Cultured

The kinetics of activation of factor IX and factor X by factor VIIa was studied in the presence of various sources of tissue factor: (1) a surface membrane of human ovarian carcinoma cell line, OC-2008 (2) the cell lysate (of OC-2008) and (3) reconstituted purified human tissue factor. The rates of activation of factors IX and X were monitored in activation peptide release assays using tritiated substrates. The results indicate that the apparent Km values for factor IX and factor X were similar for a given tissue factor, but varied with tissue factor source. The source of tissue factor greatly influenced the apparent differences in Vmax for factors IX and X. When a surface of monolayer provided tissue factor, the Vmax of factor IX was only 2-3 fold lower than factor X, but when either a cell lysate or purified tissue factor was the source of cofactor activity, the difference in Vmax rose to about 8-10 fold. Although, the tissue factor apoprotein in the cells was expressed entirely on the outer surface membrane, the activity of tissue factor on the intact cell surface was 50 to 100-fold lower than in the lysed cell preparation.

Topics: Catalysis; Cell Membrane; Drug Interactions; Factor IXa; Factor VIIa; Factor Xa; Female; Humans; In Vitro Techniques; Kinetics; Ovarian Neoplasms; Thromboplastin; Tumor Cells, Cultured

The kinetics of the binding of rVIIa to cell surface tissue factor (TF) and the resultant expression of VIIa/TF activity were studied. Binding of 125I-rVIIa (10 nM) to cell surface TF required 30-60 min for saturation, whereas VIIa/TF activity was fully expressed toward factor X (F X) on intact monolayers after only 1 min of incubation. At the time only 10-20% of the total VIIa TF complexes present at saturation had formed. Freeze-thawing the monolayers before assay increased VIIa/TF activity up to 30-fold, and the time course of its expression was similar to that of TF-specific binding of VIIa to the monolayers. Equilibrium binding revealed a single high affinity binding class of TF sites on intact monolayers for rVIIa with a Kd of 1.6 nM. Experiments with active-site inhibited rVIIa yielded evidence for two populations of VIIa. TF complexes on intact monolayers: (1) a minor population (less than 20%) that formed within 1 min of incubation and accounted for all VIIa/TF activity toward F X present on the intact monolayers, and (2) a major population that was inactive toward F X on intact monolayers but which was fully active after the monolayers were lysed. Tissue factor pathway inhibitor (TFPI).F Xa complexes inhibited the VIIa/TF activity of the first population, i.e. of the complexes active on intact monolayers, half maximally at a concentration of 0.2 nM TFPI. TFPI/Xa also bound to the second population of VIIa.TF complexes on intact monolayers and inhibited their expression of VIIa/TF activity following cell lysis with a half-maximal inhibitory concentration of 2.0 nM. The potential physiologic implications of these findings are discussed.

Topics: Amino Acid Sequence; Binding Sites; Cell Line; Cell Membrane; Factor VIIa; Factor Xa; Female; Humans; Kinetics; Lung; Molecular Sequence Data; Oligopeptides; Ovarian Neoplasms; Recombinant Proteins; Thromboplastin

Numerous incidents of thromboembolic complications have been documented in cancer patients and in recipients of mismatched organ transplants. Tumor procoagulants have also been implicated in the process of metastasis. Two protein bands of 35,000 and 28,000 daltons isolated from human ovarian carcinoma possessed procoagulant activity. The 35,000 dalton protein had an amino terminal sequence identical to that of the major histocompatibility antigen HLA-DR. Further, isolation of the protein using immunoaffinity column chromatography with monoclonal antibody to HLA-DR resulted in the isolation of procoagulant activity. The immunoaffinity purified protein enhanced thrombin generation in recalcified normal plasma approximately 20- fold. HLA-DR procoagulant activity was completely inhibited by Staphylococcal enterotoxin A. We propose that the procoagulant nature of HLA-DR may contribute to thrombotic disorders in several cancers and in association with graft rejection. The ability of enterotoxin A to inhibit this procoagulant may lead to development of future therapeutic strategies.

Topics: Antibodies, Monoclonal; Blood Coagulation; Blood Coagulation Factors; Blotting, Western; Chromatography, Affinity; Female; HLA-DR Antigens; Humans; Mitochondria; Ovarian Neoplasms; Thromboplastin

Topics: Blood Coagulation Tests; Female; Humans; Laparotomy; Neoplasm Metastasis; Ovarian Neoplasms; Thromboplastin