RESUMO
The effect of preimmunisation with cancer procoagulant (CP) on the growth of Walker 256 carcinosarcoma cells in Wistar Lew/Han/IMP rats in vivo has been analysed. One group of rats was immunised with CP purified from human amnion-chorion membranes. The rest of the animals (control groups) were injected with 0.9% NaCl in Freund's adjuvant or were not immunised at all. When the presence of polyclonal anti-CP antibody was detected in CP-immunised rats' serum, all (CP-immunised and control) animals were injected i.p. with 4.8 x 10(5) Walker 256 cells per rat. After 5 days ascitic fluid was collected and viable cells were counted. The complete lack of viable Walker 256 carcinosarcoma cells in 24% of the CP-immunised rats was observed. However, the viable neoplastic cells were present in all control (NaCl-injected and nonimmunised) animals. It seems possible that CP plays an important role in tumour progression, so immunisation with CP can reduce the risk of development of malignant disease.
Assuntos
Carcinoma 256 de Walker/terapia , Cisteína Endopeptidases/imunologia , Proteínas de Neoplasias/imunologia , Animais , Carcinoma 256 de Walker/imunologia , Carcinoma 256 de Walker/patologia , Cisteína Endopeptidases/administração & dosagem , Cisteína Endopeptidases/isolamento & purificação , Feminino , Humanos , Imunização , Masculino , Proteínas de Neoplasias/administração & dosagem , Proteínas de Neoplasias/isolamento & purificação , Gravidez , Ratos , Ratos WistarRESUMO
Hemostatic disorders mainly due to thrombocytopenia represent an important clinical problem in patients with myelodysplastic syndromes (MDS). Much less is known about the possible coagulation abnormalities. Thirty patients with MDS were studied. Activity of cancer procoagulant (CP), concentrations of activation markers of coagulation and fibrinolysis such as thrombin-antithrombin complexes (TAT), prothrombin fragment 1+2 (F1+2) and D-dimers (DD) as well as standard coagulation tests were determined. Coagulation abnormalities concerned mainly patients with RAEB and RAEB-t. In this group the mean values of TATand F1+2 concentrations were significantly higher than in control indicating chronic coagulation activation similar to that observed in acute leukemias. CP activity in MDS patients did not differ from the control group.
Assuntos
Transtornos da Coagulação Sanguínea/sangue , Cisteína Endopeptidases/sangue , Síndromes Mielodisplásicas/sangue , Proteínas de Neoplasias , Antitrombina III , Humanos , Fragmentos de Peptídeos/sangue , Peptídeo Hidrolases/sangue , Protrombina , Tempo de ProtrombinaRESUMO
Cancer procoagulant, cysteine proteinase (CP; EC 3.4.22.26) activates factor X and functions in the absence of factor VII. CP may also change the platelet function. It induces an increase of platelet adhesion to collagen and fibrinogen. Using wortmannin--the inhibitor of phosphoinositide 3-kinase (PI 3-K)--we studied the role of this enzyme in the action of cancer procoagulant on blood platelet adhesion in vitro. Wortmannin (25, 50 and 100 nM, 30 min, 37 degrees C) caused a reduction of platelet adhesion to fibrinogen (P<0.01) when blood platelets were stimulated by both 0.2 U/ml thrombin (IC(50)approximately 75 nM) and by 1 microM ADP (IC(50)approximately 60 nM). We observed that after CP treatment the adhesion of thrombin-activated and ADP-stimulated platelets to fibrinogen was augmented. The potentiated by CP adhesion of activated platelets to fibrinogen was reduced after preincubation of platelets with wortmannin (50 nM, 30 min, 37 degrees C). We conclude that in adhesion of platelets to fibrinogen stimulated by CP PI 3-K take place.
Assuntos
Cisteína Endopeptidases/farmacologia , Fibrinogênio/metabolismo , Proteínas de Neoplasias , Fosfatidilinositol 3-Quinases/fisiologia , Adesividade Plaquetária/efeitos dos fármacos , Difosfato de Adenosina/farmacologia , Androstadienos/farmacologia , Animais , Relação Dose-Resposta a Droga , Inibidores de Fosfoinositídeo-3 Quinase , Ativação Plaquetária/efeitos dos fármacos , Suínos , Trombina/farmacologia , WortmaninaRESUMO
The effects of cancer procoagulant (CP), cysteine protease (EC 3.4.22.26), on the pig blood platelet secretory process and platelet aggregation have been studied. The response of platelets to CP was compared with the response of these cells to thrombin. The obtained results show that blood platelets treated with CP (0.5, 1, 2.5, and 5 microg/ml, 2-30 min, 37 degrees C) released adenine nucleotides (P < 0.05) and proteins (P < 0.05). The secretion of compounds from blood platelets after incubation with CP does not correlate with the release of platelet lactic dehydrogenase activity (marker of cell lysis) into the extracellular medium. In comparison with thrombin action, CP stimulates secretory process to a smaller extent than thrombin alone. In the presence of CP, the thrombin action is suppressed (P < 0.05). We noticed that CP does not induce platelet aggregation.
Assuntos
Plaquetas/metabolismo , Cisteína Endopeptidases/farmacologia , Proteínas de Neoplasias , Neoplasias/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Adenina/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Suínos , Trombina/farmacologia , Fatores de TempoRESUMO
Cancer procoagulant (CP), EC3.4.22.26, is a cysteine proteinase produced by malignant or fetal tissue. It is a single chain Mr 68 kDa protein containing no carbohydrates. Prevalent amino acids are Ser (19.15), Gly (18.7%), Glu (12.5%), Lys (8.1%) and Asp (7.1%). Amino-acid sequence of CP is not determined yet. The natural substrates for CP are coagulation factor X and probably PAR-1 receptor. However, the peptidyl bond in factor X molecule hydrolyzed by CP is different from the bond recognized by other known FX-activators. Peptidyl chromogenic substrates hydrolyzed by CP are those with Pro at P2 and Arg or Lys at P1 position. Factor X-activating activity of CP is inhibited by number of various inhibitors: peptidyl diazomethyl ketones. iodoacetamide, mercuric chloride, PMSF, leupeptin and antipain. It has been demonstrated that E-64, a specific inhibitor of cysteine proteinases, inhibits CP reversible acting as a competitive inhibitor of the enzyme. The CP activity is modulated by the presence of divalent metal ions in the reaction environment. Ca2+, Cd2+, Mg2+ and Mn2+ ions potentiate the procoagulant activity when Cu2+, Fe2+, Sn2+ and Zn2+ ions inactivate CP.
Assuntos
Fatores de Coagulação Sanguínea/química , Cisteína Endopeptidases/química , Proteínas de Neoplasias , Animais , Cátions Bivalentes/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Fator X/metabolismo , Humanos , Camundongos , CoelhosAssuntos
Cisteína Endopeptidases/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Ácido Araquidônico/metabolismo , Radicais Livres/sangue , Radicais Livres/metabolismo , Radicais Livres/farmacologia , Humanos , Medições Luminescentes , Malondialdeído/metabolismo , Proteínas de Neoplasias/farmacologia , Superóxidos/sangue , Trombina/farmacologiaAssuntos
Plaquetas/citologia , Cisteína Endopeptidases/farmacologia , Proteínas de Neoplasias , Difosfato de Adenosina/farmacologia , Animais , Fatores de Coagulação Sanguínea/farmacologia , Adesão Celular/efeitos dos fármacos , Colágeno/sangue , Cisteína Endopeptidases/sangue , Fibrinogênio/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Suínos , Trombina/farmacologiaRESUMO
The activity of cancer procoagulant (CP), prothrombin time (PT), activated partial thromboplastin time (APTT), the concentration of thrombin-antithrombin complexes (TAT) and the concentration of fibrinogen were analysed in blood of breast cancer patients scheduled for surgery. The serum level of CP activity was dependent on the stage of the disease. The CP activity was increased in 72% of patients with an early stage of cancer and in only 20% of patients with an advanced stage of the disease when compared to the baseline level for non-cancer controls. In all patients PT remained at normal levels (80-120%). There was no significant change in APTT (27-39 s) in early stage cancer patients. Only one patient with advanced cancer had APTT shortened to 23 s. Also one advanced stage patient had significantly elevated level of TAT (14.96 microg/l); in all other patients the concentration of TAT remained at normal levels (1-4.1 microg/l). Forty-four percent of early stage cancer patients and 22% of advanced cancer patients had an elevated level of fibrinogen (Fg) ( > 350 mg%). However, there was no correlation between the level of Fg and the CP activity (P > 0.05). The data suggest that: (1) serum CP activity increases at the early stage of breast cancer and decreases down to the normal level in the advanced stage of the disease; (2) there is no evidence of blood clotting activation in the early stage breast cancer patients; and (3) CP does not facilitate the activation of coagulation in the breast cancer patients or the level of such activation is below the sensitivity of assays used in the experiment.
Assuntos
Fatores de Coagulação Sanguínea/análise , Coagulação Sanguínea , Neoplasias da Mama/sangue , Cisteína Endopeptidases/análise , Proteínas de Neoplasias , Antitrombina III/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Tempo de Tromboplastina Parcial , Peptídeo Hidrolases/análiseRESUMO
Hemostatic abnormalities associated with malignant disease led to the search for and discovery of a proteolytic enzyme that activated factor X in the blood coagulation cascade. It was named cancer procoagulant (CP). CP is a cysteine proteinase that is found in malignant and fetal (human amnion-chorion) tissue; it has not been found in normally differentiated tissue. It is a calcium-dependent, Mn2+ stimulated enzyme that has enhanced activity and inhibition in a reduced environment. This review presents a complete compilation and discussion of the known chemical and enzymatic characteristics of CP as well as many purification and assay procedures. Several unique properties of these procedures are described. Some problems and controversies are highlighted in each of the sections. An immunoassay for CP as a tumor marker and some of its potential applications in the diagnosis and monitoring of cancer are reviewed. Some therapeutic implications of CP are noted in light of the observation that antibodies to CP block the metastatic seeding of lung colonies in vivo and diminish the viability of tumor cells in vitro. Finally, comments about the relationship between tissue factor and CP in the malignant cells are provided.
Assuntos
Fatores de Coagulação Sanguínea/fisiologia , Cisteína Endopeptidases/fisiologia , Substâncias de Crescimento/fisiologia , Proteínas de Neoplasias/fisiologia , Animais , Biomarcadores Tumorais , Fator Xa/fisiologia , HumanosRESUMO
The effect of calcium (Ca2+), magnesium (Mg2+), manganese (Mn2+), iron (Fe2+), and zinc (Zn2+) on the factor X-activating activity of cancer procoagulant (CP) was studied. Activity of CP was evaluated with a three-stage chromogenic assay (liquid-phase assay, "native" CP) and with an immunocapture enzyme (ICE) assay (solid-phase assay, immobilized CP). In the liquid-phase assay, CP activity was Ca(2+)-dependent, and Mg2+ (5 mM) or Mn2+ (0.01-0.1 mM) could substitute for Ca2+. There was no additive effect of Mg2+ and Ca2+ on the activity of CP. Activity of CP in the liquid-phase assay, in the presence of 7 mM Ca2+, was enhanced by 0.1 mM Mn2+ to about 240% of the activity observed when only Ca2+ was present in the reaction. Zn2+ and Fe2+ did not activate CP in the absence of Ca2+; they inhibited CP activity in a concentration-dependent mode when administered in the presence of Ca2+. The activity of CP evaluated by the solid-phase assay (ICE assay) was neither Ca(2+)-dependent nor was it susceptible to potentiation by Mn2+ administered after CP was bound to IgM. CP exposed to 5 mM Mn2+ before binding to IgM expressed about 85% higher activity than without the presence of Mn2+. When CP was first preincubated with divalent ion and then immunocaptured, the signal generated in the enzyme-linked immunoadsorbent assay by Mn(2+)-containing CP was significantly different (30% greater) than signals generated by CP without Mn2+ or containing different ion. These data suggest that: (1) there is a significant conformational change of the CP molecule that takes place after capturing CP by the monoclonal IgM antibody on the solid surface; (2) the divalent ions are not directly involved in enzyme-substrate interactions in the CP moiety; and (3) the interaction of Mn2+ with CP seems to be different from that of the other divalent ions.
Assuntos
Cátions Bivalentes/farmacologia , Cisteína Endopeptidases/metabolismo , Proteínas de Neoplasias , Âmnio/química , Fatores de Coagulação Sanguínea , Cálcio/farmacologia , Córion/química , Fator X/metabolismo , Compostos Ferrosos/farmacologia , Humanos , Imunoglobulina M/metabolismo , Magnésio/farmacologia , Manganês/farmacologia , Zinco/farmacologiaRESUMO
BACKGROUND: In spite of many advances in the analytical reagents (antibodies), analytical systems, and the clinical application of tumor markers, the present markers do not detect early stage cancer. Preliminary data with an antigen specific to tumor tissue, cancer procoagulant (CP), suggest its possible role in the detection of early stage cancer. This study was aimed at determining the clinical use of CP as an early stage tumor marker. METHODS: An improved enzyme-linked immunosorbent assay (ELISA) was developed to measure CP concentration in serum. A panel of 817 blinded serum samples were examined from three groups of people: 573 cancer, 106 benign, and 139 normal. RESULTS: The sensitivity of all samples analyzed from cancer patients was 80%. The CP ELISA was able to detect ovarian, colon, and kidney cancer at a sensitivity greater than 85%; breast, prostate and small cell lung cancer was detected at a sensitivity of 80-85%. Particularly interesting was the observation that early stage cancers, regardless of site, were detected effectively. In some groups, the CP assay correctly identified 100% of the patients with stage I and II cancer. The assay was able to identify correctly noncancer patient sera at a specificity of 83% for those with benign disease and 82% for the normal individuals. CONCLUSIONS: The CP assay has potential as an aid in diagnosing early stage malignancies and thereby may significantly improve the survival rate of cancer patients.
Assuntos
Biomarcadores Tumorais/sangue , Fatores de Coagulação Sanguínea/análise , Cisteína Endopeptidases/sangue , Proteínas de Neoplasias , Adenocarcinoma/sangue , Antígenos Glicosídicos Associados a Tumores/sangue , Neoplasias da Mama/sangue , Antígeno Carcinoembrionário/sangue , Carcinoma de Células Escamosas/sangue , Neoplasias do Colo/sangue , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Feminino , Neoplasias Gastrointestinais/sangue , Humanos , Masculino , Neoplasias/sangue , Neoplasias Ovarianas/sangue , Neoplasias da Próstata/sangue , Neoplasias Retais/sangue , Sensibilidade e EspecificidadeRESUMO
A new, sensitive and specific immunocapture enzyme (ICE) assay for quantitation of the enzymatic activity of cancer procoagulant (CP) has been developed. The assay had good reproducibility (inter- and intra-assay CV were 6.4% and 5.7% respectively) and was linear for concentrations of CP from 0.5 microgram/ml to 10 micrograms/ml (r2 = 0.995). Using this assay the inhibition of CP by iodoacetamide, mercuric chloride, E-64, leupeptin and antipain was demonstrated. There was no significant effect of cystatin and natural plasma proteinase inhibitors alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 2-macroglobulin and antithrombin-III/heparin, on the activity of the CP.
Assuntos
Compostos Cromogênicos , Cisteína Endopeptidases/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas de Neoplasias , Biomarcadores Tumorais/sangue , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/imunologia , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Neoplasias/sangue , Sensibilidade e EspecificidadeRESUMO
Acute promyelocytic leukemia (APL) cells express different types of procoagulant activity (PCA), including tissue factor (TF), and cancer procoagulant (CP). The aim of this study was to investigate whether the NB4 cell line, the first ever isolated human APL line, with the typical t(15;17) chromosomal balance translocation, possess CP as well as the cells freshly isolated from APL patients. Secondly, since the NB4 line is maturation inducible by all-trans-retinoic acid (ATRA), we wanted to verify whether CP, if present, was affected by ATRA treatment. The NB4 cells were able to shorten the recalcification assay of normal human plasma (total PCA). To distinguish CP in the assay for clotting activity, two criteria were used, the independence from factor VII to trigger blood coagulation and the sensitivity to cysteine proteinase inhibitors. Forty-seven per cent of total PCA of cell extracts was found to be FVII-independent PCA. A similar proportion of FVII-independent activity (42%) was detected in the cell serum-free supernatants. The activity was significantly decreased by cysteine proteinase inhibitors, including HgCl2, lodoacetic acid and Z-Ala-AlaCHN2. Additionally CP was directly identified and quantified by an immunocapture enzyme assay. The mean +/- SD concentration of CP detected by this assay in the NB4 cells, before any treatment, was 1.89 +/- 0.5 microgram/mg protein. Treatment of NB4 cells with 10(-6) M ATRA for 5 days significantly decreased the expression of CP, which became virtually undetectable by the clotting assay, and was 64% less than the untreated control by the immunocapture enzyme assay. This study provides the first evidence that the human promyelocytic cell line NB4 possess CP. The expression of this procoagulant is modulated by ATRA.
Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Cisteína Endopeptidases/metabolismo , Leucemia Promielocítica Aguda/enzimologia , Proteínas de Neoplasias , Tretinoína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Cisteína Endopeptidases/efeitos dos fármacos , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , Indução de Remissão , Sensibilidade e Especificidade , Translocação Genética/genética , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The cysteine proteinase, cancer procoagulant (CP; EC 3.4.22.26) was isolated from human amnion-chorion and purified by precipitation with polyethylene glycol and either ion exchange or immunoaffinity chromatography. A new, sensitive, three-stage chromogenic assay was developed for determination of CP factor X-activating activity. Using this assay some properties including dose-response, effect of calcium, phospholipid and pH on the activation of factor X by CP was determined. There was an excellent linear correlation (r2 = 0.99) between concentration and the enzymatic activity of CP. The activation of factor X by purified CP was calcium dependent with an optimum calcium concentration of 7 mM. CP was not phospholipid dependent. There was a rather broad pH optimum between pH 6.9 and 7.25 for the activation of factor X by CP.
Assuntos
Testes de Coagulação Sanguínea , Compostos Cromogênicos/metabolismo , Cisteína Endopeptidases/metabolismo , Fator Xa/biossíntese , Proteínas de Neoplasias , Oligopeptídeos/metabolismo , Cálcio/farmacologia , Cisteína Endopeptidases/isolamento & purificação , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Fosfolipídeos/farmacologiaRESUMO
Procoagulant activity in extracts from human stomach and colon cancers was examined, using chromogenic substrate S-2222. The activity of direct activator of factor X varied between 6 and 96% of total procoagulant activity of the tested extracts. The direct activator of factor X from stomach cancer was sensitive to heating and was inhibited by phenylmethylsulphonylfluoride and also by iodoacetic acid and HgCl2. Such results lead to the assumption that investigated activator is of enzymatic nature.
Assuntos
Fatores de Coagulação Sanguínea/análise , Neoplasias do Colo/análise , Cisteína Endopeptidases/análise , Proteínas de Neoplasias , Neoplasias Gástricas/análise , HumanosRESUMO
The activity of cancer procoagulant (CP) during the development of Guérin epithelioma was studied in the blood of Wistar rats. Blood was collected from the carotid artery and, after clotting, proteins adsorbing on aluminum hydroxide were removed from the serum. Then procoagulant activity was determined in the test system without factor VII by means of substrate S-2222 specific for factor Xa. A statistically significant increase in the activity of CP in serum was detected, coinciding with the period of intensive tumor growth (15th-25th day of disease).
Assuntos
Biomarcadores Tumorais/sangue , Fatores de Coagulação Sanguínea/análise , Carcinoma/sangue , Cisteína Endopeptidases/sangue , Proteínas de Neoplasias , Animais , Feminino , Métodos , Transplante de Neoplasias , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Coagulation activity in the blood of rats during the development of Guerin epithelioma was studied. Clotting time, level of fibrinogen and some coagulation factors (II, V, VII + X) in the plasma were determined and thromboelastographic studies were performed. Two periods of blood hypercoagulability were observed in the process of epithelioma development. The first a short-term period, was noticed during the first days following the implantation of the neoplastic tissue. The second took place during the intensive growth of the primary tumor and metastases.
Assuntos
Coagulação Sanguínea , Carcinoma/sangue , Animais , Fator V/análise , Fator VII/análise , Fator X/análise , Feminino , Protrombina/análise , Ratos , Ratos Endogâmicos , Valores de ReferênciaRESUMO
The procoagulant activity (PCA) was examined in extracts from primary tumor and metastases of experimental Guerin epithelioma transplanted into Wistar rats. PCA was evaluated by measurement of the level of activation of coagulation factor X using specific chromogenic substrate S-2222. We observed that extracts from both primary tumor and metastases were able to activate factor X in vitro. This activity was not factor VII-dependent, it was inhibited by phenylmethylsulfonyl fluoride (PMSF) and mercuric chloride, and in a smaller degree by p-chloromercuric benzoate (p-CMB) and iodoacetic acid (IAA). No difference between procoagulant activity in extracts from primary tumor and from metastases was observed.