Plasminogen activators and their inhibitors in leukemic cell homogenates

Plasminogen activator (PA) and PA inhibitor (PAI) were measured in homogenates of leukemia cells. Both PA and PAI levels were higher in non-lymphoblastic leukemia than in lymphoblastic leukemia. The levels were below the sensitivity of determination in chronic myelocytic leukemia (CML) but showed significant increases in blast crisis (CML, bc). The level of the tissue type PA (t-PA) antigen was highest in acute myelobtestic leukemia (AML) and that of the urokinase type PA (u-PA) was highest in acute promyeiocytic leukemia (APL). The PAH antigen showed no marked cell specificity, but the PAI-II antigen was markedly increased in myelomonocytic leukemia and acute monocytic leukemia (AMoL). From these findings, various PAs and PAIs are considered to be present in leukemia cells and to be involved in hemostatic disorders, thus they are of diagnostic value in leukemia. © 1993 Wiley-Liss, Inc.     

Plasminogen activators in experimental colorectal neoplasia: a role in the adenoma-carcinoma sequence?

An important step in the transition from adenomatous polyp to invasive carcinoma is the degradation of the epithelial basement membrane. By the generation of plasmin, plasminogen activators may play an important role in regulating the extracellular protease activity required for this event to occur. The production of biofunctional urokinase and of tissue plasminogen activator was therefore investigated in the dimethylhydrazine induced rat model of colorectal neoplasia. Both adenomatous polyps (p values less than 0.001) and colorectal carcinomas (p values less than 0.001) were demonstrated to produce a significant excess of both urokinase and tissue plasminogen activator when compared with macroscopically normal colon. There was, however, no increased production of either enzyme by macroscopically normal preneoplastic colon when compared with control colon. This enhanced capacity of colorectal tumours to produce plasminogen activators and generate plasmin is thus a feature of both the premalignant as well as the malignant phenotype. These enzymes may contribute to the malignant potential of adenomatous polyps and to the invasive capacity of established carcinomas.     

Elimination of inhibition in euglobulin fibrinolysis by use of flufenamate: involvement of C1-inactivator

The fibrinolytic activity of euglobulin fractions prepared from human morning plasma and assayed on fibrin plates is strongly inhibited by the C1-inactivator present in the fractions. Flufenamate, a potent representative of the group of synthetic thrombolytic agents, eliminates this inhibition in euglobulin fractions. This elimination is an apparently irreversible reaction dependent on concentration, time and temperature. The fibrinolytic enhancing effect of flufenamate in euglobulin fractions correlated well with a similar effect of added C1s, which neutralized the C1-inactivator. The effect of flufenamate was slightly greater than that of added C1s, suggesting an additional effect of the flufenamate. The activity enhancing effect of the flufenamate at the lower molarities could be separated from an activity decreasing effect at the higher molarities. A simple technique by which inhibitory effects in euglobulin fibrinolysis are selectively eliminated is described.     

Plasminogen activator inhibitor-1 suppresses endogenous fibrinolysis in a canine model of pulmonary embolism

BACKGROUND. Plasminogen activator inhibitor-1 (PAI-1), the specific, fast-acting inhibitor of tissue-type plasminogen activator (t-PA), binds to fibrin and has been found in high concentrations within arterial thrombi. These findings suggest that the localization of PAI-1 to a thrombus protects that same thrombus from fibrinolysis. In this study, clot-bound PAI-1 was assessed for its ability to suppress clot lysis in vivo. METHODS AND RESULTS. Autologous, canine whole blood clots were formed in the presence of increasing amounts of activated PAI-1 (0-30 micrograms/ml). Approximately 6-8% of the PAI-1 bound to the clots under the experimental conditions. Control and PAI-1-enriched clots containing iodine-125-labeled fibrin (ogen) were homogenized, washed to remove nonbound elements, and delivered to the lungs of anesthetized dogs where the homogenates subsequently underwent lysis by the endogeneous fibrinolytic system. 125I-labeled fibrin degradation products appeared in the blood of control animals within 10 minutes and were maximal by 90 minutes. PAI-1 reduced fibrin degradation product release in a dose-responsive manner at all times between 30 minutes and 5 hours (greater than or equal to 76% inhibition at 30 minutes, PAI-1 greater than or equal to 6 micrograms/ml). PAI-1 also suppressed D-dimer release from clots containing small amounts of human fibrin (ogen). t-PA administration attenuated the effects of PAI-1, whereas latent PAI-1 (20 micrograms/ml) had no effect on clot lysis. Blood levels of PA and PAI activity remained unaltered during these experiments. CONCLUSIONS. The results indicate that PAI-1 markedly inhibits endogenous fibrinolysis in vivo and, moreover, suggest that the localization of PAI-1 to a forming thrombus is an important physiological mechanism for subsequent thrombus stabilization.     

Plasminogen activators in synovial fluid and plasma from patients with arthritis.

The activity of plasminogen activators and inhibitors in the synovial fluid and plasma of patients with various forms of chronic arthritis was characterised. Tissue-type plasminogen activator antigen (t-PA:Ag), urokinase-type plasminogen activator antigen (u-PA:Ag), the proenzyme single chain u-PA (scu-PA), and plasminogen activator inhibitor (PAI) were measured in the synovial fluid and plasma of 22 patients with seropositive rheumatoid arthritis (RA), 13 with seronegative RA, and 23 patients with various forms of arthritis. In all patient groups the levels of t-PA:Ag in synovial fluid were lower and the levels of u-PA:Ag and PAI higher than plasma levels. Synovial fluid u-PA was more activated than plasma u-PA. Comparison of the patient groups showed that the largest differences between fibrinolytic parameters in synovial fluid and plasma were present in patients with seropositive RA followed by patients with seronegative RA and patients with various forms of arthritis. This order paralleled the functional and radiological scores of joint destruction in the patient groups studied. The results of this study indicate that suppression of t-PA production and enhancement of u-PA synthesis and activation in arthritic joints are associated with the clinical severity of arthritis.     

Effects of dextran sulphate and flufenamic acid on euglobulin fibrinolytic activity of glass-treated or heated plasma

Treatment of human citrated plasma with glass has complex effects on the fibrinolytic system. While the spontaneous euglobulin activity is only slightly affected by the glass treatment, the activity precipitated in the presence of dextran sulphate diminishes rapidly with increasing amounts of glass. With common glass a minimum is reached, and the activity reappears when larger amounts of glass are used. With Pyrex glass the decrease continues. Addition of flufenamate to the solutions recover much of the missing activity suggesting the presence in the euglobulin precipitates of an inhibitor sensitive to flufenamic acid. Heating of plasma at 56 degrees C rapidly destroys its ability to produce spontaneously active euglobulin precipitates while the capacity to elicit fibrinolytic activity by precipitation in the presence of dextran sulphate remains largely undisturbed suggesting a relative stability of the precursors of the intrinsic fibrinolytic system.     

Plasminogen activators in normal tissue and carcinomas of the human oesophagus and stomach.

Carcinogenesis in the human colon is associated with a marked increase of urokinase type plasminogen activator and a decrease of tissue type plasminogen activator. This study was performed to determine the concentrations of urokinase type plasminogen activator and tissue type plasminogen activator in normal tissue and carcinomas along the upper part of the gastrointestinal tract. Activity and antigen levels of both activators were determined in homogenates of endoscopically obtained biopsies from normal and carcinomatous tissues. Although the concentrations of tissue type plasminogen activator and urokinase type plasminogen activator in normal squamous epithelium of the oesophagus were low compared with those in columnar epithelium from the stomach, the urokinase type plasminogen activator/tissue type plasminogen activator antigen ratio of the different locations showed hardly any difference. Significant but heterogeneous increases were found in urokinase type plasminogen activator concentrations of biopsy specimens originating from carcinomas of both epithelial cell types. A decrease in tissue type plasminogen activator concentrations, as found in human colon carcinomas, could only be shown in carcinomas of columnar epithelium origin but not in squamous cell carcinomas of the oesophagus. The increase of urokinase type plasminogen activator and urokinase type plasminogen activator/tissue type plasminogen activator antigen ratio and the decrease of tissue type plasminogen activator in the carcinomas did not show a significant correlation with known prognostic determinants as differentiation grade, TNM classification, intestinal metaplasia, inflammation, and ulceration. The heterogeneous increase of urokinase type plasminogen activator in oesophageal and stomach carcinomas, together with the recently described association of urokinase type plasminogen activator in tissue extracts of breast carcinomas with aggressiveness and prognosis, may be relevance to prognostic studies, may be of relevance to prognostic studies in oesophageal and gastric cancer.     

Tumour-associated fibrinolysis: the prognostic relevance of plasminogen activators uPA and tPA in human breast cancer

Prognostic variables in breast cancer are urgently needed to individualize adjuvant cytotoxic therapy, especially in those patients where metastases in the lymph nodes have not been detected (node-negative disease). So far histomorphological criteria, the determination of receptors for steroid hormones or EGF (epidermal growth factor), the protease cathepsin D or DNA-ploidy are used to distinguish between low- and high-risk patients. High-risk patients have a higher incidence of recurrences and/or shorter overall survival after surgery of the primary tumour than low-risk patients. High-risk patients (node-positive; hormone-receptor-negative) would receive adjuvant hormone therapy or chemotherapy. In the node-negative patient, adjuvant therapy is only recommended if a high content of cathepsin D and aneuploidy of the tumour (or high S-phase in diploid tumours) has been diagnosed. Determination of cathepsin D in tumour extracts as a variable in breast cancer patients is based on the fact that invasion and metastasis is correlated with elevated levels of tumour-associated proteases such as cathepsins B and D, collagenase IV and plasminogen activators. The urokinase-type plasminogen activator (uPA) which is secreted by tumour cells as an enzymatically inactive proenzyme (pro-uPA) seems to play a key role in mediating tumour cell invasion in cancer tissues. Receptor-bound uPA converts enzymatically inactive plasminogen into the serine protease plasmin which then degrades the extracellular matrix surrounding the tumour cells (tumour stroma). We localized pro-uPA/uPA immunohistochemically in paraffin-embedded formalin-fixed breast cancer tissue sections. Pro-uPA/uPA was detected in the cytoplasm and on the plasma membrane of the tumour cells reflecting receptor-bound pro-uPA/uPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of C1 inactivator and other proteinase inhibitors in euglobulin fractions and their influence on fibrinolytic activity.

Considerable amounts of C1 inactivator and inter-alpha-trypsin inhibitor pecipitate during euglobulin fractionation of human plasma. The amount precipitated depends on the ionic strength and the pH during the fractionation procedure. In contrast, alpha1-anti-trypsin, alpha2-macroglobulinand antithrombin II are present in euglobulin fractions in trace amounts only. The fibrinolytic activity of the euglobulin fractions is inhibited by the endogenous C1 inactivator, particularly as shown by comparison of normal and hereditary angioneurotic edema (HANE) plasma.     

Plasminogen activator inhibitor: a risk factor for myocardial infarction in diabetic patients.

OBJECTIVE--To determine whether diabetic patients admitted with acute myocardial infarction have impaired fibrinolytic activity due to raised plasminogen activator inhibitor compared with non-diabetic patients. SETTING--A district general hospital. PATIENTS--90 non-diabetic and 38 diabetic patients admitted with acute myocardial infarction. RESULTS--Both plasminogen activator inhibitor activity and antigen were significantly higher in diabetic than in non-diabetic patients (24.7 (6.8) v 18.5 (6.8) AU/ml; p = 0.0001 and 64.2 (range 13.1 to 328.8) v 38.5 (range 10.9 to 173.7 ng/ml; z = 3.3; p = 0.0008) with a positive correlation between activity and antigen (rs = 0.51; p = 0.0001). In both groups, activity and antigen concentrations were significantly higher than in diabetic and non-diabetic subjects without coronary artery disease (p = 0.002 to 0.0001 for each comparison). Plasminogen activator inhibitor activity correlated significantly with admission plasma glucose (r = 0.32; p = 0.0001), glycated haemoglobin (r = 0.32; p = 0.0001), admission plasma insulin (rs = 0.48; p = 0.001), and Killip grade of heart failure both on admission (rs = 0.27; p = 0.001) and on discharge (rs = 0.22; p = 0.006), but not with cumulative creatine kinase MB isoenzyme release (rs = -0.08). There were similar but weaker correlations between tissue plasminogen activator antigen and admission plasma glucose, glycated haemoglobin, and insulin. In 18 patients (12 non-diabetic and six diabetic) plasminogen activator inhibitor activity was measured between six and 12 months (8.3 (1.6)) after the acute infarct and remained similar to activity on admission (24.8 (1.9) AU/ml (NS) for diabetic and 17.9 (6.9) AU/ml (NS) for non-diabetic patients) and was still significantly higher in diabetic than in non-diabetic patients (p = 0.007). CONCLUSION--These results show that diabetic patients have higher plasminogen activator inhibitor activity than non-diabetic patients both on admission with acute myocardial infarction and at follow up six to 12 months later. Raised plasminogen activator inhibitor activity may predispose diabetic patients to myocardial infarction and may also impair pharmacological and spontaneous reperfusion after acute myocardial infarction thus contributing to the poor outcome in these subjects.     

Snake venom activators of factor X: an overview

Activators of blood coagulation factor X have been described in the venom of many snake species belonging to the genus Viperidae and Crotalidae as well as from a few Elapid species. Based on the structural and functional properties of purified activating principles, factor X activators are either metalloproteases or serine proteases. The best known activator is RVV-X from Russell's viper (Daboia russelli), a metalloprotease consisting of a heavy chain containing the catalytic domain and two light chains which share homology with C-type lectins and which are thought to exert a regulatory function in the Ca(2+)-dependent activation of factor X. This activator is also one of the best examples of the use of exogenous activators in coagulation research and in addition it is used in many diagnostic research kits. In this paper, an overview is given of the structural and functional properties of snake venom factor X activators thus far described in the literature.     

Synergistic fibrinolysis: combined effects of plasminogen activators and an antibody that inhibits alpha 2-antiplasmin.

To improve the efficacy of plasminogen activators, we produced a monoclonal antibody (RWR) that inhibits human alpha 2-antiplasmin (alpha 2AP). In addition to inhibiting alpha 2AP in plasma, RWR binds to and inhibits fibrin cross-linked alpha 2AP and reproduces the "spontaneous" clot lysis that is the hallmark of human alpha 2AP deficiency. By inhibiting the inactivation of plasmin by alpha 2AP, RWR interacts synergistically with plasminogen activators to increase the potency (for 50% clot lysis) of urokinase by 80-fold, tissue plasminogen activator by 27-fold, and streptokinase by 20-fold. Yet, for a given amount of fibrinolysis, the combination of RWR and lower doses of plasminogen activator leads to less fibrinogen consumption than is obtained with higher, equipotent doses of plasminogen activator alone. These results suggest a strategy for increasing the efficacy of plasminogen activators. More generally, this approach to amplifying enzymatic activity by immunoneutralizing an inhibitor may be useful in other biologic processes that are rigidly governed by inhibitors.     

A direct oral anticoagulant edoxaban accelerated fibrinolysis via enhancement of plasmin generation in human plasma: dependent on thrombin-activatable fibrinolysis inhibitor

Journal of Thrombosis and Thrombolysis - A direct oral anticoagulant, edoxaban, is as effective as vitamin K antagonists for the treatment of venous thromboembolism (VTE). However, the mechanism...