Cancer Procoagulant Research Articles

Cancer procoagulant

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.

Diagnosis of cancer-associated vascular disorders

Unexplained thromboembolism may be an early indicator of the presence of a malignant tumor before signs and symptoms of the tumor itself become obvious. A survey of the MEDLINE data-base was conducted concerning cancer-associated vascular disorders and their role in the diagnosis of hidden cancer. The spectrum of vascular disorders heralding occult cancer and the associated laboratory abnormalities were scrutinized. Deep venous thrombosis was associated with a significantly higher frequency of malignancy during the first 6 months after diagnosis. Malignancies were found using simple clinical and diagnostic methods; additional screening was not cost-efficient. Other signs associated with deep venous thrombosis that increased the probability of an occult cancer were age older than 50 years, multiple sites of venous thrombosis, associated venous and arterial thromboembolism, thromboembolism resistant to warfarin therapy, and paraneoplastic syndrome. Among vascular syndromes, only cutaneous leukocytoclastic vasculitis presenting after the age of 50 years was consistently associated with cancer. Preliminary data with an antigen specific to tumor tissue, the cancer procoagulant, suggested its possible role as a tumor marker. The sensitivity for all samples analyzed from cancer patients was 80% and the specificity was 83%. Data from the literature enabled us to outline clinical clues that might distinguish patients with cancer-associated vasculopathies from those unaffected by malignancies. Preliminary data with an antigen specific to tumor tissue, the cancer procoagulant, suggested its possible role in detecting early stage cancer. However, large-scale prospective studies are not currently available to evaluate the role of these clues and laboratory assays in the diagnosis of early stage cancer.

Diagnosis of cancer‐associated vascular disorders

BACKGROUND Unexplained thromboembolism may be an early indicator of the presence of a malignant tumor before signs and symptoms of the tumor itself become obvious. METHODS A survey of the MEDLINE data-base was conducted concerning cancer-associated vascular disorders and their role in the diagnosis of hidden cancer. The spectrum of vascular disorders hearalding occult cancer and the associated laboratory abnormalities were scrutinized. RESULTS Deep venous thrombosis was associated with a significantly higher frequency of malignancy during the first 6 months after diagnosis. Malignancies were found using simple clinical and diagnostic methods; additional screening was not cost-efficient. Other signs associated with deep venous thrombosis that increased the probability of an occult cancer were age older than 50 years, multiple sites of venous thrombosis, associated venous and arterial thromboembolism, thromboembolism resistant to warfarin therapy, and paraneoplastic syndrome. Among vascular syndromes, only cutaneous leukocytoclastic vasculitis presenting after the age of 50 years was consistently associated with cancer. Preliminary data with an antigen specific to tumor tissue, the cancer procoagulant, suggested its possible role as a tumor marker. The sensitivity for all samples analyzed from cancer patients was 80% and the specificity was 83%. CONCLUSIONS Data from the literature enabled us to outline clinical clues that might distinguish patients with cancer-associated vasculopathies from those unaffected by malignancies. Preliminary data with an antigen specific to tumor tissue, the cancer procoagulant, suggested its possible role in detecting early stage cancer. However, large-scale prospective studies are not currently available to evaluate the role of these clues and laboratory assays in the diagnosis of early stage cancer. Cancer 1996;77:1759-67.

Loss of blast cell procoagulant activity and improvement of hemostatic variables in patients with acute promyelocytic leukemia administered all-trans-retinoic acid

All-trans-retinoic acid (ATRA) induces complete remission (CR) in up to 90% of acute promyelocytic leukemia (APL) patients with rapid amelioration of the bleeding syndrome. Previous studies indicate that ATRA treatment in vitro of the APL NB4 cell line can affect their procoagulant activity (PCA). To assess whether ATRA has this effect also in vivo, we prospectively studied the PCA of bone marrow blasts from APL patients on therapy with ATRA alone or associated with chemotherapy. Samples were obtained before, during, and after ATRA. To characterize the coagulopathy, we measured a series of plasma hemostatic variables before and during the first two weeks of therapy, as follows: (1) markers of hypercoagulability; (2) natural anticoagulants; (3) fibrinolysis proteins; and (4) elastase. The results by enzymatic and immunologic methods show that both total (tissue factor-like) and factor VII-independent (cancer procoagulant- like) blast cell PCAs, present before therapy, were reduced during (69% and 65% decrement, respectively) and virtually undetectable after ATRA. The plasma hemostatic assessment of patients before treatment was elevated hypercoagulability markers, low mean protein C, normal fibrinolysis proteins, and increased elastase. After starting ATRA, hypercoagulability markers were reduced within 4 to 8 days, protein C augmented, the overall fibrinolytic balance was unmodified, and elastase remained elevated. These results were not different either with or without chemotherapy and are consistent with the clinical findings of rapid improvement of the coagulopathy.

Cancer and Thrombosis: from Phlegmasia Alba Dolens to Transgenic Mice

Thrombosis is the most frequent complication and the second cause of death in patients with overt malignant diseases. Increasing evidence suggests that thrombotic episodes may also precede the diagnosis of cancer by months or years thus representing a potential marker for occult malignancy. Recently, emphasis has been given to the potential risk of cancer therapy (both surgery and chemotherapy) in enhancing the risk for thromboembolic disease. Post-operative deep-vein thrombosis is indeed more frequent in patients operated for malignant diseases than for other disorders. On the other hand, both chemotherapy and hormone therapy are associated with an increased thrombotic risk, which can be prevented by low-dose oral anticoagulation. Possible contributory causes for thromboembolic disease in cancer include the capacity of tumor cells and their products to interact with platelets, clotting and fibrinolytic systems, as well as their interactions with endothelial cells and tumor-associated macrophages. In particular, procoagulant activities of tumor cells have been extensively studied; one of these, cancer procoagulant, could represent a novel marker of malignancy in both solid tumors and acute promyelocytic leukemia (APL). In solid tumors, CP, a vitamin K dependent enzyme could represent the selective target of the antimetastatic effects of warfarin treatment. In APL, CP may contribute to trigger the well known intravascular coagulation syndrome accompanying the early manifestations of the disease and is depressed by all-trans-retinoic acid, an agent capable to determine complete remission with a rapid amelioration of the bleeding syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Divalent Ions on the Activity of Cancer Procoagulant

The effect of calcium (Ca 2+), magnesium (Mg 2+), manganese (Mn 2+), iron (Fe 2+), and zinc (Zn 2+) 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 Mg 2+ (5 mM) or Mn 2+ (0.01-0.1 mM) could substitute for Ca 2+. There was no additive effect of Mg 2+ and Ca 2+ on the activity of CP. Activity of CP in the liquid-phase assay, in the presence of 7 mM Ca 2+, was enhanced by 0.1 mM Mn 2+ to about 240% of the activity observed when only Ca 2+ was present in the reaction, Zn 2+ and Fe 2+ did not activate CP in the absence of Ca 2+; they inhibited CP activity in a concentration-dependent mode when administered in the presence of Ca 2+. The activity of CP evaluated by the solid-phase assay (ICE assay) was neither Ca 2+-dependent nor was it susceptible to potentiation by Mn 2+ administered after CP was bound to IgM. CP exposed to 5 mM Mn 2+ before binding to IgM expressed about 85% higher activity than without the presence of Mn 2+. 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 Mn 2+ 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 Mn 2+ with CP seems to be different from that of the other divalent ions.

Application of cancer procoagulant as an early detection tumor marker

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. 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. 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. The CP assay has potential as an aid in diagnosing early stage malignancies and thereby may significantly improve the survival rate of cancer patients.

New Immunocapture Enzyme (ICE) Assay for Quantification of Cancer Procoagulant Activity: Studies of Inhibitors

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.

39] Cancer procoagulant

Cancer procoagulant is a unique cysteine proteinase. The enzyme has been purified by several procedures and many of its characteristics and enzymatic properties have been determined. Several sensitive and reproducible assays are now available. Many proteinase inhibitors have been evaluated for their effect on CP; most low molecular weight inhibitors work well in a reduced environment. In the foreseeable future, protein and gene sequence information, expression vectors, molecular probes, and highly specific antibodies and inhibitors should provide the research tools to delineate a functional understanding of CP at the molecular and cellular level.

Three-stage chromogenic assay for the analysis of activation properties of factor X by cancer procoagulant

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.

New insights into the pathogenesis of coagulation dysfunction in acute promyelocytic leukemia.

Patients with acute promyelocytic leukemia (APL) are at high risk for the development of life-threatening thrombotic and hemorrhagic complications, particularly during induction chemotherapy. This propensity has been attributed to the release of tissue factor (TF)-like procoagulants from the leukemic cells leading to disseminated intravascular coagulation (DIC). However, recent data suggest that the pathogenesis of the coagulopathy is more complicated and may involve activation of the generalized proteolytic cascade resulting in either clotting and/or excessive fibrinolysis. Furthermore, controversy exists regarding the mechanism(s) responsible for the activation of either clotting or fibrinolysis. The malignant promyelocyte may act directly to activate coagulation and/or fibrinolysis. Alternatively, reactive inflammatory cells, which express procoagulant and/or profibrinolytic activities may play an essential role. A third possibility may involve endothelial cell expression of mediators with procoagulant/profibrinolytic properties. Putative profibrinolytic mechanisms include the release of urokinase-type and tissue-type plasminogen activators, decreases in plasminogen activator inhibitor-1 and 2, and decreases in alpha-2 plasmin inhibitor. Putative procoagulant mechanisms include the release of tissue factor, Cancer Procoagulant, or cytokines such as interleukin-1, tumor necrosis factor and vascular permeability factor. Putative anticoagulant mediators include annexins, a group of proteins in human tissue which bind phospholipids and have anticoagulant activity, which have been reported in patients with APL. The current treatment of APL is rapidly evolving because of the efficacy of all-trans retinoic acid (ATRA). All-trans retinoic acid promotes terminal differentiation of leukemic promyelocytes leading to complete remission in the majority of patients with APL with rapid resolution of the coagulopathy. Although the mechanism by which this occurs has not been established, preliminary data suggest that ATRA blocks the downregulation of the thrombomodulin gene and the up-regulation of the tissue factor gene induced by tumor necrosis factor. Since APL is a relatively uncommon disorder, the collaboration of cooperative oncology groups will be important to study patients receiving ATRA or conventional chemotherapy to further elucidate the mechanism(s) of the coagulopathy.

Cancer Cell Procoagulants and Their Implications

Cancer cells induce abnormal blood coagulation through a process that probably involves a combination of increased activators and deficiencies of anticoagulants. The probable participating factors (including tissue factor-factor VII and cancer procoagulant) and their role in this process are discussed. The current theory on the role of coagulation in malignant disease is also discussed.

The purification and properties of cancer procoagulant from murine tumors

The protease, cancer procoagulant, was isolated from three murine metastatic tumors and was purified to apparent homogeneity (SDS-PAGE) from Lewis lung cells by the sequence of (NH 4) 2SO 4 precipitation, DE-53 anion-exchange chromatography, and Sephacryl 200 chromatography. The murine tumor enzyme has a molecular weight of 68,000 and Ca 2+ is required for procoagulant and proteolytic activity; thus, the murine enzyme is very similar to that isolated from rabbit tumors. Two peptidyl chromogenic substrates of cancer procoagulant were discovered, facilitating kinetic and inhibition studies with the enzyme. The peptide substrate structures and the results of inhibition studies suggest that cancer procoagulant is thrombin-like in specificity but is a thiol protease.

Blood coagulation changes in nude mice bearing human colon carcinomas

We studied several blood coagulation parameters and tumor tissue procoagulant activity (PCA) in nude mice bearing human colorectal carcinomas (HCC). In a control group of 51 tumor-free nude mice, platelet number was 1.2 +/- 0.03 x 10(6)/microliters, thrombotest activity 90% +/- 2.6 and fibrinogen 172 +/- 11 mg/dl. The same parameters were studied in nude mice (n = 71) bearing 7 different HCC lines subcutaneously (s.c.). The results did not significantly differ from those in control mice but there was broad variability among groups of mice injected with different HCC lines, ranging from 0.36 to 2.55 x 10(6)/microliters for platelets, from 100 to 28% for thrombotest activity and from 42 to 460 mg/dl for fibrinogen. The results were significantly (p less than 0.05) different from those in the tumor-free group when each group of HCC-bearing animals was analyzed individually. A malignant HCC line that grew in the liver of nude mice (n = 24) significantly (p less than 0.001) reduced thrombotest activity (58% +/- 5.9). The PCA of tissue extracts from tumors grown s.c. in nude mice was assayed. All the HCC xenografts expressed PCA which differed significantly for the various tumor lines (from 25.5 +/- 1.9 to 2.8 +/- 0.6 unit/mg in tumor tissue). Cancer procoagulant (CP), a cysteine proteinase with a direct factor-X-activating effect, was present in different amounts (84.7 +/- 4.3 to 59.5 +/- 9.0%) in the tumors. Our results indicates that the nude mouse is a suitable model for evaluating the hemostatic changes induced by human tumors and may represent a tool for investigating the underlying biochemical mechanisms.

Factor IXi-antithrombin (IXiAT) and thrombin-antithrombin (TAT) complexes in lung cancer patients.

Coagulation activation frequently occurs in cancer patients, resulting in thromboembolic complications and/or intravascular coagulation activation. The mechanisms leading to these alterations still are poorly understood. One explanation for the coagulation activation in malignant diseases is the presence of a direct factor X-activating cancer procoagulant. Coagulation activation in lung cancer patients develops at earlier stages than factor X activation; we demonstrated increased factor IXiAT complexes in addition to elevated TAT complexes. The increases of factor IXiAT complexes were not dependent upon the stage of the disease. In contrast, TAT complexes were higher in patients suffering from advanced pulmonary non-small cell carcinoma than in patients with limited disease. In conclusion, coagulation activation in pulmonary cancer patients occurs at earlier steps in the coagulation cascade than factor X activation. While this activation is not dependent upon the stage of the disease, the observation that TAT complexes showed higher elevations in patients with advanced than in those with limited pulmonary non-small cell carcinoma could be an indication of a cancer procoagulant that directly activates factor X.

The site of activation of factor X by cancer procoagulant

Cancer procoagulant (CP) is a cysteine proteinase found in a variety of malignant cells and tissues and in human amnion-chorion tissue. It initiates coagulation by activating factor X. However, the amino acid sequence of the substrate protein that determines the cleavage site of cysteine proteinases is different from that of the serine proteinases that normally activate factor X, such as factor IXa, VIIa and Russell's Viper Venom (RVV). Therefore, it was of interest to determine the site of cleavage of human factor X by CP. Purified CP was incubated with purified factor X and the reaction mixture was electrophoresed on a 10% Tris-tricine SDS-PAGE gel. The proteins were electroeluted on to a polyvinylidene difluoride (PVDF) membrane, and stained with Coomassie blue. The heavy chain of activated factor X was cut out of the PVDF membrane and sequenced with an Applied Biosystems 477A with on-line HPLC. The primary cleavage sequence was Asp-Ala-Ala-Asp-Leu-Asp-Pro-; two other secondary sequences Ser-Ile-Thr-Trp-Lys-Pro- and Glu-Asn-Pro-Phe-Asp-Leu were found. The penultimate amino acid on the carbonyl side of the hydrolysed amide bond plays a critical role for the recognition of the cleavage site of cysteine proteinases. These data indicate that the penultimate amino acid for the primary cleavage site of factor X by CP is proline-20 and for the secondary sites, proline-13 and proline-28. This is in contrast to arginine-52 that determines the specificity of the cleavage by normal serine proteinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer procoagulant in acute lymphoblastic leukemia

In a previous study we characterized cancer procoagulant (CP), a 68 kd cysteine proteinase which directly activates coagulation factor X in various subtypes (from M1 to M5) of acute non-lymphoblastic leukemia (ANLL). The aim of this study was to determine whether CP is also expressed by acute lymphoblastic leukemia (ALL) cells. Blasts from 25 ALL patients were extracted and tested for their procoagulant properties. 16 samples (64%) shortened the recalcification time of normal human plasma, and 9 (36%) did not. 8 of the 16 active samples showed properties compatible with CP, i.e. independence from factor VII in triggering blood coagulation and sensitivity to cysteine proteinase inhibitors. Selected samples also cross-reacted with a polyclonal antibody raised against purified CP. The specific activity of CP in ALL extracts was significantly lower than in most ANLL types previously studied (all but M4). These finding indicate that CP can be a property of the lymphoid phenotype although its expression may be lower than in the myeloid phenotype.

Cancer procoagulant in serum of rats during development of experimental epithelioma

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).

Cancer Procoagulant in Acute Non Lymphoid Leukemia: Relationship of Enzyme Detection to Disease Activity

Blast cell extracts from patients with acute non lymphoid leukemia (ANLL) express cancer procoagulant (CP). This factor X (FX) activator is distinct from tissue factor (TF) in that it does not require factor VII (FVII) to trigger blood coagulation, it acts as a cysteine proteinase and is not present in normal mononuclear cells. To assess whether there is any relationship between the presence of CP and the status of the disease, ANLL patients have been studied at diagnosis, during remission, at relapse. The procoagulant activity in either the presence or absence of F VII and sensitivity to cysteine proteinase inhibitors were tested on cell extracts. Immunoreactivity was explored with an anti-CP polyclonal antibody. Data obtained in 91 newly-diagnosed ANLL patients (subtypes M1 to M5, FAB classification) confirmed the presence of CP in M1 to M4 groups (mean +/- SE FVII-independent activity: M1 = 2.1 +/- 0.7 unit/mg; M2 = 5.7 +/- 1.7 unit/mg; M3 = 31.5 +/- 8 unit/mg; M4 = 1.6 +/- 1.2 unit/mg); CP was absent in the M5 type. In eight patients analyzed in a subsequent phase of partial remission, specific activity had dropped from 26.9 +/- 7.8 to 10.5 +/- 4.0 unit/mg. Activity was virtually absent (0-0.05 unit/mg) in the bone marrow of 37 patients studied at complete remission. Bone marrow samples from six subjects tested at different intervals after complete remission were repeatedly negative for CP but became positive 2 to 5 months before relapse. Upon relapse, the FVII independent activity rose to 24.2 +/- 8.2 unit/mg.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of serum cancer procoagulant activity and its possible use as a tumor marker

The two objectives of the study were to determine whether a procedure could be developed for measuring cancer procoagulant (CP) activity in human serum and if this procedure provided a method for distinguishing people with cancer from those without cancer. A procedure was developed for processing human serum such that the activity of other coagulation enzymes would be minimized and the activity of cancer procoagulant could be measured. In a blinded study, we collected serum from 61 individuals in serum separator tubes, removed the clot by centrifugation, extracted the serum with a simple, single step procedure and analyzed the extract for CP activity. The results indicate that this test could correctly identify about 92% of the cancer patient serum samples and about 75% of the non-cancer patients serum samples, for an overall accuracy of about 85%.