warfarin and Carcinoma--Hepatocellular

Topics: Animals; BCG Vaccine; Brain Neoplasms; Carcinoma, Hepatocellular; Culture Techniques; DNA, Neoplasm; Fluorouracil; Glioma; Humans; Kinetics; Liver Neoplasms; Mice; Mitosis; Neoplasms, Experimental; Neuroblastoma; RNA, Neoplasm; Warfarin

Hepatocellular carcinoma (HCC) is a long-term complication of Fontan-associated liver disease (FALD). However, risk factors for HCC in patients with FALD remain unclear. This study aimed to identify factors associated with HCC development post-Fontan procedure.. We retrospectively examined 103 post-Fontan patients who underwent hepatic imaging at our institution. HCC incidence and patient characteristics were analyzed. A Cox proportional hazards model was used to identify risk factors for HCC.. The median interval between Fontan surgery and final hepatic imaging was 19.6 (1.0-37.7) years. Among 103 patients, nine developed HCC. The cumulative incidence rates of HCC at 10, 20, and 30 years postoperatively were 0%, 7%, and 13%, respectively. In the univariate analysis, age at Fontan surgery, situs inversus, and warfarin absence were associated with HCC occurrence. The multivariate analysis identified the warfarin absence (adjusted hazard ratio [aHR], 22.71; 95% confidence interval: 3.29-507.1; p = 0.0005) and situs inversus (aHR, 14.36; 95% confidence interval: 2.75-105.5; p = 0.002) as risk factors. The prevalence of situs inversus and the warfarin absence was 12% and 50%, respectively. The 20- and 30-year incidence rates of HCC among patients who received warfarin were 0% and 7%, respectively, while those among patients who did not receive warfarin were 14% and 21%, respectively. HCC incidence was significantly higher in the non-warfarin group than in the warfarin group (p = 0.006) and among patients with situs inversus than among those with situs solitus (p = 0.004).. Warfarin absence and situs inversus were associated with HCC development post-Fontan procedure.

Topics: Carcinoma, Hepatocellular; Fontan Procedure; Humans; Liver Neoplasms; Retrospective Studies; Situs Inversus; Warfarin

Topics: Anticoagulants; Biomarkers; Blood Coagulation; Carcinoma, Hepatocellular; Case-Control Studies; Chromatography, High Pressure Liquid; Drug Monitoring; Healthy Volunteers; Hemorrhage; Humans; Liver Neoplasms; Prothrombin; Tandem Mass Spectrometry; Warfarin

A 69-year-old man was admitted to hospital with abdominal pain. In the four years prior to his presentation, he had undergone repeated transarterial chemoembolizations and injections for hepatocellular carcinoma. He underwent his 8th transcatheter arterial therapy one month prior to admission. Abdominal X-rays and contrast-enhanced computed tomography showed large amounts of small intestinal gas and venous thrombosis from the portal vein to the superior mesenteric vein, respectively. The thrombosis was reduced after anticoagulation therapy (heparin, antithrombin III, danaparoid sodium and warfarin). This is the first case report of paralytic ileus due to superior mesenteric venous thrombosis after transcatheter arterial therapy for hepatocellular carcinoma with an arterioportal shunt.

Topics: Abdominal Pain; Aged; Anticoagulants; Carcinoma, Hepatocellular; Embolization, Therapeutic; Humans; Intestinal Pseudo-Obstruction; Liver Neoplasms; Male; Mesenteric Ischemia; Mesenteric Veins; Portal Vein; Radiography; Thrombophlebitis; Treatment Outcome; Venous Thrombosis; Warfarin

Serum des-γ-carboxy prothrombin (DCP) levels using a newly developed electrochemiluminescence immunoassay (ECLIA, novel DCP [NX-DCP]) were measured, and the utility of NX-DCP and DCP/NX-DCP ratio for the diagnosis of hepatocellular carcinoma (HCC) was investigated. Antigenic differences in DCP between HCC and non-HCC patients were elucidated.. The subjects included 170 patients with HCC, 61 with benign liver disease, 12 with obstructive jaundice, and 10 warfarin users. NX-DCP was quantitated by sandwich ECLIA employing novel anti-DCP monoclonal antibodies, P11 and P16. Conventional DCP was quantitated by standard ECLIA. DCP extracted from serum by affinity-chromatography was analyzed by Western blotting.. Conventional serum DCP levels were high in patients with HCC and obstructive jaundice, and in warfarin users, consistent with previous reports. Serum NX-DCP levels were high only in warfarin users and obstructive jaundice patients (vitamin K-deficient patients) but not in HCC patients. The DCP/NX-DCP ratio was significantly higher in the HCC group than in the benign liver disease, obstructive jaundice, and warfarin groups (P < 0.001). Receiver operating characteristic analysis showed significant superiority of the DCP/NX-DCP ratio over conventional DCP as a marker for HCC diagnosis (P < 0.05). Western blot analysis showed that P11 and P16 reacted strongly with DCP from a warfarin user and an obstructive jaundice patient but very faintly with DCP from an HCC patient. Immunohistochemistry on HCC samples and autopsied normal liver tissues from warfarin users showed similar results.. The DCP/NX-DCP ratio is very useful for diagnosing HCC. DCP in HCC patients is distinct from that in vitamin K-deficient patients.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Biomarkers, Tumor; Carcinoma, Hepatocellular; Diagnosis, Differential; Female; Humans; Immunoassay; Jaundice, Obstructive; Liver Neoplasms; Male; Middle Aged; Protein Precursors; Prothrombin; Vitamin K Deficiency; Warfarin

Des-gamma-carboxy prothrombin (DCP) is a useful tumor marker for hepatocellular carcinoma (HCC), but its utility is limited in patients taking vitamin K antagonists. We evaluated the NX-DCP ratio, a newly developed method to measure serum DCP, for its ability to identify DCP elevation induced by HCC in this patient subpopulation. Conventional DCP measurements and the NX-DCP ratio were compared in patients with and without HCC, all of whom were taking the vitamin K antagonist warfarin. We found no differences in conventional DCP measurements between patients with and without HCC due to warfarin treatment. In contrast, the NX-DCP ratio was significantly higher in patients with HCC; the NX-DCP ratio in all patients without HCC was <1.50. When the cut-off was fixed at 1.50, sensitivity and specificity for HCC diagnosis were 60.0% and 100.0%, respectively, which are comparable to those of conventional DCP measurements in patients not taking warfarin. The novel NX-DCP ratio identifies patients on warfarin with elevated DCP due to HCC and is useful as a tumor marker for HCC in this patient subpopulation.

Topics: Aged; Aged, 80 and over; Biomarkers; Biomarkers, Tumor; Blood Chemical Analysis; Carcinoma, Hepatocellular; Female; Humans; Liver Neoplasms; Male; Middle Aged; Protein Precursors; Prothrombin; Sensitivity and Specificity; Warfarin

A 60-year-old man with liver cirrhosis caused by hepatitis C, who was receiving warfarin anticoagulation following acute myocardial infarction, was diagnosed with advanced hepatocellular carcinoma and multiple lung metastases, and began treatment with sorafenib 200 mg daily. From the treatment's start to 14 and 63 days later, sorafenib was increased to 400 mg and 600 mg, respectively. After increasing the quantity to 600 mg, he had an increase in PT-INR values and experienced a lower-extremity hemorrhage. For the patient with liver cirrhosis, who is receiving warfarin, PT-INR values might be elevated during the early period of sorafenib treatment dosage as for the increase in quantity. Therefore, when increasing dosage, a frequent measurement of PT-INR and a careful follow-up for PT-INR is necessary.

Topics: Anticoagulants; Antineoplastic Agents; Benzenesulfonates; Carcinoma, Hepatocellular; Drug Therapy, Combination; Gastrointestinal Hemorrhage; Humans; Liver Neoplasms; Male; Middle Aged; Niacinamide; Phenylurea Compounds; Pyridines; Sorafenib; Tomography, X-Ray Computed; Warfarin

The case of a patient who experienced elevated International Normalized Ratio (INR) values and hemorrhage after sorafenib was added to his warfarin regimen is presented.. A 70-year-old Caucasian man with a history of hypertension, congestive heart failure, gastroesophageal reflux disease, chronic obstructive pulmonary disease, and chronic hepatitis C was treated with warfarin for anticoagulation subsequent to atrial fibrillation. He began anticoagulation management by a clinical pharmacist in April 2007 and was stabilized on warfarin 36 mg weekly. He was diagnosed with hepatocellular carcinoma in June 2007 and began treatment with sorafenib 200 mg daily in September 2007. The patient arrived at the emergency room one month later with a prothrombin time (PT) of 84.8 and an INR value of 39.5. He was admitted for lower-extremity hemorrhage and diagnosed with warfarin toxicity. Sorafenib was discontinued, and warfarin was held during this hospital stay. The patient was discharged on warfarin 3 mg daily. In November, warfarin was increased to 36 mg weekly, and his INR values stabilized. In late November, he was restarted on sorafenib 200 mg daily presumably due to multiple new hepatic hypodense lesions indicating progression of the metastatic disease. Approximately two weeks later, the patient's INR value increased to 4.7. Sorafenib was discontinued permanently. Both the Naranjo et al. probability scale score and the drug interaction probability scale score suggest that there was a probable interaction between warfarin and sorafenib.. A 70-year-old man diagnosed with hepatocellular carcinoma experienced an increase in INR values after the addition of sorafenib to his warfarin regimen.

Topics: Aged; Anticoagulants; Benzenesulfonates; Carcinoma, Hepatocellular; Drug Interactions; Hemorrhage; Humans; International Normalized Ratio; Liver Neoplasms; Male; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Pyridines; Sorafenib; Warfarin

This study describes a generic biological screening assay designed to detect anticoagulant rodenticides based on their inhibitory action on the vitamin K epoxide reductase protein complex, resulting in an accumulation of under-carboxylated prothrombin or proteins induced by vitamin K antagonism (PIVKA-II). A combined cell culture/ELISA assay was optimized to measure PIVKA-II production by the human hepatoma HepG2 cell line cultured in the presence of anticoagulant rodenticides. The specificity and sensitivity of the assay was validated using 41 grain extracts containing representative concentrations of rodenticide or appropriate nonrodenticide control compounds. In all cases, PIVKA-II produced by HepG2 cells in response to grain extracts spiked with rodenticides was detected by ELISA, while PIVKA-II was not detected in supernatants collected from cells exposed to nonrodenticide controls. This represents a novel, class-specific biological assay for the detection of anticoagulant rodenticides present in contaminated grain.

Topics: Anticoagulants; Biological Assay; Biomarkers; Carcinoma, Hepatocellular; Cell Line, Tumor; Coumarins; Edible Grain; Enzyme-Linked Immunosorbent Assay; Food Contamination; Humans; Indans; Liver Neoplasms; Organophosphates; Protein Precursors; Prothrombin; Rodenticides; Warfarin

In the absence of vitamin K (VK) or in the presence of VK antagonists, hepatic VK-dependent carboxylase activity is inhibited and des-gamma-carboxyprothrombin (DCP) is released into the blood. We analyzed the number of glutamic acid (Glu) residues and their positions in the Gla domain (GD) of DCP to investigate the gamma-carboxylation mechanism of VK-dependent carboxylase. Several DCPs were found in each subject studied. The 10 Gla residues of human prothrombin were carboxylated in order from the N-terminal (residues 26, 25, 16, 29, 20, 19, 14, 32, 7 and 6). The process of Glu carboxylation seemed to proceed three-dimensionally from inside to outside the molecule.

Topics: Adult; Aged; Amino Acid Motifs; Anticoagulants; Biomarkers; Biomarkers, Tumor; Carbon-Carbon Ligases; Carcinoma, Hepatocellular; Female; Glutamic Acid; Humans; Liver Neoplasms; Male; Peptide Fragments; Protein Precursors; Prothrombin; Vitamin K; Warfarin

We experienced two patients with a prosthetic heart valve, who underwent hepatic resection for hepatoma while on anticoagulation therapy. Patients with a prosthetic heart valve have the following characteristics; an increased risk of thromboembolism due to diminished anticoagulation in the perioperative period, a greater risk of endocarditis due to the artificial material in the heart, and impaired cardiopulmonary function including possible arrhythmia and heart failure. Furthermore, when such patients also have liver cirrhosis with a hepatoma, there is an increased risk of perioperative bleeding while on anticoagulation due to coagulopathy and also a risk of infection due to decreased cellular immunity. Patients with a prosthetic heart valve therefore require special care and attention whenever they have to undergo hepatic resection. With respect to anticoagulation, a minimal level is required to prevent bleeding and thromboembolism. Warfarin being administered preoperatively may be switched to heparin while closely monitoring the activated clotting time (biomaterial valve: 130-150 sec, non-biomaterial valve: 150-180 sec); the heparin should then be changed back to warfarin immediately after starting oral intake following operation. For the prevention of infection, a broad spectrum antibiotic should be used prophylactically both intra-operatively and postoperatively. The cardiopulmonary function must also be carefully monitored. For the assessment of postoperative liver function, lecithin: cholesterol acyltransferase, serum bilirubin and albumin are useful because there is no relevance of coagulation parameters such as prothrombin time under anticoagulation.

Topics: Carcinoma, Hepatocellular; Clinical Protocols; Combined Modality Therapy; Heart Valve Diseases; Heart Valve Prosthesis; Heparin; Hepatectomy; Humans; Liver Neoplasms; Male; Middle Aged; Mitral Valve; Postoperative Complications; Preoperative Care; Treatment Outcome; Tricuspid Valve; Warfarin

Vitamin K is a substrate for the enzyme catalyzing the carboxylation of specific glutamyl residues to gamma-carboxyglutamyl residues in hepatic precursors of a limited number of plasma proteins, including prothrombin. The gamma-carboxylation of these proteins can be blocked by the anticoagulant warfarin; and in the bovine and human, warfarin treatment results in the secretion of under-gamma-carboxylated forms of prothrombin into plasma. In the rat, this response is not seen, but plasma prothrombin concentrations are drastically decreased. This response has now been studied in rat hepatoma (H-35) cells in which prothrombin secretion is decreased 90% by incubation in the presence of warfarin. Neither prothrombin mRNA levels nor the apparent rate of prothrombin message translation were decreased when cells were cultured in the presence of warfarin rather than of vitamin K. The pool of intracellular prothrombin precursors is increased threefold by warfarin treatment, and this pool is rapidly secreted when vitamin K is administered. In contrast, continued incubation in the presence of warfarin resulted in the degradation of 60% of this pool in 24 hours. When transport of secretory proteins to the golgi apparatus was blocked with Brefeldin A, this precursor pool was gamma-carboxylated in the presence of vitamin K and no degradation occurred. Lysosomal enzyme inhibitors did not block the degradation, and the data suggest that, in rat hepatocytes, under-gamma-carboxylated prothrombin is specifically targeted to a pathway of protein degradation located in the endoplasmic reticulum.

Topics: Animals; Carcinoma, Hepatocellular; Liver Neoplasms, Experimental; Lysosomes; Protein Precursors; Prothrombin; Rats; RNA, Messenger; Tumor Cells, Cultured; Warfarin

Cultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 micrograms vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 micrograms/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 micrograms/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 micrograms/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240-350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on gamma-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.

Topics: Antigens; Blotting, Northern; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Methionine; Nucleic Acid Hybridization; Precipitin Tests; Prothrombin; RNA, Messenger; Sulfur Radioisotopes; Transcription, Genetic; Tumor Cells, Cultured; Vitamin K 1; Warfarin

Guidelines for the management of patients receiving chronic anticoagulation therapy who require liver biopsy are not clearly defined. In patients with normal coagulation, liver biopsy is a relatively safe procedure with a morbidity of less than 0.1% and a mortality of less than 0.01%. We report a patient with a prosthetic aortic valve who developed a large subcapsular hematoma 12 days after a percutaneous liver biopsy as a consequence of warfarin toxicity. Based on the experience with this patient, reinstitution of anticoagulant therapy should be avoided for at least 72 h after a percutaneous liver biopsy. Intravenous heparin should be resumed first, and warfarin added if no bleeding has occurred after an additional 48-72 h. The prothrombin time should be maintained at 1.5 times the baseline.

Topics: Biopsy, Needle; Carcinoma, Hepatocellular; Chemical and Drug Induced Liver Injury; Hematoma; Humans; Liver Diseases; Liver Neoplasms; Male; Middle Aged; Prothrombin Time; Time Factors; Warfarin

About 30% of human plasma protein C is smaller than the predominant form as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has been suggested that this species, referred to as beta protein C, is a degraded molecule. However, beta protein C is secreted in culture by the HepG2 cell line and is present in plasma collected directly into numerous proteinase inhibitors; the percentage of beta protein C does not change with time during culture or after blood collection. Neither thrombin, activated protein C, nor activated factor X converts the alpha form to beta in the presence or absence of calcium and phospholipids. The NH2-terminal sequences of the heavy chains of both forms are identical, and both release the same dodecapeptide and develop a functional active site when cleaved by thrombin. Both also react with antibodies to a synthetic COOH-terminal peptide. Timed digests with N-glycosidase are consistent with the interpretation that beta protein C has three N-linked oligosaccharide chains whereas alpha protein C has four. It is asparagine 329 that is not glycosylated in beta protein C since antibodies to a synthetic peptide based on the sequence around this amino acid react only with beta protein C. This site is unique in having cysteine instead of serine or threonine 2 residues distal. It is likely that the sulfhydryl group can substitute for the usual hydroxyl group as a hydrogen bond acceptor for the glycosylation reaction only until it forms a disulfide bond. The percentage of protein C that is glycosylated at this site may therefore depend at least in part on the rate of disulfide bond formation which may in turn be related to the rate of protein synthesis.

Topics: Amino Acid Sequence; Antibodies; Asparagine; Binding Sites; Blotting, Western; Carcinoma, Hepatocellular; Cysteine; Epitopes; Glycoside Hydrolases; Glycosylation; Humans; Liver Neoplasms; Molecular Sequence Data; Oligosaccharides; Protein Biosynthesis; Protein C; Thrombin; Tumor Cells, Cultured; Warfarin

We measured des-gamma-carboxyglutamic acid prothrombin (protein induced by vitamin K absence or antagonist-Factor II: [PIVKA-II]) in plasmas of normal subjects, patients with thrombotic disease, those with hepatic disease including hepatocellular carcinoma, and those with carcinoma of other tissues, and compared the results with results of blood coagulation tests used for the examination of hepatic function. In addition, in the patients with hepatic disease, PIVKA-II and alpha-fetoprotein (AFP) levels were compared. The PIVKA-II level was frequently high in patients with thrombotic disease given warfarin therapy and those with hepatocellular carcinoma. However, in patients with thrombotic disease who were not given warfarin therapy, no significant correlation was seen between the PIVKA-II value and the results of the thrombotest or hepaplastin test, suggesting no association between the PIVKA-II level and the degree of impairment of hepatic function. In 70 patients with hepatocellular carcinoma, the percentage of patients positive for PIVKA-II (greater than or equal to 0.1 micrograms/ml) and those positive for AFP (greater than or equal to 20 ng/ml) were similar (77% and 74%, respectively). Pearson's correlation of coefficient between the PIVKA-II value and the AFP value in the 70 patients was 0.463. However, false-positive rates in patients with hepatic disease other than hepatocellular carcinoma were lower for PIVKA-II. Combined assessment of PIVKA-II and AFP increased positive rates and allowed exclusion of false-positive patients. The plasma PIVKA-II level is suggested to be useful as an indicator of warfarin control in patients with thrombotic disease, as a marker of hepatocellular carcinoma, and is particularly of value when assessed in combination with AFP.

Topics: Biomarkers; Biomarkers, Tumor; Blood Coagulation Factors; Carcinoma, Hepatocellular; Humans; Liver Diseases; Liver Neoplasms; Protein Precursors; Prothrombin; Warfarin

In order to examine the control of human factor X biosynthesis we have molecularly cloned the cDNA and investigated the expression of the Factor X gene. A recombinant clone of approximately 1100 base pairs in length containing the sequence of factor X was identified in a lambda gt11 human liver cDNA library by screening with polyclonal antibodies. One plaque was selected and confirmed for specificity with a mixture of five factor X specific monoclonal antibodies (MoAbs). A partial nucleic acid sequence of the 5' end of the cDNA corresponded to the described amino acid sequence between residues 41 and 56 of the light chain of factor X. Northern blot analysis of RNA from human liver and the hepatoma cell line, Hep G2, identified the factor X mRNA as a single molecular species of approximately 1700 bases. Cell lines which do not secrete factor X did not contain factor X mRNA indicating restriction of transcription to hepatocytes. Slot-blot hybridization analysis of factor X and actin mRNA demonstrated no change in the levels of total or specific factor X mRNA in Hep G2 cells following treatment with warfarin or vitamin K. We conclude that modulation of factor X production by these drugs, known to influence gamma-carboxylation and total factor X secretion by these cells, is mediated by changes in posttranscriptional events rather than by effects on the steady state levels of factor X mRNA.

Topics: Actins; Carcinoma, Hepatocellular; Cell Line; DNA; Factor X; Gene Expression Regulation; Humans; Liver; Liver Neoplasms; RNA, Messenger; Vitamin K; Warfarin

Prothrombin synthesis and secretion were studied in a human hepatoma cell line (Hep G2) incubated with 35S-methionine for 2 to 24 hours at 37 degrees C. Extracellular and intracellular prothrombin were detected by immunoprecipitation with affinity-purified antiprothrombin antibody. Incorporation of 35S-methionine into prothrombin was monitored by counting specific bands excised from 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Prothrombin represented 0.3% to 0.7% of total newly synthesized protein secreted into the media. Warfarin had no effect on total prothrombin synthesis (extracellular plus intracellular). However, warfarin inhibited secretion of newly synthesized prothrombin by 58% to 73% over a 2 to 4 hour period. This was accompanied by the intracellular accumulation of an immunoprecipitable species of prothrombin of 78 kd, 6 kd less than extracellular prothrombin. At the end of the 4-hour incubation with warfarin, intracellular prothrombin increased from 44% to 82% (twofold) of total prothrombin, whereas extracellular prothrombin decreased from 56% to 19% (threefold) of total prothrombin. After 24-hour incubation with warfarin, intracellular and extracellular immunoprecipitable prothrombin approached control values. Deglycosylation of extracellular and intracellular prothrombin with hydrofluoric acid (HF) resulted in a decrease in mol wt for both species to 66 kd. Endoglycosidase-H treatment, which digests "early mannosyl" residues, resulted in a decrease in the mol wt of the intracellular species of 8 kd with no effect on the extracellular species. Thus, the lower mol wt intracellular species that accumulates following early warfarin treatment is due to the presence of incompletely processed carbohydrate chain. The data are compatible with the hypothesis that optimum glycosylation and secretion require Vitamin K-dependent carboxylation.

Topics: Acetylglucosaminidase; Carcinoma, Hepatocellular; Cell Line; Cycloheximide; Glycosylation; Humans; Liver Neoplasms; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Molecular Weight; Protein Biosynthesis; Prothrombin; Time Factors; Warfarin

Using specific radioimmunoassays, 8 day cultures of Hep G2 cells were shown to contain in their supernatants 16, 74, and 828 ng/mL and in their cell lysates, 8, 55, and 48 ng/2 X 10(8) cells of factor VII, protein C, and protein S, respectively. These proteins and the protein C inhibitor were functionally active, and each of these activities was neutralized by their respective polyclonal antibodies. Although vitamin K had a modest effect, warfarin decreased the activity of secreted factor VII, protein C, and protein S by 50% to 90%. Protein C and protein S antigens were reduced three- to fourfold by warfarin. The protein C inhibitor antigen and activity were unaffected by vitamin K or warfarin treatment. Intrinsic labeling and immunoprecipitation indicated that factor VII, protein S, and the protein C inhibitor were secreted as 52,000, 77,000, and 58,000 molecular weight (mol wt) proteins, respectively. Protein C was secreted as a single-chain protein of about 65,000 mol wt, indicating that all of the vitamin K-dependent proteins are translated and secreted as single-chain molecules. Each of the four proteins studied represented their plasma protein counterparts structurally, functionally, and immunochemically. Thus, all of the known soluble components of the protein C pathway are produced by liver parenchymal cells.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; Factor VII; Glycoproteins; Humans; Liver Neoplasms; Molecular Weight; Protein C; Protein S; Rabbits; Radioimmunoassay; Vitamin K; Warfarin

Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on Western blotting stained with an apparent intensity 5-8% that of Factor VII. This glycoprotein, tentatively called VII*, has a molecular weight 4,500 D less than Factor VII, lacks detectable Factor VII functional activity, does not bind to barium citrate, and is not recognized by a monoclonal antibody that recognizes Factor VII but not alpha-chymotrypsin-treated Factor VII. VII* was not proteolytically produced from Factor VII during in vitro coagulation or after infusion of human Factor VII into rabbits. As determined by Western blotting, the human hepatoma cell line, HepG2, cultured in the presence of vitamin K, secreted relatively greater levels of VII* in proportion to VII (75%) than that found in human plasma. Warfarin treatment of HepG2 cells decreased the quantity of VII secreted by 77%, whereas it only inhibited the secretion of VII* by 14%. Immunologic studies of the plasmas from a patient on chronic warfarin therapy and an individual given a short course of high dose warfarin therapy corroborated the in vitro synthetic studies obtained with HepG2 cells. The data are consistent with the production of VII* by posttranslational, proteolytic, modification of VII, that, at least in the HepG2 cells studied, occurs intracellularly. However, other mechanisms for the production of VII*, in particular, alternative RNA splicing of the transcript from a single gene, cannot be excluded.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Atrial Fibrillation; Carcinoma, Hepatocellular; Collodion; Cross Reactions; Electrophoresis, Polyacrylamide Gel; Factor VII; Factor VII Deficiency; Humans; Isoantigens; Liver Neoplasms; Mice; Rabbits; Warfarin

The human hepatoma cell line, Hep G2, was analyzed for the ability to synthesize and secrete several coagulation proteins. Using specific radioimmunoassays, factor X, prothrombin, and antithrombin III were present in 8-day culture supernatants at 62, 405, and 1,220 ng/mL, respectively. Factor IX was not detected, either in supernatants or in cell extracts. Intrinsically labeled factor X was secreted as a single-chain polypeptide of 66,000 daltons, as measured by sodium dodecylsulfate-polyacrylamide gels under nonreduced and reduced conditions. Immunoblots of Hep G2 supernatants and normal human plasma also indicate the presence of single-chain factor X. These findings support the hypothesis of a postsecretion proteolytic cleavage of factor X into the two-chain form. Prothrombin and antithrombin represented their plasma protein counterparts structurally, with molecular weights of 73,000 and 61,000, respectively. Secreted factor X, prothrombin, and antithrombin III were biologically active, as determined in coagulation or chromogenic assays, and all three activities were neutralized by monospecific antibodies. Vitamin K increased the quantity of prothrombin secreted by twofold, without affecting the rate of secretion over a five-day culture period, and had an apparent transient inhibitory effect on secretion of antithrombin III. Warfarin caused a three to fourfold decrease in the rate and quantity of secreted prothrombin, but did not affect intracellular concentrations. The intracellular and extracellular concentrations and rate of secretion of antithrombin III were not modulated by warfarin. These data suggest that the Hep G2 cell line may provide a useful model for assessing the regulation of biosynthesis and secretion of human coagulation proteins.

Topics: Antithrombin III; Carcinoma, Hepatocellular; Cell Line; Chromogenic Compounds; Factor X; Humans; Liver Neoplasms; Prothrombin; Tissue Extracts; Vitamin K; Warfarin

Topics: Animals; Carbon Radioisotopes; Carcinoma, Bronchogenic; Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Cell Division; Culture Techniques; Drug Synergism; Fluorouracil; Glioblastoma; HeLa Cells; Humans; L Cells; Liver; Liver Neoplasms; Mice; Mice, Inbred Strains; Microsomes, Liver; Neoplasm Transplantation; Neoplasms; Neoplasms, Experimental; Neuroglia; Pancreatic Neoplasms; RNA, Ribosomal; Warfarin

Topics: Animals; Carbon Isotopes; Carcinoma, Hepatocellular; Cell Nucleus; Cytoplasm; Liver; Liver Neoplasms; Male; Mice; Microsomes; Orotic Acid; Rats; Rats, Inbred Strains; RNA, Ribosomal; Warfarin

Topics: Animals; Barium Sulfate; Blood Coagulation Factors; Carcinoma, Hepatocellular; Chromatography, Gel; Clone Cells; Culture Media; Factor V; Factor VII; Humans; Liver Neoplasms; Methods; Neoplasms, Experimental; Rats; Vitamin K; Warfarin

Topics: Adolescent; Carcinoma, Hepatocellular; Diagnosis, Differential; Female; Humans; Hypersensitivity; Liver Neoplasms; Necrosis; Skin Diseases; Thigh; Thrombophlebitis; Warfarin