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The relationship between coagulation cascade activation and glioma cell proliferation was examined. The human glioma cell lines T98G, TM-1 and normal human astrocyte cell strain (NHA) were examined. Using anti-tissue factor (TF) antibody, immunocytochemical detection of TF antigen was obtained in both cell lines and cell strain. TF antigen in cell lysates was also measured by enzyme linked immunosorbent assay (ELISA). In a one-stage clotting assay, T98G, TM-1 and NHA revealed procoagulant activity (PCA) in normal human plasma and factor VII deficient plasma. PCA in normal human plasma was significantly inhibited by both inhibitory anti-TF antibody and cysteine protease inhibitor HgCl2. This result indicates that T98G, TM-1 and NHA cells express not only TF but also cancer procoagulant (CP) at the same time. In a cell proliferation assay, thrombin induced proliferation in T98G and TM-1 cells in a dose-dependent fashion and in NHA cell in a bell-shaped fashion. This mitogenic stimulant was inhibited by the specific thrombin inhibitor hirudin. The combinations of coagulation factors II, V, and X with or without factor VII induced proliferation in T98G, TM-1, and NHA cells. The maximal mitogenic stimulatory effects were larger in glioma cells than in NHA. These mitogenic stimulatory effects were also inhibited by hirudin. Each coagulation factor on its own or in any other combination of coagulation factors had no proliferative effect. Thus, these mitogenic stimulatory effects were considered to be the effect of thrombin. In conclusion, T98G and TM-1 human glioma cells express two different types of procoagulants TF and CP. In the presence of coagulation factors, these glioma cells can generate thrombin and this thrombin generation is capable of inducing glioma cell proliferation in vitro.

Elevated plasma concentrations of endogenous thrombin generation markers and thrombotic events have been reported in children with leukemia. The aim of this study was to evaluate the effects of cancer and its treatment on thrombin generation (TAT levels) in children with acute lymphoblastic leukemia (ALL). The authors evaluated 32 children (23 M, 9 F) aged between 1 and 15 years (mean 6) affected by ALL (immunophenotypic subgroups: 16 common, 7 T, and 9 pre-B type). In all patients TAT levels at onset and after 5-6 doses of L-asparaginase were evaluated. TAT levels were higher in patients both at onset (13.04 +/- 10.90 ng/L) and after the 5-6 doses of L-asp (19.41 +/- 11.05 ng/L) with respect to controls (4 +/- 1 ng/L) (p < .001 and p < .001). TAT levels after 5-6 doses of L-asp were higher than those at onset (p < .001). Factorial ANOVA showed that at onset there was a significant effect of leukemia immunophenotypic subgroups upon TAT levels (p < .05) and no effect of inherited thrombotic risk factors. These results indicate that in children with ALL an important role is played by acquired thrombotic risk factors, among which the indirect cancer procoagulant activity has its importance.

BACKGROUND Tumor procoagulant is one of the factors responsible for disseminated intravascular coagulation and metastasis. The authors found procoagulant activity in LK52 human squamous cell carcinoma cells, which they designated cancer cell-derived blood coagulating activity 1 (CCA-1). A monoclonal antibody (MoAb) was generated to characterize this CCA-1 procoagulant activity. To date, antibodies that show an inhibitory effect on procoagulant activity as well as high reactivity in cancer cells are well known for their tissue factor specificity. METHODS Characterization of the procoagulant activity of CCA-1 was performed and an anti-CCA-1 MoAb, FS01, was generated. CCA-1 expression on the cancer cell surface was examined by flow cytometry. Procoagulant activity of various cancer cell lines and the inhibitory effect of the FS01 MoAb on this procoagulant activity was monitored by a clot timer. RESULTS The enzymologic character differed from that of cancer procoagulant (CP). The FS01 MoAb inhibited the procoagulant activity of CCA-1, but did not inhibit that of tissue factor. A positive correlation was observed between the expression intensity of CCA-1 and the inhibitory effect of the FS01 MoAb on the procoagulant activity of cancer cell lines. Expression of CCA-l was observed more frequently than that of tissue factor in human cancer cell lines. CONCLUSIONS The FS01 MoAb generated in the current study is a new antibody that reacts with various cancer cell lines, but not with normal cells. FS01 inhibits cancer cell-derived procoagulant activity and does not react with tissue factor and CP. CCA-1, which is recognized by the FS01 MoAb, appears to play a major role in cancer cell-derived procoagulant activity. Cancer 1998;82:1563-9. © 1998 American Cancer Society.

In this study we demonstrate that prothrombin activates the cell proliferation of the lung adenocarcinoma A549 cells. The A549 cell expresses factor Xa-like prothrombinase activity on its surface and prothrombin was converted to thrombin on the cell surface. Furthermore, thrombin induced the activation of PKC, increased [Ca2+]i and potentiated MAP kinase activity through thrombin receptor. The mitogenic activity of prothrombin and the conversion to thrombin were completely abolished by the synthetic coagulation factor Xa inhibitor, DX9065a. These findings suggest that DX9065a is an effective agent for a therapeutic strategy against cancer itself and prothrombotic complications associated with malignancy.

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.

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.

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)

Hematology-Oncology Department Ospedali Riuniti Largo Barozzi, 1 24128 Bergamo (Italy) Tel. 39 035 269492, Fax 39 035 266659 E-Mail annafalanga@yahoo.com All-trans-retinoic acid (ATRA) inhibits cell growth and proliferation by inducing cyto-differentiation and/or apoptosis in several cell types. These effects have become a therapeutic objective in human cancers. In addition to these functions, ATRA has been shown to affect cellular hemostatic properties [1]. Much understanding in this field has come from the clinical and experimental studies of human acute promyelocytic leukemia (APL), a distinct subtype of acute myelogenous leukemia (AML-M3), cytogenetically characterized by the balanced reciprocal translocation between chromosomes 15 and 17. In these cells, the fusion of the nuclear retinoic acid receptor (RAR ) gene on chromosome 17 with part of the PML gene on chromosome 15 results in the expression of a chimeric PML/RAR protein, which is involved in the leukemogenesis and is the target for the myeloid differentiation effect induced by ATRA. The disease typically presents with a life-threatening hemorrhagic diathesis, which is worsened by cytotoxic chemotherapy. The bleeding disorder is particularly severe in the microgranular variant of APL (M3v), which is characterized by marked hyperleukocytosis. Before the introduction of ATRA for the management of APL patients, fatal hemorrhages due to the associated coagulopathy were a major cause of induction remission failure. In a large retrospective study, the overall remission rate was 62% and the prevalence of hemorrhagic deaths in induction 14% [2]. The use of ATRA for the remission induction therapy of APL has raised the complete remission rate to greater than 90%, together with a rapid resolution of the coagulopathy, without causing bone marrow hypoplasia. ATRA promotes the terminal differentiation of leukemic promyelocytes, which is accompanied by prompt improvement of the coagulopathy typical of this disease [3]. Consistent with that, normalization in the plasma levels of hypercoagulation markers and downregulation of the two major blast cellassociated procoagulants (i.e. tissue factor, TF, and cancer procoagulant, CP) were observed [4]. A number of laboratory studies have subsequently confirmed the decrease or normalization of clotting and fibrinolytic variables during the first 1 or 2 weeks of therapy with ATRA. Inspite of that, the impact of ATRA therapy on early hemorrhagic deaths and CR rate in APL remains uncertain compared to chemotherapy alone. In nonrandomized studies, APL patients administered ATRA showed 9% to 20% improvement of CR rate and 5% to 6% reduction of early hemorrhagic deaths, compared to historical controls receiving conventional chemotherapy [1, 5–8]. These preliminary findings have been more recently confirmed by randomized clinical trials. APL patients treated with different combinations of ATRA plus chemotherapy show a prevalence of early hemorrhagic deaths from 2.4% to 6.5% [9–13]. However, both nonrandomized and randomized clinical trials clearly show an improvement of event free and overall survival in patients receiving induction therapy All-Trans-Retinoic Acid and Bleeding/Thrombosis

SummaryCancer increases the risk of venous thromboembolism (VTE). Here, we investigated the contribution of microparticle (MP)-dependent procoagulant activity to the prothrombotic state in these patients. In 43 cancer patients without VTE at study entry and 22 healthy volunteers, markers of in vivo and MP-dependent coagulation were measured and patients were prospectively followed for six months for the development of VTE. Procoagulant activity of MPs was measured in vitro using a tissue factor (TF)-independent phospholipid dependent test, a factor Xa-generation assay with and without anti-TF, and a fibrin generation test (FGT) with and without anti-factor VII(a). Markers of in vivo coagulation activation and total number of MPs at baseline were significantly elevated in cancer patients compared to controls (F1+2 246 vs. 156 pM, thrombin-antithrombin complexes 4.1 vs. 3.0 mg/l, D-dimer 0.76 vs. 0.22 mg/l and 5.53 x 106 vs. 3.37 x 106 MPs/ml). Five patients (11.6%) developed VTE. Patients with VTE had comparable levels of coagulation activation markers and phospholipid-dependent MP pro-coagulant activity. However, median TF-mediated Xa-generation (0.82 vs. 0.21 pg/ml, p=0.016) and median VIIa-dependent FGT (13% vs. 0%, p=0.036) were higher in the VTE group compared with the non-VTE group. In this exploratory study the overall hypercoagulable state in cancer patients was not associated directly with the MP phospholipid-dependent procoagulant activity. However, in the patients who developed VTE within six months when compared to those who did not, an increased MP procoagulant activity was present already at baseline, suggesting this activity can be used to predict VTE.

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.

Cancer procoagulant is present only in malignant tumours and the undifferentiated tissues of human placenta. Its possible role in angiogenesis and metastasis was investigated. Cancer procoagulant increased the steady-state mRNA level of L1 cell adhesion molecule (L1CAM) in MCF-7 breast cancer cells and E14 mouse embryonic stem cells (MESCs), while an increase in angiogenin mRNA was observed in MDA-MB-231 breast cancer cells. Furthermore, production of vascular endothelial growth factor (VEGF) protein in MCF-7 breast cancer cells and E14 MESCs, but decreased in MDA-MB-231 breast cancer cells. We conclude that cancer procoagulant could potentially play a part in angiogenesis in cancer and vascular development during embryonic development.

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 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.

Nearly all patients with cancer manifest laboratory evidence of hypercoagulability and some develop clinical thromboembolic disease (TED). Routine laboratory studies of blood coagulation have been performed in several large, prospective trials of the use of anticoagulant drugs in cancer treatment. The results of these studies, as well as data from several smaller studies of more sensitive tests of hypercoagulability [e.g. fibrinopeptide A (FPA); thrombin-antithrombin (TAT) complexes; prothrombin fragment F1 + 2)], indicate that the levels of some clotting proteins parallel disease activity. However, no studies of sound methodologic design have yet been performed to indicate that any of these tests of blood coagulation can serve as adequate predictors of TED in patients with cancer. In addition to the important role played by tumor-related procoagulants, several other mechanisms may be involved in the pathogenesis of thromboembolic events in patients with cancer, including stasis and endothelial damage. Considerable variability in the relative importance of these mechanisms in the pathogenesis of TED may exist among patients with different types of cancer. The risk for TED associated with surgical procedures in cancer patients is substantial and prophylactic antithrombotic therapy should be considered for most of these patients. Chemotherapy and hormonal therapy of cancer probably increases the likelihood of TED, particularly in those subjects with indwelling venous catheters. This risk has been particularly well-studied in patients with breast cancer treated with tamoxifen plus cytotoxic drugs. The pathogenic mechanisms may be complex but vascular injury is likely as a proximate cause of venous access catheter thrombosis and can be prevented with low dose coumadin therapy. The utility of low dose coumadin anticoagulation in reducing the risk for TED during breast cancer treatment is unknown but is currently being tested in a large, multiinstitutional study. Since chronic coumadin anticoagulation of cancer patients, and single pulse dose heparin prior to intravenous chemotherapy, both prevent thrombin generation, these agents may be of use in reducing the risk of chemotherapy-associated thrombosis. Prophylactic anticoagulation should be considered for high risk patients.

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.

Prostate cancer is the most prevalent malignancy in men and the third leading cause of cancer deaths worldwide. Disorders of hemostasis are commonplace in patients with prostate cancer and include disseminated intravascular coagulation, venous thromboembolism, acute coronary syndrome, and postsurgical bleeding. These hemostatic disorders contribute to the mortality and morbidity of prostate cancer. The leading mechanisms proposed to underlie prostate cancer-related coagulopathies are thought to be a hyperexpression of tissue factor, cancer procoagulant, and platelet-activating factor, which is then accompanied by release of large amounts of both prothrombotic and profibrinolytic substances into the bloodstream. Given the generally accepted notion that prostate-specific antigen (PSA) represents an important biomarker in prostate cancer diagnostics, large population screenings were initiated for early detection of cancer. However, recent clinical and economic drawbacks have been recently raised, including evidence that screening exposes patients to a significant risk of both overdiagnosis and overtreatment. Nevertheless, several lines of evidence suggest that PSA may have tumor-suppressing activities. Despite being a member of the vast kallikrein family, which actively interplays with the coagulation cascade, the role of PSA in the pathogenesis of hemostatic disorders observed in prostate cancer patients remains circumstantial and speculative. However, observations that the levels of this cancer marker tend to correlate positively with those of several markers of thrombin generation, and with postsurgical bleeding as well as with coronary atherosclerosis and negative outcomes of myocardial infarction, raise a new and intriguing scenario regarding the pathophysiological role of this serine protease.

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)

Neoplastic cells produce procoagulants responsible for hypercoagulation states frequently observed in cancer patients. It is accepted that two major procoagulants from malignant tissue are tissue factor (TF) and a direct activator of coagulation factor X called cancer procoagulant (CP). Direct factor X-activating activity of cultured human malignant melanoma WM 115 cells has been analyzed in the cell extracts, whole cells and in the medium after the cell culture. The factor X-activating activity was detected in the malignant cell lysates but not in the cultured medium or intact malignant cells. The lysates contained no TF as determined by Western blotting and enzyme-linked immunosorbent assay (ELISA) using anti-TF monoclonal antibody. The enzymatic characteristics of the activity was typical for CP. The results suggest that cancer procoagulant is an intracellular protein.

The diagnosis of multiple myeloma (MM) has been associated to an increased risk of venous thromboembolic events (VTE). Described risk factors that are not exclusive to MM include old age, chemotherapy, immobility, high levels of vascular endothelial growth factor, cancer procoagulant and paraproteinemia. Disease-specific risk factors unique to MM are production of procoagulant autoantibodies, a high incidence of acquired activated protein C resistance, increased levels of factor VIII and von Willebrand factor, and increased production of inflammatory cytokines, mainly IL-6, TNF and C-reactive protein. Treatment regimens that include thalidomide or related compounds such as lenalidomide combined with glucocorticoids and/or cytotoxic chemotherapy were associated with an increased risk of VTE. The risk appears to be particularly high when these immunomodulatory agents are combined with anthracyclines as treatment of newly-diagnosed disease. Combinations including thalidomide plus dexamethasone and/or alkylating agents are associated with an intermediate risk. The same regimens for relapsed/refractory myeloma seem to be associated with a lower risk. The use of newer immunomodulators such as brotezomib seem to reduce the thrombogenic potential. Several different thromboprophylaxis strategies have been effective in lowering the risk of VTE but the data are disputable. None of these VTE prevention strategies have been prospectively compared head-to-head.

The modern pathobiochemical explanation of the old clinical finding, i.e. that the occurrence of the thromboembolic complications in cancer patients is significantly higher, starts to be clarified on the basis of recent knowledge regarding the role of cancer procoagulant, the expressed tissue factor and the changes in the clotting and fibrinolytic systems. Furthermore, the presently used chemotherapeutic agents have activating effects of the coagulation system as well. The details of these phenomena, the frequently used drugs and their pathobiochemical effects are detailed in this publication. Finally, it gives an outlook regarding the new adjuvant, beneficial effects of the direct anticoagulants in malignancies.

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.

SummaryBlast cell extracts from patients with acute non lymphoid leukemia (ANLL) express cancer procoagulant (cp). Thii 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. Tb 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 proteinaie inhibitors were tested on cell extracts. Immunoreactivity was explored with an anti-cP polyclonal antibody. Data obtained in gL newlydiagnosed ANLL patients (subtypes M1 to M5, EAB classification) confirmed the presence of cp in M1- to M4 groups (mean + sE Fvll-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 analy zedin a subsequent phase of partial remission, specific activity had dropped from 26.9 ± 7.8 to 10.5 ± 4.0 unit/mg. Activiiy 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 indbpendent activity rose to 24.2 ± 8.2 unit/mg.These results suggest that CP activity may be closely associated with the presence of myeloid malignant cells in the bone marrow, a finding of potential relevance not only to the coagulation disorders of acute leukemia, but also to the early deteition of blast cells in ANLL.

The relationship between increased clotting and malignancy is well recognized, though the bidirectional development of this association is often overlooked. In the challenging cancer biology, transforming genes often act in concert with numerous epigenetic factors, including hypoxia, inflammation, contact between blood and cancer cells, and emission of procoagulant vesicles from tumors, to determine a net imbalance of the hemostatic potential which is detectable by a variety of laboratory tests. Procoagulant factors, in particular, are intimately involved in all aspects of hemostatic, cell proliferation and cellular signalling systems. However, the biggest as yet unresolved question is why cancer patients develop thrombosis? Since the thrombus itself does not apparently contributes directly to the tumor biology, enhanced hemostasis activation in cancer patients may be interpreted according to the most recent biological evidences. Coagulation and cancer biology interact bidirectionally in a "vicious cycle", in which greater tumor burden supplies greater procoagulants (tissue factor, cancer procoagulant) and thrombin, which would in turn act as strong promoters of cancer growth and spread. In this perspective, thrombosis may be interpreted as a epiphenomenon of an intricate an effective biological feedback to maintain or promote cancer progression. In this review article, we briefly analyze the pathogenesis, laboratory, clinical and therapeutic features of cancer and thrombosis.

Venous thromboembolism (VTE) is a frequent and potentially life-threatening complication associated with hematological and solid tumor malignancies. In patients with cancer, VTE portends a poor prognosis; in fact, only 12% of those who suffer an event will survive beyond one year. There are several different risk factors for the development of VTE in cancer patients that are well-described in the literature. One that has become increasingly recognized over the past two decades is the independent risk factor of chemotherapy. The annual incidence of VTE in patients receiving chemotherapy is estimated at 11%. This risk can climb to 20% or higher depending on the type of drug(s) being administered. In addition to chemotherapy, there are many other anti-neoplastic and supportive therapies that are also associated with an increased risk for the development of VTE. At present, several original basic science studies and clinical trials are underway in an effort to enhance our understanding of the mechanisms by which different chemotherapeutic agents can generate a prothrombotic state. The purpose of this article is to review the pertinent literature related to VTE in malignancy, and more specifically, chemotherapy and other cancer-related treatments associated with VTE.

The procoagulant activity (PCA) of four T-lymphoblastoid cell lines (CEM-CCRF, Jurkat, Molt-4 and A3.01) at different stages of differentiation has been characterized and compared with that of a monocytoid cell line (THP-1). Four assay systems were employed; the activated partial thromboplastin time (APTT); prothrombin time/tissue factor (TF) activity; a purified factor (F)Xa generation system and cancer procoagulant. High levels of TF activity were seen only with the monocytic cells. However the more differentiated of the T-lymphoblastoid cells (Molt-4 and A3.01) were more active than monocytic cells in supporting FXa generation. This pattern was not repeated for the APTT assay, which was related to cell-surface TF activity, since it was partially inhibited by antiTF antibody. Annexin V totally inhibited the activity observed in all three assay systems, indicating that the PCA of T-lymphoblastoid cells is primarily due to expression of negatively charged phospholipids. However, antiphosphatidylserine antibody even at a high concentration gave only partial inhibition of the activity observed in the APTT and FXa generation systems for the cells compared with almost total inhibition for the phospholipid standard, suggesting either that cellular phosphatidylserine (PS) is less accessible to the antibody, or that PS is not the sole negatively charged phospholipid responsible for this activity. Flow cytometry studies using propidium iodide and annexin V showed that the PCA, although linked to PS exposure, was not the result of apoptosis.

Cancer procoagulant (CP) and tissue factor (TF; only in complex with Factor VIIa (FVIIa)) can activate FX to FXa. Controversy still exists whether or not CP is an entity different from TF, or whether CP activity is due to contamination of CP preparations with TF/FVIIa complex. We therefore looked for proteins in CP preparations that were detected by anti-TF antibodies and then sequenced these proteins. One- and two-dimensional gels of CP and TF were used to identify proteins immunoreactive to monoclonal anti-CP and anti-TF antibodies (Mabs). Those proteins in the CP preparation recognized by anti-TF antibodies were sequenced. Angiotensinogen precursor, alpha-1-antitrypsin precursor, and vitamin D-binding protein were identified along with one so far unidentified sequence; however, no TF-sequences were identified. Also, no proteins with the correct molecular weight for TF were identified using anti-TF antibodies. It seems possible that CP preparations contain proteins that have some epitopes similar to the epitopes recognized in TF by anti-TF Mab. However, these proteins do neither have the molecular weight nor the amino acid sequence of TF.

Tumor cells produce tissue factor, cancer procoagulant, plasminogen activators and other factors that interact with the coagulation system, the fibrinolytic system and vascular or blood cells such that they can upset the normal homeostasis and balance between activation and inhibition of the coagulation and fibrinolytic systems. These activities play a role in tumor cell growth and metastasis, vascular wall function, and hemostasis. Proteases and their inhibitors are intimately involved in all aspects of the hemostatic, cell proliferation and cellular signalling systems. This review provides a brief examination of recent observations in this complex interaction of cellular and hemostatic factors.

VTE is a common complication in cancer patients and may even be the first sign of malignancy. Cancer induces activation of the blood coagulation in most cancer patients. Tissue factor and cancer procoagulant are expressed on tumour cells, leading to activation of clotting factors VII and X. Cytokines released from tumour cells activate coagulant activity on monocytes, thrombocytes, and endothelial cells. This process of activated clotting enhances clinical VTE. Low-molecular weight heparins are effective and safe for the prevention and treatment of VTE in cancer patients. Activated coagulation supports tumour growth and angiogenesis. This concept may offer interesting new strategies for the treatment of cancer [28].

The "hypercoagulable state" of malignancy is due to a complex interaction of tumor cells and their products with host cells, leading to various degrees of impairment of the normal defense mechanisms that ordinarily protect the host against thrombogenesis. Tumor cells can activate directly the blood clotting cascade and cause thrombosis or can induce procoagulant properties and inhibit anticoagulant properties of vascular endothelial cells, platelets, and monocytes and macrophages. In the setting of the local and systemic effects of cancer (e.g., stasis induced by prolonged bed rest and/or vascular invasion by tumor), together with iatrogenic complications of the treatment of cancer (e.g., the use of central vein catheters and angiopathic chemotherapy), this basic pathophysiology conspires to make cancer perhaps the best example of "acquired thrombophilia." In this brief review, we have attempted to describe what is currently known about the mechanisms for the hypercoagulable state of cancer and provide a summary of the evidence that indicates the many levels of defects in patients with malignancies that predispose them to thrombosis. A better understanding of the pathophysiology of thrombophilia in cancer should provide clinicians with an improved rationale for more aggressive and specific anticoagulant strategies in selected patients.

Activity of cancer procoagulant (CP) was studied in blood serum of 90 patients with cancer of lung, breast, oesophagus and colorectum, and of 15 healthy people. The activity of CP was determined by the coagulation method. Sera of patients with cancer showed higher mean activity of CP than sera of healthy control. Of the 90 cancer patients 78 were identified correctly by this test as having cancer (sensitivity 85%). In the case of lung and colorectal cancers the higher CP activity was observed the more advanced was the clinical stage of cancer, and the test was positive in 100%. After radical removal of malignant tumor of lung, decreased CP activity was found.

Publisher Summary This chapter describes cancer procoagulant (CP) and its physical and chemical properties. CP has been identified in a broad spectrum of malignant tissues however has not been found in normally differentiated tissues. One of the unique features of CP protein is that there is a high degree of enzymatic and structural similarity between CP isolated from rabbit V2 carcinoma, rat Walker 256 carcinosarcoma, mouse B 16 melanoma, and human amnion–chorion tissue. CP from all these sources have the same properties including molecular mass, isoelectric point, activation of human factor X, inhibition characteristics, and immunoreactivity to anti-CP antibodies. CP may also have a primary function in the undifferentiated or dedifferentiated cells. The classic methods for measuring CP activity employ measurement of the recalcified clotting time of citrated plasma. Because of its hydrophobic nature, CP associates with other proteins in tissue extracts and in serum. In high concentrations, CP probably self-associates or associates with other proteins, so it appears to be a high molecular weight aggregate and elutes from a sizing column in the 107 molecular weight region.

Abstract 3346 Abnormal hemostatic function in acute promyelocytic leukemia (APL) contributes to the bleeding and thrombotic complications, which account for much of the morbidity and mortality. Increased procoagulants (tissue factor (TF) and cancer procoagulant) and increased fibrinolytic components (annexin A2 and tPA) have been found in the blood of APL patients and in APL cell line NB4. However, their role in the pathogenesis of these complications is not clear. In recent years, increased numbers of circulating microparticles (MP) containing tissue factor have been observed in many cancer patients. This abstract describes the first study of MP in APL. Plasma samples were collected from 23 APL patients with 37 age-matched controls from healthy subjects. The APL patients were treated with a regimen of ATRA and anthracyclin according to the protocol of the International Consortium for APL (Sem. Thromb. Hemost. 2010, 369:17–924).The first sample was collected at diagnosis and second after induction therapy when molecular remission was obtained in all patients in this study. Platelet poor plasma (PPP) was obtained by centrifuging at 1,500 G for 20 minutes. 250 μL of PPP was centrifuged at 20,000 G for 10 minutes. The sediment containing MP was resuspended in 100μL of trisHCL buffer for fluorochrome-labelling with antibodies respectively against (1) human TF, (2) human annexin A2, (3) CD33, (4) human tPA, (5) human PAI-1 and (6) human annexin A5. Following incubation for 45 min, buffer was added to the suspension to a volume of 0.5 ml for FAC analysis using Coulter CyAn with Summit software. Electronic triggering was done on side-scatter, and samples were gated for size using enumeration beads (0.3 to 1.0 micron) and for annexin A5 positivity. Using MP sediment, the activities of TF, tPA and PAI-1 were respectively measured using chromogenic assays (Actichrome™ TF ELISA, American Diagnostica),(Spectrolyze™ tPA) and (Spectrolyze™ PAI-1). At diagnosis, FAC analysis showed tissue factor, PAI-1 and tPA in the MPs were significantly higher than controls with respective P values of 0.008, 0.0001 and 0.002. MPs with annexin A2 and CD33 in APL samples were not different from controls. After achieving molecular remission, the levels of tissue factor, CD33, and annexin A2 were significantly reduced with respective P values of 0.0001, 0.0007 and 0.05, while those of PAI-1 and tPA were not. Of the MP bearing tissue factor, 83% were associated with MP with CD33. This fraction was high at diagnosis and significantly reduced at remission (P=0.016). On the other hand, when assayed for activity, the TF was functional in all samples, but elevated in only one patient at diagnosis. Likewise, PAI-1 and tPA were functional but their respective activities were not elevated. Western blot showed formation of tPA/PAI-1 complex. These results showed that the increased number of TF-bearing MPs in APL plasma must be viewed with caution. Although the TF was functional, but its activity was not higher than in the controls. The same is true with PAI-1 and tPA. We hypothesize that inhibitors of TF, such as tissue factor pathway inhibitor, are present in APL patients mitigating the hypercoagulability, and that PAI-1 is complexed with tPA, reducing both their activities. Further studies testing this hypothesis are in progress. Disclosures: No relevant conflicts of interest to declare.

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.

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 (NH4)2SO4 precipitation, DE-53 anion-exchange chromatography, and Sephacryl 200 chromatography. The murine tumor enzyme has a molecular weight of 68,000 and Ca2+ 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.

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.

Abstract 3986 Poster Board III-922 Introduction A deregulated expression of procoagulant and anticoagulant activities characterises the phenotype of APL cells and contributes to the coagulopathy of this disease. The APL molecular remission induced by differentiation therapy with ATRA or ATO significantly affects specific cellular hemostatic properties of APL blasts. It is not known whether these variations translate into a comparable reduction of the overall procoagulant activity of APL cells. In this study we characterize the overall APL cell hemostatic potential by the calibrated automated method (CAT), a standardized global assay which reflects the net results of pro- and anti-coagulant forces. The endogenous thrombin potential (ETP), measured as the area under the thrombin generation (TG) curve, is a good indicator of overall plasma prothrombotic and hemorrhagic tendency. We evaluated 1) the sensitivity of NB4 cell TG potential to treatment with ATRA or ATO; 2) the correlation of CAT parameters to the levels of two known procoagulants, i.e. tissue factor (TF) and cancer procoagulant (CP), and anticoagulants (i.e. thrombomodulin, TM) and 3) the association of global TG to cell differentiation, proliferation, and apoptosis/necrosis. Methods NB4 cells were incubated 24h with either 0.1 μM ATO, or 1 μM ATRA, or the combination 0.1 μM ATO/1 μM ATRA, or the vehicle (control). The TG potential of NB4 cells was evaluated in normal pool plasma (NPP) by CAT assay; TF by chromogenic, ELISA, and cytofluorimetric assays; TFmRNA by RT-PCR; CP activity by chromogenic assay; TM by ELISA, cell differentiation by cytofluorimetric analysis of surface CD11b expression; and cell apoptosis/necrosis by annexin V/propidium iodide staining. Results TG potential of control NB4 cells (1350±70 nM*min) was significantly higher compared to normal granulocytes (p Conclusions ATRA alone or in combination with ATO is more effective in reducing the NB4-associated TG potential compared to ATO alone. While the ATRA±ATO-induced reduction of NB4-TG potential is associated to a downregulation of procoagulant factors (i.e. TF and CP) and to a parallel increase of the anticoagulant TM, differently, the ATO-induced decrease of NB4-TG potential is associated with the downregulation of TF and CP, without TM modification. CAT is a reliable method to characterize the TG potential of APL cells, and is sensitive to cell pharmacological treatments. Studies are needed to confirm the utility of the CAT to understand the role of different TG phenotypes in predicting the APL coagulopathy. Disclosures: No relevant conflicts of interest to declare.

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.

Background: Two procoagulant factors are associated with the tumor cell: tissue factor (TF) and cancer procoagulant (CP). The activation of the coagulation system can cause thrombosis and it has also been involved in the development of metastasis. Aims: 1- to establish the relationship between the presence and the levels of both procoagulants and the development of thrombosis or disease progression. 2- to analyse if the treatment with chemotherapy increases the levels of TF or CP. Methods: Patients with recent diagnosis of cancer were included in this prospective study. TF and CP determinations were done at diagnosis, at three months after chemotherapy, and at either event of progression or thrombosis. TF evaluation was done through ELISA test (normal value 159 pg/ml) and CP evaluation by a coagulometric method (Gordon et al, Thromb Res1989;56:431–40). In the last test negative values indicate high CP activity. Statistical analysis of the data was performed by two-tailed Fisher exact test and Mann-Withney/Wilcoxon test. Results: 31 patients, 61% female sex, age median 54 years. Digestive cancer 49%, breast 32%, genitourinary 13%, lung 2%. Stages of the disease were: early (I and II) 48% and advanced (III and IV) 52%. Median follow-up was 11.6 months. High levels of TF and CP at diagnosis were seen in 48% and 84% respectively. No patients developed thrombosis. Nine patients had disease progression (29%) and 4 (13%) died. Eigthy eight percent patients of this last group had advanced stages of the disease. The type of tumor more frequently associated with the event was digestive cancer (45%) and genitourinary cancer (33%). TF and CP values were increased by 40% and 50% respectively during chemotherapy treatment. Level of CP at diagnosis predicted progression (-26 sec versus -8 sec in patients with and without progression p 0.006) and also mortality (-34 sec versus -10 sec p0.003). TF at diagnosis predicted neither progression nor mortality. All patients who had progressive disease showed increased levels of TF (388 pg/ml vs 217 pg/ml p0.002) and CP (-63 sec vs -23 sec p.006) with respect to the values at diagnosis. Conclusions: Higher levels of CP at diagnosis are associated with a greater frequency of progression and mortality. No patients developed thrombosis. TF and CP levels were increased at the time of progression with respect to the values obtained at diagnosis. Chemotherapy increases the values of both procoagulants. A larger follow-up and a large number of patients could clarify the role of these procoagulants in the development of disease progression (metastasis).

Many tumors express procoagulant activities that contribute to the hemostatic complications associated with cancer. Tumor coagulant activities include tissue factor (TF) and cancer procoagulant (CP), a cysteine protease that activates Factor X. In current models of hemostasis, TF is critical in the initiation of coagulation following vascular injury as TF expressed on cells within the vessel wall complexes with blood-borne coagulation Factor VIIa to activate FX and FIX. In addition, TF transported to a growing thrombus by blood-borne microparticles binds FVIIa and contributes to thrombus propagation and stabilization. Recently, TF as well as other coagulation factors have been implicated in a variety of non-hemostatic processes including inflammation, angiogenesis, vascular development and cancer. To study the role of TF in tumor development we down-regulated TF expression in a prostate tumor cell line developed from the Lobund-Wistar (LW) rat. The LW rat combines histologic features of prostate cancer with the clinical features resembling clinical human disease; androgen-modulated growth, age-dependent spontaneous onset, and metastatic potential. Autochthonous tumors develop spontaneously or can be induced by treatment with MNU and testosterone. In addition, a cloned cell line from a spontaneous LW prostate tumor, PA-3, provides a transplantable tumor model. PA-3 cells were transfected with a series of plasmids expressing hairpin siRNAs designed to interfere with TF expression. The plasmids contained a U6-hairpin siRNA expression cassette, a neoR gene and EGFP. Cloned stable transformants of PA-3 cells expressing a siRNA corresponding to positions 260–278 of rat TF mRNA, PA-3[797], reliably reduced TF expression by 65% compared to control clonal PA-3 transformants, PA-3[776], expressing neoR and EGFP but no siRNA. To test the consequences of reduced TF on tumor development in vivo, 4 independently isolated PA-3[797] cell lines and 4 independently isolated PA-3[776] control lines were injected subcutaneously into LW rats (3 rats per cell line). Ten of the 12 rats injected with control cells developed detectable tumors 4 weeks post transplantation (tumor mass = 2.2g +/− 1.8g). In contrast, only 1 of the 12 rats receiving the low TF expressing PA-3[797] cells developed a small tumor (0.07 gm) suggesting TF expression is required for tumorigenesis.

The clinical usefulness of our assay can be demonstrated by two typical cases: Fig. 1 gives the course of a patient who was investigated three times during CR. While the proportion of leukaemic clones was below 20% during maintenance chemotherapy it had increased to 60% 10 months later during CR. 3 months later, the patient relapsed clinically. In contrast, another patient maintained a proportion of 5-30% of phenotypically leukaemic clones at repeated investigations over a period of nearly 3 years without relapsing clinically (Fig. 2). Our data clearly demonstrate that “complete remission” of adult AML represents a balance of leukaemic and normal hematopoiesis rather than eradication of leukaemia. They argue for the action of mechanisms which suppress the outgrowth of leukaemic progenitor cells in viva. Our in vitro culture system is applicable to all cases of AML and not restricted to certain immunological constellations as the investigation of uncultured bm cells in ALL [5] and much more sensitive than the Southern blot methodology in those cases which have a gene rearrangement as a clonal marker [4, 61. Since it is relatively easy to perform it can be a valuable tool in clinical trials of postremission therapy including alternative approaches (e.g. cytokines like interleukin-2 or autologous bone marrow transplantation) as well as for testing the effectivity of in vitro purging methods.

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).

The procoagulant activity of cells from some experimental tumours isolated in culture or in single-cell suspensions from ascitic fluid was investigated. Cells from Lewis lung carcinoma (primary and metastasis), Ehrlich carcinoma ascites and JW sarcoma ascites were able to shorten markedly the recalcification time of normal, Factor VIII- and Factor VII-deficient but not of Factor X-deficient human plasma. The same cells generated thrombin when mixed with a source of prothrombin and Factor X, absorbed bovine serum (as a source of Factor V), phospholipid and calcium chloride. Thrombin formation was not influenced by the presence of Factor VII. Cells from Sarcoma 180 ascites were completely inactive in both test systems. It is concluded that cells from some experimental tumours have the capacity to activate Coagulation Factor X directly. These findings suggest the existence of an alternative "cellular" pathway in the initiation of blood clotting distinct from both the intrinsic and extrinsic mechanisms.

A proteolytic procoagulant has been identified in extracts of human and animal tumors and in cultured malignant cells. It directly activated Factor X but its similarity to other Factor S-activating serine proteases was not clear. This study describes work done to determine whether this enzyme, cancer procoagulant, is a serine or cysteine protease. Purified cancer procoagulant from rabbit V2 carcinoma was bound to a p-chloromercurialbenzoate-agarose affinity column and was eluted with dithiothreitol. The initiation of recalcified, citrated plasma coagulation activity by cancer procoagulant was inhibited by 5 mM diisopropylfluorophosphate, 1 mM phenylmethylsulfonylfluoride, 0.1 mM HgCl2, and 1 mM iodoacetamide. Activity was restored in the diisopropylfluorophosphate-, phenylmethylsulfonylfluoride-, and HgCl2-inhibited samples by 5 mM dithiothreitol; iodoacetamide inhibition was irreversible. Russell's viper venom, a control Factor X-activating serine protease, was not inhibited by either 0.1 mM HgCl2 or 1 mM iodoacetamide. The direct activation of Factor X by cancer procoagulant in a two-stage assay was inhibited by diisopropylfluorophosphate and iodoacetamide. Diisopropylfluorophosphate inhibits serine proteases, and an undefined impurity in most commercial preparations inhibits cysteine proteases. Hydrolysis of diisopropylfluorophosphate with CuSO4 and imidazole virtually eliminated inhibition of thrombin, but cancer procoagulant inhibition remained complete, suggesting that cancer procoagulant was inhibited by the undefined impurity. These results suggest that cancer procoagulant is a cysteine endopeptidase, which distinguishes it from other coagulation factors including tissue factor. This and other data suggest that neoplastic cells produce this unique cysteine protease which may initiate blood coagulation.