thromboplastin and Urinary-Bladder-Neoplasms

To examine the hypothesis that increased monocyte tissue factor (mTF) levels may reflect urological tumour presence and progression.. Using a two-stage kinetic chromogenic assay, mTF levels were measured in 60 controls (normal subjects [60] and patients awaiting hernia repair or cholecystectomy [60]), patients with benign and malignant disease of the bladder (73), or prostate (81), and in patients with and without recurrent malignant disease of the bladder (30). The levels were assessed under fresh resting conditions (baseline) and after incubation for 6 h without (unstimulated) and with (stimulated) Escherichia coli endotoxin. Each benign disease group was subdivided into inflammatory and non-inflammatory categories.. Patients with bladder and prostate malignancy showed significantly higher mTF levels than did each control for baseline and stimulated cells. The benign inflammatory groups for both organs had significantly higher mTF levels than had each control for baseline cells. There was no difference between malignant and benign inflammatory groups. Stimulated mTF levels showed better discrimination between the study groups. The mTF levels were associated with histological tumour progression, serum prostate specific antigen level and static bone scan images. Levels were also higher in patients with bladder cancer recurrence than in those with a normal check cystoscopy.. Stimulated mTF levels are raised in malignant and inflammatory disease compared with controls and patients with non-inflammatory conditions, and give maximal discrimination between these groups. mTF levels showed an association with tumour grade and other markers of tumour progression.

Topics: Biomarkers, Tumor; Disease Progression; Female; Humans; Male; Monocytes; Neoplasm Recurrence, Local; Prognosis; Prostatic Neoplasms; Thromboplastin; Urinary Bladder Neoplasms

Procoagulant activity attributed to tissue factor (TF, CD142) bound to lipid microvesicles has previously been shown to be elevated in urine of patients with various solid cancers. The phosphorylation of the C-terminal signal transduction peptide (STP) at Ser253 and Ser258 has been determined to be important for the formation of TF-microvesicles. The purpose of this work was to investigate the marker potential of the TF-STP domain in urine of patients with cancer using immunologic methods to quantitate unphosphorylated TF and TF phosphorylated at Ser253 and Ser258.. We developed monoclonal and polyclonal antibodies directed against the 3 C-terminal STP species of TF and constructed 3 enzyme-linked immunosorbent assays (ELISAs) that specifically recognize unphosphorylated TF and TF phosphorylated at either Ser253 or Ser258. As proof of principle, a preliminary pilot study with stored Biobank-sourced urinary specimens from 45 healthy individuals and 38 patients with bladder cancer were studied using these ELISAs.. We report that all 3 species of TF were found in the urine. Two species, TF-pSer258 and unphosphorylated TF, were significantly elevated in the cohort with bladder cancer. The sensitivity of TF-pSer258 by the receiver operator characteristic technique was 86.8%, with a specificity of 97.8% at a cutoff value of 0.55 ng/mL. Using a simplified sample preparation method for the ELISAs on the same clinical specimens, the sensitivity of TF-pSer258 was 86.8%, with a specificity of 93.3% at a cutoff value of 0.53 ng/mL. The unphosphorylated TF species was significantly elevated in later stage bladder cancer with best results seen for the unfractionated preparation technique (95% confidence interval, 10.55-15.74; N = 20) but not early stage non-muscle-invasive bladder cancer (95% confidence interval, 4.71-10.73; N = 18; P < .02).. The development of these new ELISAs allows the quantitation of the urinary biomarkers TF-pSer258 and unphosphorylated TF, which may lead to a new diagnostic approach to the early detection of bladder cancer and warrant further investigation in a prospective trial.

Topics: Biomarkers, Tumor; Case-Control Studies; Female; Humans; Male; Neoplasm Staging; Phosphorylation; Pilot Projects; Prospective Studies; Protein Sorting Signals; Serine; Thromboplastin; Urinary Bladder Neoplasms

Tissue factor (TF), the principal initiator of the extrinsic coagulation pathway, is expressed by many tumors and can be released into the bloodstream on plasma microparticles (MPs). Experimental studies indicate that TF may facilitate hematogenous metastasis by promoting tumor cell-induced microvascular thrombosis, but clinical data supporting this hypothesis is sparse.. Here, we report 2 unusual cases of rapidly progressive solid malignancies (gastric and urothelial carcinoma). In both patients, cancer cell dissemination with diffuse bone marrow involvement was either strongly suggested by leukoerythroblastic changes on peripheral blood smear or directly proven by positive findings on aspiration cytology. Furthermore, laboratory evidence of thrombotic microangiopathy (TMA) and disseminated intravascular coagulation was accompanied by new-onset severe pulmonary hypertension and a hemolytic uremic syndrome-like disorder in the gastric and the urothelial carcinoma patient, respectively. TF-specific procoagulant activity of isolated plasma MPs, as assessed by single-stage clotting assay, was dramatically increased in both patients compared to healthy controls (21- and 55-fold), and primary tumor samples stained strongly positive for TF by immunohistochemistry.. TMA was likely caused by TF-triggered tumor cell embolization in both patients. Further clinical evidence is thus provided that TF directly links coagulation activation to cancer cell dissemination.

Topics: Aged; Aged, 80 and over; Female; Gastrointestinal Neoplasms; Humans; Male; Neoplasm Invasiveness; Thromboplastin; Thrombotic Microangiopathies; Urinary Bladder Neoplasms

Tissue factor (TF) is the major physiological initiator of the coagulation cascade and has an important function in the morbidity and mortality associated with many disease states, including cancer-associated thrombosis and atherosclerosis. TF normally exists in a partially encrypted state and its de-encryption on circulating monocytes, platelets or endothelial cells by inflammatory mediators can lead to thrombosis. Furthermore, many cancer cells express large amounts of TF and these cells communicate readily with the circulation through the fenestrated tumor endothelium. To assess agents or conditions that modulate the encryption state of TF, we developed a continuous assay for the determination of TF procoagulant activity (PCA) in a cell-based system. We have shown the use of this assay at detecting agents that de-encrypt TF thereby leading to an increase in TF PCA in three cancer cell lines, namely, T24/83 bladder carcinoma cells and PC-3 and DU145 prostate cancer cells. Further, through use of this assay, we have shown that the endoplasmic reticulum calcium pump inhibitor, thapsigargin, stimulates the de-encryption of TF. The continuous assay for the determination of TF PCA proved to have inherently less intra- and inter-assay variability than the widely used discontinuous assay and is considerably less labor intensive. Further, the continuous assay produced progress curves that were compatible with curve fitting to allow for the determination of the nature of reaction as well as rate constants for the underlying enzymes, TF/FVIIa and FXa. The continuous assay for the assessment of TF PCA on intact cells is applicable for high-throughput screening to allow for the determination of compounds that modulate TF PCA.

Topics: Biomarkers, Tumor; Carcinoma; Cell Line, Tumor; Cell Physiological Phenomena; Cell Survival; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Immunoblotting; Neoplasm Proteins; Thapsigargin; Thromboplastin; Urinary Bladder Neoplasms

Tissue factor (TF), a transmembrane glycoprotein responsible for initiating the extrinsic pathway of blood coagulation plays a key role in cancer growth, metastasis and angiogenesis. Various studies have demonstrated the prognostic potential of TF expression in several cancers. However, its role in bladder cancer is unclear. This study evaluated the prognostic potential of TF expression in muscle-invasive bladder tumors from patients treated with radical cystectomies. Immunohistochemical staining using a monoclonal antibody (mAb) anti-TF was carried out on sections of tissue microarray blocks containing cores of muscle-invasive bladder tumors (4 cores/tumor) from 218 patients. The intensity of the staining was evaluated on a scale from 0 to 3 by two independent observers who were both unaware of the clinicopathological characteristics of the samples. TF was expressed in 77.6% of tumors, independently from baseline characteristics (age, gender, stage and grade) as assessed using the chi(2) and Student t tests. During follow-up (median: 2.6 years), 45.4% of the patients died from the progression of their cancer. Kaplan-Meier survival showed that among the 103 patients with node-negative (N0) transitional cell carcinoma (TCC), those with TF-positive tumors had shorter bladder cancer-specific survival (p = 0.0276). Moreover, multivariate Cox regression analysis showed they had a 3.15-fold greater risk of dying from bladder cancer (95% CI: 1.1-9.0; p = 0.032). In conclusion, TF expression was an independent predictor of disease-specific survival in N0 muscle-invasive TCCs treated by radical cystectomy and therefore, might help identify patients at higher risk of disease progression. These patients could potentially benefit from adjuvant chemotherapy.

Topics: Aged; Analysis of Variance; Biomarkers, Tumor; Carcinoma, Transitional Cell; Cystectomy; Disease-Free Survival; Female; Follow-Up Studies; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Neoplasms, Muscle Tissue; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Protein Array Analysis; Thromboplastin; Urinary Bladder Neoplasms

Thromboembolic disorders are common in cancer patients. Two major contributing factors are central venous catheters for drug delivery and the use of l-aparaginase, which decreases the plasma antithrombin level, but the causes of the hypercoagulable state in these patients are not fully understood. In this study, the T24/83 cell line was used as a model to investigate the effects of chemotherapeutic agents on cell surface thrombin regulation. Plasma thrombin generation and prothrombin consumption was increased in most of the treated cells, particularly vincristine- and adriamycin-treated cells (P < 0.05), compared with controls. However, no free thrombin generation or prothrombin consumption was observed in factor VII (FVII)-depleted plasma. No significant differences in the levels of thrombin-alpha2-macroglobulin (IIa-alpha2M) and thrombin-anti-thrombin (TAT) were observed between controls and any of the treatments, except for vincristine- and adriamycin-treated cells, which showed a significant difference in TAT production (P < 0.05). Also, there was an upregulation in tissue factor (TF) mRNA expression in etoposide-, methotrexate- and vincristine-treated monolayers compared with controls, as well as an upregulation in TF protein production in vincristine-treated cells. The data suggests that thrombin generation occurs via the extrinsic (TF-dependent) coagulation pathway on cell surfaces and that some chemotherapeutic agents are able to upregulate TF mRNA and protein expression in T24/83 cells.

Topics: alpha-Macroglobulins; Antineoplastic Agents; Antithrombins; Cell Membrane; Doxorubicin; Etoposide; Humans; Methotrexate; Prothrombin; Thrombin; Thromboplastin; Thrombosis; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vincristine

Human tissue factor (TF) is involved in tumor angiogenesis and metastasis. However, little is known about the distribution of TF in urological cancer. In this study we investigated the TF expression in tumor tissue and autologous non-malignant tissue as well as in serum of patients with renal cell carcinoma (RCC), bladder cancer, and prostate cancer (PCa). To study the distribution of TF in tumor tissue and in the surrounding non-malignant tissue, we measured TF protein by ELISA in tissue specimens obtained intraoperatively from 18 RCC, seven bladder cancer and six PCa patients. Differences in TF expression were found between tumor tissue and nonmalignant tissue for the three tumor types at the protein level (in the order RCC < bladder cancer < PCa). In all but one of the 18 RCC patients (94 %) higher TF protein level was observed in non-malignant tissue as compared to the tumor tissue. In addition, the relative TF mRNA expression analyzed by a quantitative RT-PCR assay in the same RCC tissue sample pairs was higher in 78% of non-malignant tissues in comparison to the tumor tissue specimens. Moreover, using enzyme linked immunosorbent assay the TF protein content was measured in serum samples of 66 patients with bladder cancer, 75 RCC patients and 157 PCa patients, and was compared with the TF serum level of 92 healthy volunteers. Whereas no differences were detected between normal volunteers and patients with PCa or RCC, patients with bladder cancer showed a significantly increased level of serum TF (P=0.0076). However, no causal association between TF levels in serum and TF content in tissue extracts for all three tumor types of urological tumors was found. Our results suggest that TF in non-malignant renal tissues was expressed at a higher level compared to the supposed de novo TF expression in RCC tissue specimens. This indicates a tumor-associated induction of TF expression in the TF-negative RCC progenitor cells. The increased serum TF levels are alike the reported higher urinary TF levels found in bladder cancer patients. The potential clinical relevance of this finding should be further elucidated.

Topics: Carcinoma, Renal Cell; Enzyme-Linked Immunosorbent Assay; Humans; Male; Polymerase Chain Reaction; Prostatic Neoplasms; RNA, Messenger; Thromboplastin; Tissue Distribution; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Urologic Neoplasms

The aim of study was to evaluate TF activity and TFPI concentration in patients with haematological malignancies and urinary tract tumors. TFPI concentration and activity and TF concentration were measured in 20 patients suffering from acute myeloblastic leukaemia (AML), 21 patients with chronic myelogenous leukaemia (CML), 17 patients with chronic lymphatic leukaemia (CLL), 16 patients with multiple myeloma (MM) and 65 healthy adults. TFPI and TF concentrations were measured also in patients with renal cell carcinoma (n = 12) and bladder cancer (n = 17) and patients with benign prostatic hyperplasia (BPH) (n = 15). Patients with AML, CML, CLL, and cancer revealed elevated TFPI concentrations. Patients with AML, CML, CLL, MM showed decreased TFPI activity. However TFPI concentration correlated inversely with TFPI activity only in the AML group. No significant changes were observed in TF concentrations in all investigated groups.

Topics: Biomarkers, Tumor; Carcinoma, Renal Cell; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; Humans; Kidney Neoplasms; Leukemia; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Lipoproteins; Male; Multiple Myeloma; Prostatic Hyperplasia; Thromboplastin; Urinary Bladder Neoplasms; Urologic Neoplasms

Tissue factor (TF) is a transmembrane glycoprotein that was originally recognized for its ability to initiate the extrinsic pathway of coagulation. More recently, additional functions of TF in cellular signalling have emerged, notably the role of TF in vasculogenesis and angiogenesis. We have described previously the ability of a peptide derived from the apolipoprotein B100 (apoB100) moiety of low-density lipoproteins (KRAD14) to inhibit the procoagulant function of TF. In this study, we demonstrate the ability of the KRAD14 peptide to attenuate the density of cellular network structures of T24 cells grown on specialized matrix (Matrigel). In addition, an alternative inhibitor of TF activity, the TF8 5G9 antibody, also reduces the density of cellular network formation. Targeted use of a stable structural equivalent of the KRAD14 peptide may thus prove useful in the prophylactic treatment of diseases whose pathologies feature the formation of neovascular tissue, e.g. tumour growth and metastasis, rupture of atherosclerotic plaques and retinopathy secondary to diabetes.

Topics: Amino Acid Sequence; Antibodies; Binding Sites; Factor VIIa; Humans; In Vitro Techniques; Models, Molecular; Neovascularization, Pathologic; Peptide Fragments; Protein Structure, Tertiary; Thromboplastin; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Tissue Factor (TF) is a transmembrane glycoprotein that complexes with factor VII/activated factor VII to initiate blood coagulation. TF may be expressed on the surface of various cells including monocytes and endothelial cells. Over-expression of TF in human tumor cell lines promotes metastasis. We recently showed that hemoglobin (Hb) forms a specific complex with TF purified from human malignant melanoma cells and enhances its procoagulant activity (PCA). To further study this interaction, we examined the effect of Hb on the expression of TF on human malignant (TF+) cells and KG1 myeloid leukemia (TF-) cells. Human melanoma A375 and J82 bladder carcinoma cells, which express TF at moderate and relatively high levels, respectively, were incubated with varying concentrations (0-1.5 mg/ml) of Hb. After washing, cells were analyzed for Hb binding and TF expression using flow cytometry and confocal microscopy. Hb bound to the cells in a concentration-dependent manner, and increased both TF expression and PCA. The human A375 malignant melanoma cells incubated with Hb (1 mg/ml) expressed up to six times more TF antigen than cells without Hb. This increase in TF expression and PCA of intact cells incubated with Hb was significantly inhibited by cycloheximide at a concentration of 10 microg/ml (P < 0.01). An increase in total cellular TF antigen content was demonstrated by specific immunoassay. In contrast, Hb (5 mg/ml) did not induce TF expression and PCA on KG1 cells as determined by flow cytometry and TF (FXAA) activity. We conclude that Hb specifically binds to TF-bearing malignant cells and increases their PCA. This effect seems to be at least partly due to de novo synthesis of TF and increased surface expression. However, the exact mechanism by which Hb binds and upregulates TF expression remains to be determined.

Topics: Carcinoma, Transitional Cell; Cycloheximide; Factor Xa; Flow Cytometry; Gene Expression Regulation, Neoplastic; Hemoglobins; Humans; Melanoma; Microscopy, Confocal; Neoplasm Proteins; Protein Synthesis Inhibitors; Stimulation, Chemical; Thromboplastin; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Coagulation activation is a recognized complication of cancer in which increased tissue factor (TF) is implicated. TF can be detected in urine (uTF). This study assesses uTF levels in benign and malignant urological disease and correlates the results with conventional markers of tumour progression.. Using a simple and reproducible kinetic chromogenic assay, we determined uTF levels in controls (normal volunteers (n = 57) and patients with renal stones (n = 30)), benign and malignant bladder (n = 75) or prostate (n = 106) disease and in patients with or without recurrent bladder cancer (n=30). Each benign disease group was stratified as inflammatory (cystitis or prostatitis) or non-inflammatory (negative cystoscopy following haematuria or benign prostatic hypertrophy).. The controls and the benign non-inflammatory results were indistinguishable. The malignant and inflammatory groups showed raised uTF levels over controls (P<0.001 bladder and P<0.01 prostate). The difference between malignant and benign inflammatory disease was only significant for the bladder group. uTF levels were significantly related to histological tumour grading, prostate serum specific antigen, static bone scan images and recurrence status.. uTF levels can distinguish, statistically but not without overlap, patients with malignancy from normal controls and benign non-inflammatory conditions. Discrimination between inflammatory and malignant disease has only been demonstrated in the bladder. uTF levels showed a significant association with markers of tumour progression or metastasis and may be useful in predicting bladder tumour recurrence.

Topics: Adult; Aged; Biomarkers, Tumor; Bone Neoplasms; Case-Control Studies; Cystitis; Disease Progression; Humans; Kidney Calculi; Male; Middle Aged; Prostate-Specific Antigen; Prostatic Hyperplasia; Prostatic Neoplasms; Prostatitis; Thromboplastin; Urinary Bladder Neoplasms

The association between cancer and thromboembolic disease has been known for over a century. Increased tissue factor expression by endothelial cells, monocytes or macrophages is implicated. Thus, monocyte tissue factor measurements may reflect disease presence or progression.. Using a 2 stage kinetic chromogenic assay, monocyte tissue factor levels were assessed in normal controls (n=60), patient controls (hernia or cholecystectomy, n=60) and in patients with benign and malignant disease of the bladder (n=73), prostate (n=81), breast (n=83) and colorectum (n=62). This was performed as baseline (resting cells) and after 6 hours incubation with (stimulated) and without (unstimulated) lipopolysaccharide. Each benign disease group was sub-divided into inflammatory and non-inflammatory categories.. The relative operating characteristic curve for the lipopolysaccharide-stimulated monocyte tissue factor assay showed sensitivity and specificity for cancer, the area under the curve being 0.71. The control groups and the benign non-inflammatory groups gave similar results and were pooled for further analysis. Each malignant group showed higher monocyte tissue factor levels than the control groups for baseline (P< 0.05) and lipopolysaccharide-stimulated cells (P< 0.05). All benign inflammatory groups apart from breast, showed increased monocyte tissue factor levels over controls for baseline (P< 0.05) and lipopolysaccharide-stimulated cells (P< 0.05). In all cases there was no significant difference between the malignant and the benign inflammatory groups. Monocyte tissue factor levels were related to tumor grade or stage, patients' survival time, serum prostate specific antigen and static bone scan images. Levels were also higher in patients with bladder cancer recurrence and in those who subsequently died.. Lipopolysaccharide-stimulated monocyte tissue factor assay showed sensitivity and specificity for cancer compared to controls. Monocyte tissue factor levels are raised in malignant groups compared to controls and non-inflammatory diseases but not when compared with inflammatory conditions. Stimulated cells give better discrimination between the groups and may be useful in identifying high risk individuals. Monocyte tissue factor levels were related to tumor progression.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Diseases; Breast Neoplasms; Case-Control Studies; Colonic Diseases; Colorectal Neoplasms; Discriminant Analysis; Disease Progression; Female; Humans; Inflammation; Male; Middle Aged; Monocytes; Prostatic Diseases; Prostatic Neoplasms; Risk Factors; Sensitivity and Specificity; Thromboembolism; Thromboplastin; Urinary Bladder Diseases; Urinary Bladder Neoplasms

Tissue factor (TF), the cell-surface receptor for coagulation factor VIIa, supports metastasis. Equally important for this process are (a) interactions of the TF cytoplasmic domain, which binds the mobility-enhancing actin-binding protein 280, and (b) the formation of a proteolytically active TF-VIIa complex on the tumor cell surface. In primary bladder carcinoma cells, we find that this complex localizes to the invasive edge, in proximity to tumor-infiltrating vessels that stain intensely for TF pathway inhibitor (TFPI-1), the major inhibitor of the protease activity of the complex. In culture, binding of VIIa to TF-expressing tumor cells is sufficient to allow cell adhesion, migration, and intracellular signaling on immobilized TFPI-1. Immobilized heparin, a mimic for extracellular matrix-associated proteoglycans, binds physiological concentrations of TFPI-1 in a conformation that supports TF-VIIa-dependent cell adhesion. Consistent with a functional role of TFPI-1 in complex extracellular matrices, we show that TF cooperates with integrin-mediated adhesion and migration on composite matrices that contain ligands for both integrins and the TF-VIIa complex. This study thus provides evidence for a novel mechanism of protease-supported migration that is independent of proteolytic matrix degradation but rather involves protease-dependent bridging of TF's extracellular domain to an ECM-associated inhibitor.

Topics: Carcinoma; Cell Adhesion; Cell Movement; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Endopeptidases; Epithelium; Factor VIIa; Fibronectins; Glycoproteins; Heparin; Humans; Immunohistochemistry; Lipoproteins; Neoplasm Proteins; Pregnancy Proteins; Signal Transduction; Thromboplastin; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Monocyte and urinary tissue factors (mTF and uTF) are both elevated in a number of pathologic conditions, including cancer. This study validates the best available uTF and mTF assays as diagnostic tools for cancer and examines if uTF levels reflect monocyte activation. Using kinetic chromogenic assays for uTF and mTF (measured on fresh resting cells [baseline], unstimulated cells, and lipopolysaccharide [LPS]-stimulated cells), we assessed TF levels in normal individuals, surgical controls, and patients with benign and malignant diseases. Each benign disease group was stratified as inflammatory or noninflammatory. Controls and benign noninflammatory results were indistinguishable. The malignant and inflammatory groups showed raised uTF levels over controls (p < 0.001). mTF levels differ similarly. For mTF and uTF assays, there was no significant difference between the malignant and inflammatory groups. The relative operating characteristic (ROC) curve plots sensitivity against false positive rate (1-specificity) for all possible cutoff values of a diagnostic test. Assay performance is assessed as the area under the curve (AUC). The ROC curve for the uTF assay displayed both sensitivity and specificity for cancer, the AUC being 0.83. Of the three mTF levels, LPS-stimulated cells gave the optimum curve (AUC = 0.71). uTF showed a weak to moderate association with mTF levels but correlated best and was statistically significant when compared with levels in the LPS-stimulated cells. uTF represents an intrinsic, kidney-derived, physiologic concentration rather than that of preactivated or postactivated monocytes. In conclusion, both uTF and LPS-stimulated mTF levels showed sensitivity and specificity in detecting cancer and inflammatory diseases. However, the two forms of TF appear to be independently derived.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Area Under Curve; Biomarkers, Tumor; Breast Neoplasms; Child; Child, Preschool; Cholelithiasis; Colorectal Neoplasms; Diagnosis, Differential; False Positive Reactions; Female; Hernia, Inguinal; Humans; Inflammation; Kidney Calculi; Lipopolysaccharides; Male; Middle Aged; Monocytes; Neoplasms; Prostatic Neoplasms; ROC Curve; Sensitivity and Specificity; Thromboplastin; Urinary Bladder Neoplasms

Recent studies showed that the administration of active site-inhibited factor VIIa blocked factor VIIa/tissue factor-induced fibrin and thrombus formation in ex vivo and in vivo model systems. These studies suggest that inactivated factor VIIa competes efficiently with plasma factor VII(a) for a limited number of tissue factor sites. In the present study, we compared the interactions of factor VIIa and active site-inhibited factor VIIa with tissue factor. Competition studies of factor VIIa and active site-inhibited factor VIIa in a factor X activation assay showed that the affinity of the latter for relipidated tissue factor was 5-fold higher than that of factor VIIa. Radioligand binding studies with a human bladder carcinoma cell line (J82) and surface plasmon resonance studies using soluble tissue factor demonstrated a faster association and a slower dissociation for the active site-inhibited factor VIIa. Studies of equilibrium binding to cell surface tissue factor showed that the affinity of active site-inhibited VIIa was 5-fold higher than that of factor VIIa to non-functional tissue factor sites, whereas both inactivated factor VIIa and factor VIIa bound to functional tissue factor sites with the same high affinity. Comparison of the CD spectra of factor VIIa and active site-inactivated factor VIIa revealed structural differences in the protease domain. The potential physiological implications of these findings are discussed.

Topics: Amino Acid Chloromethyl Ketones; Binding Sites; Binding, Competitive; Cell Line; Cell Membrane; Circular Dichroism; Factor VIIa; Fibrin; Humans; Kinetics; Protein Conformation; Recombinant Proteins; Thromboplastin; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Previous studies have shown that antithrombin III-heparin effectively inhibited the factor VIIa-tissue factor complex. Herein, we show that the neutralization of factor VIIa in complex with the cell surface tissue factor by antithrombin III-heparin was markedly enhanced by plasma levels of factor X. Active site-mutated factor X (S376A factor X) and factor Xa previously inactivated with dansyl-Glu-Gly-Arg-chloromethyl ketone were as effective as plasma-derived factor X in this reaction, indicating that the active site serine residue of factor Xa was not involved in this mechanism. Furthermore, Gla-domainless factor X had no effect in this system, emphasizing the importance of the factor X Gladomain in this reaction. Antibody experiments revealed that this effect was not due to trace levels of a tissue factor pathway inhibitor contaminating either the factor X or antithrombin III preparations. The presence of heparin in this system was essential, as deletion of heparin resulted in a factor VIIa-tissue factor neutralization rate essentially identical to that observed for antithrombin III alone. Plasma levels of factor IX also accelerated the inhibition of factor VIIa-tissue factor by antithrombin III-heparin, although its effect was not as pronounced as that of factor X. Other vitamin K-dependent plasma proteins including protein S, protein C and prothrombin failed to augment the inhibition of factor VIIa-tissue factor by antithrombin III-heparin. Factor X did not enhance the neutralization rate of factor VIIa-tissue factor by antithrombin III-heparin when a carboxyl-terminal truncated tissue factor construct (TF1-219) was used, even in the presence of mixed phospholipids. Our collective finding suggest that antithrombin III and factor X bind to heparin at distinct sites on the heparin molecule resulting in a transient ternary complex of antithrombin III-heparin-factor X that represents the anticoagulant species. Factor X conceivably guides complex to a phosphatidylserine-rich site on the cell surface in close proximity to the factor VIIa-tissue factor complex and facilitates rapid neutralization of factor VIIa. Our findings also suggest that the effect of heparin on the regulation of the extrinsic pathway of blood coagulation may be more profound than previously recognized.

Topics: Antithrombin III; Drug Synergism; Factor VIIa; Factor X; Heparin; Humans; Thromboplastin; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Topics: Cell Line; Colony-Stimulating Factors; Culture Media, Conditioned; Cytokines; Gene Expression; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Promyelocytic, Acute; Thromboplastin; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Endothelial cells grown on filters developed junctional complexes that reduced diffusional transport and increased electrical resistance over the cell layer. Induction of tissue factor by recombinant interleukin-1 beta led to a highly polarized tissue factor expression on the apical cell surface only. After prolonged growth to allow deposition of matrix, removal of the endothelial cells by collagenase or by 0.1 mol/L NH4OH left behind some cellular material as well as tissue factor, which was only detectable in the upper compartment. A human bladder carcinoma cell line, which does not form tight junctions and expresses tissue factor constitutively, showed essentially no polarity. Endothelial cell secretory compounds like von Willebrand factor, tissue plasminogen activator, and plasminogen activator inhibitor-1 were constitutively released to both sides. The added secretion due to recombinant interleukin-1 beta stimulation of the endothelial cells observed for von Willebrand factor and tissue plasminogen activator was, however, localized to the apical surface. The availability of tissue factor on the luminal surface of endothelial cells, ie, allowing contact with factor VII in the flowing blood, has potentially very significant pathophysiological consequences.

Topics: Cell Polarity; Cells, Cultured; Endothelium, Vascular; Extracellular Matrix; Humans; Interleukin-1; Recombinant Proteins; Thromboplastin; Umbilical Veins; Urinary Bladder Neoplasms

Production of procoagulant activity by host and tumour cells may be increased in patients with cancer. Using a simple chromogenic assay, we have determined urinary tissue factor (TF) levels in patients presenting with transitional cell carcinoma of the bladder (TCC, n = 63), normal controls (n = 20) and patients with benign prostatic hypertrophy (BPH, n = 35). In addition, a separate cohort of patients undergoing endoscopic surveillance for superficial bladder cancer were studied to determine whether there was any difference in levels in those with recurrent disease compared to those with normal cystoscopies. Urinary TF activity was higher in TCC compared to controls (p less than 0.001) and patients with BPH (p less than 0.05). In patients undergoing check cystoscopy, those with recurrent disease (n = 32) had higher levels (p less than 0.01) than those with normal examinations (n = 21). It is concluded that urinary TF levels are elevated in bladder cancer and that this reflects disease activity in those at risk of recurrent superficial disease.

Topics: Carcinoma, Transitional Cell; Cystoscopy; Humans; Male; Prostatic Hyperplasia; Thromboplastin; Urinary Bladder Neoplasms

Tissue factor (TF) functions as the receptor and cofactor for factor VIIa (VIIa) to form a proteolytically active TF.VIIa complex on cell surfaces. We here demonstrate that most MAbs against human TF were poor inhibitors of TF function in plasma and that they inhibited preformed TF.VIIa complex at a slow rate which was dependent on dissociation of VIIa from the cell surface TF. An exception was defined by one MAb (TF8-5G9) which was an effective immediate anticoagulant in plasma. Binding of TF8-5G9 to TF.VIIa inhibited catalytic function prior to dissociation of the TF.VIIa complex. This analysis thus establishes two distinct mechanisms by which MAbs interfere with TF function. The MAb TF8-5G9 introduces a therapeutic principle for rapid arrest of inappropriate triggering of coagulation by TF as well as the TF.VIIa complex in vivo.

Topics: Antibodies, Monoclonal; Anticoagulants; Cell Line; Factor IX; Factor VII; Factor VIIa; Factor X; Humans; Kinetics; Protein Binding; Thromboplastin; Urinary Bladder Neoplasms

The possible self-association of tissue factor molecules was investigated by treating cells expressing tissue factor with bifunctional cross-linking agents. The two reagents chosen were 3,3'-dithiobis(sulfosuccinimidylpropionate) and sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate, both of which are membrane-impermeable and thiol-cleavable. A human bladder carcinoma cell line, J82, and a transfected human kidney cell line expressing high amounts of recombinant tissue factor were used in these studies. Exposure of the intact cells to the crosslinking reagents was found to result in the formation of multimeric tissue factor-containing complexes, the extent of which appeared to be dependent upon the amount of tissue factor expressed by the cell. The self-association of tissue factor was prevented in a variant tissue factor molecule harboring a non-homologous transmembrane domain.

Topics: Amino Acid Sequence; Autoradiography; Blotting, Western; Cells, Cultured; Cross-Linking Reagents; Humans; Kidney; Molecular Sequence Data; Plasmids; Precipitin Tests; Recombinant Proteins; Thromboplastin; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Conditioned medium of a human bladder carcinoma cell line (J82) was found to induce tissue factor synthesis in cultured human umbilical vein endothelial cells (HUVEC). A protein present in the J82 conditioned medium was partially purified by FPLC using a combination of MONO Q and Superose 6 columns. The bladder carcinoma-derived cytokine (BCDC) exhibited a Mr of 22 kDa by gel permeation HPLC. Polyclonal antibody against either interleukin-1, tumor necrosis factor, or transforming growth factor-beta failed to inhibit the ability of the conditioned medium to induce HUVEC tissue factor activity, suggesting that this tumor cell line secretes a novel cytokine responsible for HUVEC tissue factor induction.

Topics: Biological Factors; Chromatography, High Pressure Liquid; Cytokines; Endothelium, Vascular; Humans; Molecular Weight; Neoplasm Proteins; Thromboplastin; Tumor Cells, Cultured; Umbilical Veins; Urinary Bladder Neoplasms

Tissue factor (TF) is an integral membrane glycoprotein which, as the receptor and essential cofactor for coagulation factors VII and VIIa (FVII and FVIIa, respectively), is the primary cellular activator of the coagulation protease cascade. Previous studies on the procoagulant activity of a variety of cell types (either lysed or in the intact state) have variously been interpreted as showing that TF is either stored intracellularly or is present in a cryptic form in the surface membrane. Using mAbs to TF, we have directly investigated the subcellular localization and functional activity of TF in lipopolysaccharide-stimulated blood monocytes and J82 bladder carcinoma cells. Blocking of surface TF of viable cells with inhibitory anti-TF mAbs abolished greater than 90% of TF activity of the intact cells as well as of lysed cells. Furthermore, quantitative analysis of the binding of FVII and anti-TF mAb to J82 cells demonstrated that all surface-expressed TF molecules were capable of binding the ligand, FVII. By immunoelectron microscopy, TF was present only in the surface membrane of monocytes and J82 cells, although the latter also contained apparently inactive TF antigen in multivesicular bodies. On the intact cell surface the catalytic activity of the TF-FVIIa complex was investigated and found to be markedly less relative to cell lysates. Membrane alterations that affect the cofactor activity of TF may be a means of regulating the extent of initiation of the coagulation protease cascade in various cellular settings.

Topics: Antibodies, Monoclonal; Carcinoma; Cell Compartmentation; Cell Membrane; Factor VII; Factor Xa; Humans; Immunoenzyme Techniques; In Vitro Techniques; Lipopolysaccharides; Microscopy, Electron; Monocytes; Serine Endopeptidases; Thromboplastin; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Assembly of the extrinsic pathway on cell surfaces was investigated by studying the binding and activity of factor VII on the bladder carcinoma cell line J82 which expressed 18,800 milliunits of tissue factor activity/10(6) cells. In binding studies, the association of factor VII to monolayers of cells was time-, temperature-, and calcium-dependent. The ligand binding was specific, reversible, and saturable. This interaction was inhibited by a monoclonal antibody to human brain tissue factor. Factor VII added to the cells was recovered as factor VII rather than factor VIIa when incubated in the presence of factor X neutralizing antibodies, suggesting that these cells produced factor X. Specific factor VII binding to the cell revealed a sigmoidal binding isotherm with half-maximal binding occurring at 314 +/- 145 pM to 38,300 +/- 14,300 sites/cell. Hill plots of the binding data indicated an average slope of 2.1. Binding parameters were also determined kinetically. At maximal factor VII-tissue factor complex formation the apparent Km for factor X was 274 nM, the Vmax was 4.15 nM/min, and the kcat was estimated to be 14 s-1. In the presence of excess tissue factor and factor X, increasing amounts of factor VII added to the J82 cells demonstrated a sigmoidal relationship with the rate of factor Xa formation. Hill plots indicated a slope of 2.0 at the lower factor VII concentrations which changed to 1.0 at the higher input amounts of factor VII. Hanes plots were used to determine the apparent dissociation constant of the interaction (222 +/- 85 pM). The Vmax was 5.54 +/- 1.04 nM/min for the cleavage of factor X. These data are consistent with factor VII binding to at least two sites on tissue factor (receptor) with positive cooperativity. Because at saturation the stoichiometry of the factor VII-tissue factor complex is 1:1, tissue factor must be expressed as a dimer on the surface of the J82 cells.

Topics: Antibodies, Monoclonal; Blood Coagulation; Cell Line; Culture Techniques; Factor VII; Factor X; Humans; Kinetics; Temperature; Thromboplastin; Time Factors; Urinary Bladder Neoplasms

In this study we have looked for differences in coagulation parameters before and after surgical removal of benign or malignant tumours. A striking increase in thromboplastin activity of the blood monocytes was seen 1 d after surgery. This paralleled a fall in factor VII activity. At the same time, the sensitivity of the blood monocytes to stimulation by endotoxin increased significantly. We propose from this study that monocyte thromboplastin may be a postoperative thrombogenic factor. Increased levels of factor VIII and fibrinogen 2-3 d postoperatively were found, probably caused by inflammation reactions induced by the surgery. No difference in coagulation parameters could be demonstrated between patients with benign, noninvasive lesions and patients with invasive, carcinomatous lesions.

Topics: Blood Coagulation; Breast Neoplasms; Factor VII; Factor VIII; Female; Fibrinogen; Humans; Male; Monocytes; Neoplasms; Postoperative Period; Prostatic Hyperplasia; Thromboplastin; Thyroid Neoplasms; Time Factors; Urinary Bladder Neoplasms

Thromboplastic and fibrinolytic activities of 14 lines of cultured human cancer cells were estimated by modified Astrup's methods. High tissue thromboplastic activity was found in one line of urinary-bladder cancer, 2 lines of gastric cancer and one line of lung cancer, but no activity was found in 6 lines of lung cancer. High fibrinolytic activity was noted in one line of gastric cancer and 2 lines of lung cancer, but no activity was seen in 6 lines of lung cancer and one line of gastric cancer. No plasmin activity was found. The tumour cell lines could be classified into 3 groups on the basis of the 2 activities. Cancer cell lines could also be classified into 2 groups: with high or low release of thromboplastin into culture media. Fibrinolytic activity was found in the culture media of all cell lines with high fibrinolytic activity. Fibrinolytic activity, but not thromboplastic activity, seemed to be influenced by the constituents of culture media. No definite correlation was found between the 2 activities and the histological types of the parent tumours of the cultured cells.

Topics: Blood Coagulation; Cell Line; Factor IX; Factor VII; Fibrinolysis; Humans; Lung Neoplasms; Neoplasms; Stomach Neoplasms; Thromboplastin; Urinary Bladder Neoplasms

Topics: Cells, Cultured; Humans; Plasminogen Activators; Thromboplastin; Urinary Bladder Neoplasms