Effects of all-trans retinoic acid or chemotherapy on the molecular regulation of systemic blood coagulation and fibrinolysis in patients with acute promyelocytic leukemia.

We studied the pathogenesis of the bleeding disorder in acute promyelocytic leukemia by measuring procoagulant, profibrinolytic, and proinflammatory mediators in peripheral blood and bone marrow cells from 25 previously untreated patients. Patients were induced with either all-trans retinoic acid (ATRA) or chemotherapy. Plasma levels of fibrinopeptide A (FPA), fibrin d-dimer, thrombin antithrombin (TAT) complex, prothrombin fragment 1.2 (F1.2), urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (t-PA) and plasminogen activator-inhibitor 1 (PAI-1) were measured before and after therapy, as was the cellular expression of the genes for tissue factor (TF) and interleukin-1 beta (IL-1 beta). The mean plasma levels of fibrin d-dimer, F1.2, TAT and FPA were markedly elevated prior to therapy and declined during the first 30 days of treatment with either ATRA or chemotherapy, but more rapidly and to a greater extent in patients treated with ATRA. ATRA treatment was associated with a significant decrease in TF gene expression in bone marrow cells during the first 30 days of treatment, whereas IL-1 beta gene expression, which decreased in the cells of six patients treated with either chemotherapy or ATRA, actually increased in the remaining six patients treated with either chemotherapy or ATRA. In patients with APL, treatment with either chemotherapy or ATRA rapidly ameliorates the coagulopathy, as indicated by an abrupt decline in markers of clotting activation. An increase in cytokine gene expression (e.g. IL-1 beta) may provide an explanation for the persistent hypercoagulability observed in some patients with APL, regardless of therapeutic approach. Our data confirms and extends earlier observations by others that ATRA is more effective than chemotherapy alone in rapidly reducing the procoagulant burden of APL tumor cells. However, our data also suggests that cytokine expression in some patients may be accelerated by either chemotherapy or ATRA. The implications of this observation for understanding the retinoic acid syndrome will require further studies.

Expression of receptors for plasminogen activators on endothelial cell surface depends on their origin.

Receptors for plasminogen activators present on endothelial cell (EC) surface regulate local plasmin activity. Plasmin generation by human ECs, derived from cerebral cortex, skin and lung, iliac artery, iliac vein, aorta and coronary artery, was studied. The respective ECs were treated with recombinant tissue plasminogen activator (rt-PA) or with recombinant urokinase-type plasminogen activator (ru-PA), washed, plasminogen added and the plasmin generated then assayed. The largest amounts of plasmin were generated by cerebral ECs, under baseline conditions or after exposure to rt-PA or ru-PA (P < 0.0001). Exposure to rt-PA also resulted in more plasmin generation than ru-PA in the cerebral ECs (P < 0.0001) but not in the other ECs. Heparin enhanced plasmin generation by both rt-PA and ru-PA. Specific antibody against annexin II, a t-PA receptor, blocked plasmin generation by rt-PA. Western blotting showed higher amounts of annexin II on the cell membrane in cerebral ECs. This suggests that expression of annexin II in ECs depends on their location, being greatest in cerebral ECs. In contrast, expression of u-PA receptor was the same for all ECs. This has implications for higher risk of intracranial bleeding during thrombolytic therapy, and for a role of t-PA in neurological development and function.

Thrombogenic role of cells undergoing apoptosis.

Apoptosis is involved in many biological processes, especially during chemotherapy in cancer patients. Chemotherapy is also associated with an increased risk of thrombosis. The relationship between thrombogenicity and apoptosis was studied in various human tumour cell lines and non-tumour cell lines. Apoptosis was induced by the chemotherapeutic agent camptothecin and by Fas ligand, then quantified by staining with fluorescein isothiocyanate-conjugated annexin V and propidium iodide. A significant correlation between thrombin generation and degree of apoptosis was observed (P < 0.0005). Addition of anti-tissue factor antibody in excess or of tissue factor pathway inhibitor partially inhibited thrombin generation, suggesting that tissue factor activation was responsible for this process. A statistical correlation between tissue factor activity and degree of apoptosis was also found (P < 0.005). Both thrombin generation and tissue factor activity were blocked by the addition of annexin V, which binds and inhibits phosphatidylserine. This indicates that the exteriorization and exposure of phosphatidylserine on the cell surface membrane during apoptosis were essential for both thrombin generation and tissue factor activation.

Thrombotic microangiopathy in the cancer patient.

Thrombotic microangiopathy, manifesting as thrombotic thrombocytopenic purpura (TTP) or hemolytic uremic syndrome, is a common complication in cancer patients. It shares the pathogenic microvascular occlusive lesion and many clinical manifestations as the classical TTP, but the spectrum of complications varies widely. Several subsets are seen, including a microangiopathic hemolytic anemia in advanced cancer, chemotherapeutic drug-associated microangiopathy and those with the transplant setting. The prognosis is not as favorable as in classical TTP. Anecdotal reports indicate that responses are seen with plasma exchange and with immunoadsorption.

The Pathogenetic Role of Apoptosis in Hypercoagulable States.

Hypercoagulable states are common disorders with high risk of thrombosis associated with cardiovascular and malignant diseases. The pathogenesis of hypercoagulability is multifactorial. The basic physiological mechanism is the imbalance between anticoagulant activities and procoagulant activities in hemostatic system. In this review, we discuss the correlation between apoptosis and thrombogenesis in hypercoagulable states. Some cell-associated cofactors in coagulation system, including phosphatidylserine, tissue factor, thrombomodulin and cancer procoagulant, are regulated during apoptosis of various cell types. Vascular endothelial cells may act as one of the most important aspects affecting the balance of anticoagulant and procoagulant activities. When endothelial cells are activated or induced to undergo apoptosis by a number of physiological factors, such as inflammatory cytokines and bacterial lipopolysaccharide, the procoagulant activities of endothelial cells are enhanced. Other cell types such as apoptotic vascular smooth muscle cells, monocytes and macrophages may also contribute to the pathogenesis in atherosclerosis. Apoptotic tumor cells, which express high level of procoagulant activities, may act as a direct trigger for coagulation activation.

Atypical hairy cell leukemia.

The morphologic differential diagnosis of mature B-cell neoplasms with cytoplasmic projections includes splenic lymphoma with villous lymphocytes and hairy cell leukemia. Although the classification of hairy cell leukemia is not universally recognized, 3 variants have been described, namely, classic, variant, and Japanese variant, each of which has different clinical and immunophenotypic features. Classic hairy cell leukemia is virtually always CD11c(+), CD25(+), and CD103(+). Variant and Japanese variant hairy cell leukemias are usually CD11c(+), always CD25(-), and occasionally CD103(+). Each variant is characteristically CD10(-). We present a case of hairy cell leukemia with a unique immunoprofile in that the cells were CD10(+), CD25(+), and CD103(-), and we review the criteria helpful in differentiating "hairy" B-cell neoplasms. This case emphasizes the variability of hairy cell leukemia and the need to correlate all clinical and pathologic data in reaching a diagnosis.

A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo.

Urokinase plasminogen activator (uPA) plays an important role in the progression of several malignancies including breast cancer. We have identified a noncompetitive antagonist of the uPA-uPAR interaction derived from a nonreceptor binding region of uPA (amino acids 136-143). This 8-mer capped peptide (A6) inhibited breast cancer cell invasion and endothelial cell migration in a dose-dependent manner in vitro without altering cell doubling time. Intraperitoneal administration of A6 resulted in a significant inhibition of tumor growth and suppressed the development of lymph node metastases in several models of breast cancer cell growth and metastasis. Large areas of tumor necrosis and extensive positive staining by TUNEL were observed on histological and immunohistochemical analysis of experimental tumor sections from A6-treated animals. A6 treatment also resulted in a decrease in factor VIII-positive tumor microvessel hot-spots. These results identify a new epitope in uPA that is involved in the uPA-uPAR interaction and indicate that an antagonist based on this epitope is able to inhibit tumor progression by modulating the tumor microenvironment in the absence of direct cytotoxic effects in vivo.

Plasminogen activator inhibitor 1 may promote tumour growth through inhibition of apoptosis.

Plasminogen activator inhibitor 1 (PAI-1) has been found to be a bad prognostic factor in a number of tumours but the reason has not been fully explained. The human prostate cancer cell line PC-3 and the human promyelocytic leukaemia cell line HL-60 were used in this study to determine the effect of PAI-1 on spontaneous and induced apoptosis in culture. Apoptosis was induced with camptothecin or etoposide. Addition of a stable variant of PAI-1 or wild-type PAI-1 to these cells resulted in a significant inhibition of apoptosis. In contrast, both the latent form of PAI-1 and the stable variant of PAI-1 inactivated by a specific neutralizing monoclonal antibody, or the stable variant of PAI-1 in a complex with recombinant urokinase did not inhibit apoptosis. This indicated that the inhibitory activity of PAI-1 was required for its anti-apoptotic effect but the urokinase-type plasminogen activator receptor was not involved. These findings provide an explanation for the bad prognostic correlation of high PAI-1 levels in tumours. The anti-apoptotic effect was also found in non-tumoural cells including human umbilical vein endothelial cells and the benign human breast epithelial cell line MCF-10A, suggesting that this is a novel physiologic function of PAI-1.

Hereditary defects in fibrinolysis associated with thrombosis.

The plasminogen-plasmin system involves proteolytic enzymes which are primarily responsible for the degradation of fibrin deposits in blood vessels. Through intricate interactions between the various components and inhibitors, a balance is maintained between profibrinolysis and impaired fibrinolytic activity. Several hereditary defects have been described affecting functional plasminogen concentrations, plasminogen activator levels, and plasminogen activator inhibitor activity. These defects have been implicated as risk factors for thrombosis based on a multitude of case reports associating impaired fibrinolysis with thrombosis. However, under close scrutiny, the role of decreased fibrinolysis as an etiologic factor in thrombosis has not been firmly established. Rather, dysfibrinolysis may manifest itself through an accentuation of an underlying thrombophilic state such as recurrent thrombotic episodes. Further evaluation of impaired fibrinolytic activity in conjunction with an underlying thrombophilic condition is warranted.

The hyperviscosity syndromes.

Impaired blood flow due to abnormal rheologic characteristics results in a multiplicity of clinical manifestations, collectively termed the hyperviscosity syndrome. A basic knowledge of the principles of rheology is important in the understanding of its pathophysiology, especially the relationship between viscosity and flow conditions. The flow characteristics in different types of blood vessels are also determinants in the location of the clinical manifestation. The syndrome can occur in a wide variety of diseases and is best grouped according to the causative element or elements in blood. Abnormalities in the cellular components of blood can occur in the quantity and the quality of erythrocytes, leukocytes, and platelets. Abnormal plasma components can also be in both the quantity and quality of the plasma proteins. Clinical manifestations are the result of vascular occlusion, especially in the microcirculation. The altered rheologic characteristics of either the cellular or the protein component may be temperature dependent, being abnormal only at temperatures below 37 degrees C, so that only the cooler parts of the body are affected. The management of these conditions should be primarily directed at the removal of the abnormal component. At the same time, it should be accompanied by measures that can control the production of the causative element.

Thrombotic microangiopathy manifesting as thrombotic thrombocytopenic purpura/hemolytic uremic syndrome in the cancer patient.

The complication of thrombotic thrombocytopenic purpura or hemolytic uremic syndrome (TTP/HUS) can occur in cancer patients. It is characterized by a microangiopathic hemolytic anemia, severe thrombocytopenia, and renal failure. Pulmonary manifestations, especially pulmonary edema, are a common observation. Neurologic changes are also frequently seen. The etiology is unknown at this time. It has been observed in many different types of cancer and is most commonly seen in gastric adenocarcinoma followed by carcinoma of the breast, colon, and small cell lung carcinoma. The hemolysis can be massive and is due to red cell fragmentation, as schistocytes are present in all the cases. Though immune complexes are present in the plasma, the antiglobulin (Coomb's) test is negative. Chemotherapeutic agents, especially mitomycin C, have been implicated as causative factors. There is a correlation of this complication with the cumulative dose. However, chemotherapy cannot account for all the cases as the syndrome can occur in untreated patients. It can be differentiated from disseminated intravascular coagulation by the absence of a coagulopathy. Management should consist of plasma exchange, use of a Staphylococcus aureus column (Prosorba), and control of hypertension. Because of the susceptibility to pulmonary edema, blood volume overloading should be avoided.

Endothelial cell hyperplasia contributes to thrombosis in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT), a well recognized complication of heparin administration, is associated with thrombotic complications. An immunologic mechanism is believed to be responsible for the thrombocytopenia, but the cause of thrombosis is not fully understood. We report a histological and immunohistochemical study of thrombosed vessels in surgically removed ischemic tissues in patients with HIT complicated by gangrene of the lower limbs. Tissue sections were studied by: (1) hematoxylin and eosin staining and immunoperoxidase staining using a monoclonal antibody against platelet surface glycoprotein Ib(GPIb) for the identification of platelets, and (2) polyclonal antibodies against tissue-type plasminogen activator (tPA) and factor VIII for the identification of endothelial cells. We observed small arteries occluded by multiple small platelet thrombi surrounded by proliferative endothelial cells. In addition, depositions of IgG, IgA, and IgM were found in the occluded arteries. We postulate that the endothelial cell hyperplasia is caused by immunologic injury to endothelial cells as a result of immunoglobulin deposition, and by various mitogens derived from the activated platelets in the thrombi. Such endothelial cell hyperplasia is a major contributory factor, in addition to the microthrombi, of the occlusive vasculature in this disease.

Current Clinical Practice: Current Management of Thrombotic Thrombocytopenic Purpura.

Historically, the mortality rate of thrombotic thrombocytopenic purpura (TTP) approached 100%. However, by the 1980's, new therapy was instituted with a vast improvement in survival to 90%. The exact pathogenesis of TTP remains elusive. Yet, despite incomplete understanding of the pathophysiology, outcome has improved due to increased awareness of the symptomatology leading to earlier diagnosis and better supportive care, in addition to effective therapy with plasma exchange. TTP represents a disease in which prompt diagnosis and treatment can lead to a critical difference in clinical outcome.

Oestradiol regulation of the components of the plasminogen-plasmin system in MDA-MB-231 human breast cancer cells stably expressing the oestrogen receptor.

To understand the hormonal regulation of the components of the plasminogen-plasmin system in human breast cancer, we examined the oestradiol (E2) regulation of plasminogen activators (PAs), namely urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1) and uPA receptor (uPAR), in our model system. We used stable transfectants of the MDA-MB-231 human breast cancer cells that express either the wild-type (S30 cells) or the mutant 351asp-->tyr oestrogen receptor (ER) (BC-2 cells). Northern blot analysis showed that there was a concentration-dependent down-regulation of uPA, tPA and PAI-1 mRNAs by E2. In contrast, uPAR mRNA was not modulated by E2. The pure anti-oestrogen ICI 182,780 was able to block E2 action, indicating that the regulation of these genes is ER mediated. The E2 also inhibited the expression and secretion of uPA, tPA and PAI-1 proteins as determined by enzyme-linked immunosorbent assay (ELISA) in cell extracts (CEs) and conditioned media (CM). Zymography of the CM confirmed the inhibitory effect of E2 on uPA activity. Thus, we now report the regulation of uPA, PAI-1 and tPA by E2 in both mRNA and protein levels in ER transfectants. The association between down-regulation of the uPA by E2 and known E2-mediated growth inhibition of these cells was also explored. Our findings indicate that down-regulation of uPA by E2 is an upstream event of inhibitory effects of E2 on growth of these cells as the addition of exogenous uPA did not block the growth inhibition by E2.

Thrombotic thrombocytopenic purpura associated with ticlopidine. A review of 60 cases.

BACKGROUND: Thrombotic thrombocytopenic purpura, a life-threatening multisystem disease, has been infrequently associated with use of ticlopidine, a platelet anti-aggregating agent. PURPOSE: To review 60 cases of ticlopidine-associated thrombotic thrombocytopenic purpura. DATA SOURCES: Medical records, published case reports, and case reports submitted to the U.S. Food and Drug Administration. STUDY SELECTION: Instances of ticlopidine-associated thrombotic thrombocytopenic purpura were identified. DATA SYNTHESIS: Ticlopidine had been prescribed for less than 1 month in 80% of the patients, and normal platelet counts had been found within 2 weeks of the onset of thrombotic thrombocytopenic purpura in most patients. Mortality rates were higher among patients who were not treated with plasmapheresis than among those who underwent plasmapheresis (50% compared with 24%; P < 0.05). CONCLUSIONS: Ticlopidine use may be associated with the development of thrombotic thrombocytopenic purpura, usually within 1 month of initiation of therapy. The onset of ticlopidine-associated thrombotic thrombocytopenic purpura is difficult to predict, despite close monitoring of platelet counts.

Agonist activity of antiestrogen-receptor complexes to regulate urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) endogenous gene expression in breast cancer cells.

We have shown that 4-hydroxytamoxifen (4-OHT) has estrogen-like effects on induction of TGFalpha mRNA in estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells, transfected with either wildtype (S30 cells) or a codon 351asp-->tyr mutant ER (BC-2 cells). The mutant receptor used to produce the stable transfectants was identified in a tamoxifen-stimulated human breast tumor. We have also demonstrated that raloxifene exhibits a gene-specific estrogen-like effect with mutant ER (BC-2 cells) but not with wildtype ER (S30 cells) (Levenson, A.S., Catherino, W.H. and Jordan, V.C. (1997) Estrogenic activity is increased for an antiestrogen by a natural mutation of the estrogen receptor. J. Steroid Biochem. Mol. Biol., 60, 261-268). We now describe the regulation of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) endogenous gene expression by estradiol (E2) and different antiestrogens in BC-2 cells. Northern blot analyses revealed that 4-OHT and raloxifene have concentration-dependent agonistic (E2-like) effects on the regulation of these genes. In contrast, the pure antiestrogen ICI 182780 alone had no effect but could block the action of E2, 4-OHT and raloxifene. The E2-like effects of non-steroidal antiestrogens in this model system cannot be explained by the mutation in the ER alone because 4-OHT acts as an agonist with wildtype receptor as well. We propose that the clear cut biological expression of estrogen-like qualities with different antiestrogens will in the future serve as an important model to dissect the signal transduction pathway.

The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin.

Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissue-type plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA/FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases. These findings define a direct mechanism for cancer-cell-mediated angiostatin generation and permit large-scale production of bioactive angiostatin for investigation and potential therapeutic application.