Plasminogen activator activity in cultures from human tissues. An immunological and histochemical study.

Human tissues and cells from pre- and postnatal life were cultivated and studied for plasminogen activator activity. Cultures were obtained from kidney, renal blood vessels, ureter, bladder, lung, and heart. Local activator activity of cells was demonstrated by histochemical techniques. Activator released by cells into the supernatant culture media was assayed by fibrin plate techniques and was investigated for immunological identity using specific antisera to an activator of human origin, urokinase (UK). Plasminogen activator was produced in primary cultures where cells retain specific functions and generally reflect the enzyme pattern of the tissues of origin. Cells from fetal and adult sources were found to yield activator antigenically identical to UK, as well as activator activity which differed from that of UK in immunoassays and which may represent tissue type activator. Such activity was released after injury or death of cells while UK was produced in cultures containing live, metabolizing cells. Primary cultures of kidney confirmed that this organ is a rich source of UK and demonstrated, in addition, that UK is produced from the early stages of gestation and in increasing amounts thereafter. However, primary cultures also demonstrated that the ability to produce UK is not limited to the kidney but is a function of cells which are distributed widely in body tissues. Thus, activator antigenically identical to UK accumulated progressively after many refeedings in culture supernates of fetal lung and ureter, as well as in supernates of renal blood vessels of adults. These findings indicate continuous formation of UK by the cultured cells and, furthermore, provide evidence of UK production in blood vessels. In cultures from other tissues, particularly those from fetal heart and adult lung and bladder, investigation of activator was hindered by inhibitory activity which accumulated in the supernates. Such activity was derived from cells in culture and was directed selectively against UK, indicating that inhibitor as well as UK are produced by cells of various organs of the body. Plasminogen activator also was produced by serially propagated cells, diploid and heteroploid. However, only diploid cell lines retained activator activity of the original tissues and continued to produce activator antigenically identical to UK. In contrast, heteroploid line appeared to have lost the ability to form UK by yielded activator activity that differed from that of UK in immunoassays. Serially propagated cells thus provide an additional tool for in vitro study of plasminogen activator and may facilitate investigation of the fibrinolytic system in man.

Expression of plasminogen activator inhibitor type 1 by human prostate carcinoma cells inhibits primary tumor growth, tumor-associated angiogenesis, and metastasis to lung and liver in an athymic mouse model.

Expression of urokinase-type plasminogen activator (uPA) by malignant cells correlates with an aggressive phenotype, including increased invasiveness, tumor-associated angiogenesis, and metastases. Plasminogen activator inhibitor type 1 (PAI-1) is undetectable in cells of some aggressive malignancies, but present in the stroma of tumor-associated microvasculature. This analysis of an athymic mouse model of prostate carcinoma further defines the role of the uPA/PAI-1/plasmin system in primary growth and metastasis. A marked increase in PAI-1 expression was induced in clones of the aggressive human prostate carcinoma line, PC-3, by stable transfection. Primary PC-3 tumors, in mice, were significantly smaller when derived from PAI-1 expressing versus control cells. PAI-1 expression reduced the density of tumor-associated microvasculature by 22-38%. Microscopic metastases were quantitated using stable expression of the chromogenic label (beta-galactosidase) in control and PAI-1 expressing cells. PAI-1 expression resulted in a significant inhibition of lung metastases, and liver metastases. Expression of PAI-1 by malignant prostate cells resulted in a less aggressive phenotype, presumably by inhibition of uPA activity, suggesting pharmacologic or molecular inhibition of uPA activity as a potential therapeutic target.

Effect of defibrination on tumor growth and response to chemotherapy.

Other investigators have demonstrated fibrin deposition in tumors. Experiments were therefore designed to test whether systemic defibrination would alter tumor growth or tumor response to chemotherapy with cyclophosphamide. Defibrination with Ancrod, a venom extract of Agkistrodon rhodostoma, did not significantly affect tumor sensitivity to chemotherapy. Similarly, defibrination plus fibrinolytic therapy with streptokinase did not affect responsiveness to cyclophosphamide. Long-term defibrination did not affect tumor growth. These results suggest three possible interpretations: (a) the coagulation system may not be important in tumor growth and response to chemotherapy; (b) adequate clearing of fibrin from the tumor was not accomplished in our experiments; or (c) other factors such as platelet deposition may be involved and platelet function was not inhibited by the therapies used in our experiments.

Enhancement of the expression of urokinase-type plasminogen activator from PC-3 human prostate cancer cells by thrombin.

The presence of procoagulants and fibrin deposition have been demonstrated in malignant tumors. Although thrombin, a key enzyme in coagulation, has other various biological functions, the significance of its presence in tumors is not known. We studied the effects of thrombin on the expression of urokinase-type plasminogen activator (uPA) which is known to play a role in tumor invasion, using a human prostate cancer cell line PC-3. Human alpha-thrombin added to cultures of PC-3 produced a dose-dependent and time-dependent increased secretion of uPA that was greatest at 3-6 h after exposure to thrombin. Increase in uPA antigen paralleled the increase in mRNA level, which reached a maximum at 4 h. Thrombin showed the maximum effect on uPA expression at a concentration 1-2 units/ml. Zymography showed that transient exposure to thrombin induced an increase in fibrinolytic activity which could be quenched by anti-uPA antibody. The thrombin receptor-activating peptide also caused an increase in uPA protein and mRNA level, indicating the presence of the same thrombin specific receptor on PC-3 cells as on platelets and endothelial cells. Thrombin did not affect the expression of other components of the plasminogen activation system, tissue-type plasminogen activator and type-1 plasminogen activator inhibitor, and uPA receptor. These results indicate that thrombin increases uPA expression selectively by the stimulation of a functional thrombin receptor on PC-3 cells. Since uPA is known to play a role in pericellular proteolysis of extracellular matrix, thrombin may be involved in the regulation of tumor invasion and metastasis.

Elevated levels of urokinase-type plasminogen activator and plasminogen activator inhibitor type-1 in malignant human brain tumors.

The plasminogen-plasmin system has been found to modulate neoplastic spread and angiogenesis in tumors outside the central nervous system (CNS), but there have been no quantitative studies on the invasive and vascular tumors of the CNS. Quantitative zymography and enzyme-linked immunosorbent assay were used to determine the amounts of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator, and plasminogen activator inhibitors type 1 and type 2 (PAI-1 and PAI-2) in benign and malignant primary brain tumors (n = 28) as well as nonneoplastic brain (n = 5). u-PA and PAI-1 antigen were undetectable in normal brain but significantly elevated in glioblastoma multiforme (u-PA, 2.86 +/- 3.01 ng/mg; PAI-1, 8.19 +/- 5.57 ng/mg; P < 0.001). There was no difference, however, in tissue-type plasminogen activator antigen levels among control, benign, or malignant tissues except for a 4- to 7-fold increase in acoustic neuroma. PAI-2 was detected at low levels in 2 of the 33 specimens. These findings indicate that malignancy in primary CNS neoplasms is associated with elevated levels of u-PA and PAI-1, supporting the role of the plasminogen-plasmin system in the pathogenesis of CNS malignancy and as a potential biomarker and therapeutic target.

Human prostate carcinoma cells express enzymatic activity that converts human plasminogen to the angiogenesis inhibitor, angiostatin.

Angiostatin is an inhibitor of angiogenesis and metastatic growth that is found in tumor-bearing animals and can be generated in vitro by the proteolytic cleavage of plasminogen. The mechanism by which angiostatin is produced in vivo has not been defined. We now demonstrate that human prostate carcinoma cell lines (PC-3, DU-145, and LN-CaP) express enzymatic activity that can generate bioactive angiostatin from purified human plasminogen or plasmin. Affinity purified PC-3-derived angiostatin inhibited human endothelial cell proliferation, basic fibroblast growth factor-induced migration, endothelial cell tube formation, and basic fibroblast growth factor-induced corneal angiogenesis. Studies with proteinase inhibitors demonstrated that a serine proteinase is necessary for angiostatin generation. These data indicate that bioactive angiostatin can be generated directly by human prostate cancer cells and that serine proteinase activity is necessary for angiostatin generation.

Changes in blood coagulation, platelet function, and plasminogen-plasmin system in diabetes.

The increased risk of thromboembolism in people with diabetes mellitus is in part due to changes in the hemostatic mechanism including abnormal platelet function leading to platelet activation, increase in several coagulation factors, decrease in natural anticoagulants, and impaired fibrinolytic activity. Both microangiopathy and atherosclerosis in people with diabetes will enhance the thrombotic potential of these abnormal hemostatic changes. The recent recognition of a role of the components of the plasminogen-plasmin system in many biologic functions at the cellular level has led to studies showing that the angiopathic complications of diabetes may also be caused by impaired plasminogen activator function.

Current concepts of warfarin therapy.

Oral anticoagulants are used extensively, although their risks are not always fully recognized. The prophylaxis of venous thrombosis after hip surgery, the prevention of deep venous thrombosis and pulmonary emboli after an acute episode of these, the prevention of arterial emboli from the heart in patients at risk, and the prophylaxis of thrombosis in patients with congenital deficiency of antithrombin III, protein C, or protein S are some of the indications for oral anticoagulant use. Warfarin sodium is contraindicated in pregnancy, however. The recommended prothrombin time is 1 1/2 to two times control, lower than previously. The major risk of oral anticoagulant therapy, bleeding, is treated with vitamin K or plasma, depending on its severity. Warfarin necrosis and the "purple-toe" syndrome are seen more frequently than realized.

Veterans Administration Cooperative Study on antiplatelet agents in diabetic patients after amputation for gangrene: II. Effects of aspirin and dipyridamole on atherosclerotic vascular disease rates.

We report the results of a randomized multicenter clinical trial on the effects of aspirin plus dipyridamole versus placebo on major vascular end points in 231 non-insulin-dependent diabetic men with either a recent amputation for gangrene or active gangrene. Primary end points were death from atherosclerotic vascular disease plus amputation of the opposite extremity for gangrene. There were 24 atherosclerotic deaths in the drug treatment group (21.8%) and 23 in the placebo group (19.0%). There were 22 patients in the drug treatment group (20.0%) and 29 patients in the placebo group (24.0%) with opposite-side amputations. Survival curve analyses revealed little difference between these groups for major vascular end points, total mortality, all amputations, or myocardial infarctions. The most noteworthy group difference was observed for cerebrovascular end points (strokes and transient ischemic attacks), with an incidence of 8.2% (9 patients) in the drug treatment group and 19.0% (23 patients) in the placebo group. We conclude from this study that antiplatelet agents have no effect on the primary vascular end points, vascular deaths and/or amputation of the opposite extremity, in this population. Similarly, no effects were seen on secondary vascular end points, except for a suggestion of protection versus strokes and transient ischemic attacks. However, this finding must be interpreted with caution, since it is a secondary end point and was found only after multiple analyses of the data.

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.

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.

Neurovascular fibrinolytic activity in normal Lewis rats and rats with cell-transferred experimental allergic encephalomyelitis.

Fibrinolytic activity in the form of plasminogen activator (PA) was assessed using a histochemical fibrin slide technique in spinal cords of normal Lewis rats and rats with the cell-transferred form of experimental allergic encephalomyelitis (EAE). PA was localized exclusively to blood vessels. Vessels in the leptomeninges had maximum activity. A precipitous decrease in PA activity occurred in recipient rats which coincided with onset of clinical neurologic signs. A subsequent return in activity occurred in association with clinical remission of disease but remained well below the activity level of normal rats for as long as the recipient animals were followed. Vessels containing perivascular cellular infiltrates of EAE had little or no detectable PA activity. Furthermore, PA could not be demonstrated to be associated with infiltrating inflammatory cells, including macrophages. These findings provide further support for involvement of the coagulation and fibrinolytic systems in the early clinical manifestations of EAE in Lewis rats.

An acquired factor VIII inhibitor responsive to high-dose gamma globulin.

A 55-year-old previously well woman noted easy bruising and developed a swollen, painful leg after minimal trauma. A compartment syndrome was diagnosed, and medial and lateral fasciotomies were performed with evacuation of a massive hematoma. However, blood rapidly reaccumulated in the wound. The VII:C level was 2%, and 4 Bethesda units of factor VIII inhibitor were detected. After initial treatment with clotting factor concentrates and corticosteroids failed to control bleeding or reduce inhibitor titers, gamma globulin, 25 g daily for 5 days, was administered. The inhibitor became undetectable, VIII:C levels rose, and bleeding stopped. However, 5 days later VIII:C levels were again low and bleeding recurred. A second course of gamma globulin, 50 g daily for 2 days, was accompanied by a prompt increase in VIII:C, and uneventful recovery. In conclusion, in this patient with an autoantibody to VIII:C, a response to gamma globulin was observed on two occasions, and the second response came when steroids were being tapered and the patient was on no other medication.

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.

Protein C antigen deficiency and warfarin necrosis.

Recent reports have suggested a correlation between congenital protein C deficiency and tissue necrosis complicating oral anticoagulants (warfarin necrosis). The authors studied blood samples of 13 patients, obtained two weeks to more than six months after warfarin necrosis. Protein C antigen levels were assayed by an enzyme-linked immunosorbent assay method (Diagnostica Stago, France). Factors II, VII, IX, and X were assayed by functional methods, and IX and X additionally by immunologic methods. The results show that 11 of 13 patients with a history of tissue necrosis had low protein C levels, ranging from 23 to 69%, with the normal range being 70-140%. These results confirm that low protein C antigen levels are implicated in the pathogenesis of warfarin necrosis.

Antithrombin III activity in women with hypertension during pregnancy.

Antithrombin III activity was measured prospectively in 127 pregnant women within four weeks of delivery, and the results were tabulated with respect to the clinical diagnosis of their hypertensive disorder. Plasma antithrombin III activity was significantly lower than controls in women with preeclampsia and in women with chronic hypertension and superimposed preeclampsia (P less than .001). In contrast, women with chronic hypertension alone had antithrombin III activities similar to controls. Based on discriminant analysis, an antithrombin III activity of less than 70% was selected as indicative of the preeclampsia-eclampsia syndrome. The sensitivity and specificity of plasma antithrombin III for preeclampsia was 76 and 91%, respectively. Importantly, an antithrombin III activity in excess of 70% accurately predicted the absence of preeclampsia in 89% of study patients. Although gestational ages at delivery were similar in preeclamptic women with antithrombin III activity above and below 70%, women with antithrombin III activity above 70% delivered larger infants and experienced less fetal distress during labor. These findings suggest antithrombin III measurement may be useful in the management of hypertensive pregnant patients who are unresponsive to bedrest.

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.

Reproducibility of two in vivo tests of platelet function.

We correlated repeat determinations of platelet aggregate ratios (PAR) and plasma beta-thromboglobulin (BTG) in the right and left arms of thirty-four subjects in order to measure test reproducibility and to evaluate the effects of venipuncture. The reproducibility of PAR on the same blood samples was relatively low but similar to that obtained when the right arm and left arm values were compared, thus indicating little variation secondary to venipuncture. For BTG, the reproducibility on identical blood samples was quite high, but fell significantly when plasma samples from the two sides were correlated. This finding and increased BTG values when obvious blood collection problems occurred, indicated that plasma BTG levels are quite sensitive to venipuncture technique.

The concomitant augmentation of urokinase-type plasminogen activator and collagenase-like proteinase activities in X-ray irradiated cells of a human metastatic carcinomatous line.

The enzymatic activities of uPA, and a collagenase-like proteinase in the post-nuclear fraction of cell homogenates of a metastatic carcinomatous cell line following X-ray irradiation were examined by the use of chromogenic substrates and by casein- or gelatin-containing zymographies and electrophoretic gel stained with avidin-conjugated peroxidase. Enhanced activities were observed in these cells, while those of 5'nucleotidase and Na(+)-K(+)-ATPase were attenuated. A partial purification and characterization of the collagenase showed that it was able to hydrolyze the heat-denatured type-I collagen more efficiently than the native one. The activation of both uPA and collagenase enables an efficient degradation of matrix barrier proteins. These findings suggest that following a certain dose range of X-ray irradiation, tumor cells may increase their ability to migrate and invade through the enhancement of uPA and collagenase activities.

Aneurysm formation after low power carbon dioxide laser-assisted vascular anastomosis.

A series of 125 adult rats was operated upon to perform end-to-end anastomosis of the femoral artery using either a carbon dioxide laser or conventional suture technique. Vessels were inspected at varying time intervals grossly and microscopically. Overall, the rate of aneurysm formation for the laser group was 18.6% (21/113). Late aneurysm formation (1 week or longer after operation) was seen in 29.8% (20/67) of the laser group. No aneurysms were noted in the suture group either early or late. Histological examination of the laser-joined vessels revealed widespread necrosis and loss of elastic elements in the media. In time, abnormal spindle-shaped cells appeared in this damaged layer. Histologically, the aneurysms were indistinguishable from those reported in human cerebral aneurysm cases. This technique provides an experimental aneurysm model and lends support to the acquired/degenerative theory of human cerebral aneurysm formation.