Sensitive and direct assays for functionally active plasminogen activators (tissue-type and urokinase-type) in plasma

A previously described bioimmunoassay for tissue-type plasminogen activator (t-PA) has been modified in terms of antibody concentration and conditions of incubation to achieve a sensitivity range of approximately 5-500 IU/L of t-PA. A similar assay has been developed for urinary-type plasminogen activator (U-PA), also known as urokinase (UK), achieving a sensitivity range of approximately 5-500 X 10(-1) IU/L. The direct sensitive t-PA assay has been demonstrated to be a quantitative alternative to the traditional semiquantitative euglobulin clot lysis time (ECLT) assay, with correlation coefficients of 0.967 and 0.914, depending on the laboratory where the ECLT was carried out. This assay has the added advantages of avoiding loss of t-PA or UK during the formation of the euglobulin fraction and of calibration in terms of International Standards for both t-PA and UK. With both these assays, it has been demonstrated that the enhanced fibrinolytic activity observed after exercise results from increased levels of t-PA while the level of UK in plasma remains unaltered. Free t-PA (10-60 IU/L) has been demonstrated in resting plasma, whereas the level of free UK activity was 50-100 IU/L. Venous occlusion of matched groups of subjects with and without deep venous thrombosis (DVT) showed a wide variation of response in t-PA levels in each group and no change in UK levels. There was no difference between the DVT and non-DVT groups, and this suggests that the notion of the "nonresponder" being more prone to thrombosis needs to be reexamined.     

Deleterious effects of plasminogen activators in neonatal cerebral hypoxia-ischemia

The immature brains of newborns often respond differently from the brains of adults when exposed to similar insults. Previous studies have indicated that although hypoxia-ischemia (HI) induces persistent thrombosis in adult brains, it only modestly impairs blood perfusion in newborn brains. Here, we used the Vannucci model of HI encephalopathy to study age-related responses to cerebral HI in rat pups. We found that HI triggered fibrin deposition and impaired blood perfusion in both neonatal and adult brains. However, these effects were only transient in neonatal brains (<4 hours) and were accompanied by acute induction of both tissue-type and urinary-type plasminogen activators (tPA and uPA), which was not observed in adult brains subjected to the same insult. Interestingly, activation of the plasminogen system persisted up to 24 hours in neonatal brains, long after the clearance of fibrin-rich thrombi. Furthermore, astrocytes and macrophages outside blood vessels expressed tPA after HI, suggesting the possibility of tPA/plasmin-mediated cytotoxicity. Consistent with this hypothesis, injection of alpha2-antiplasmin into cerebral ventricles markedly ameliorated HI-induced damage to neurofilaments and white matter oligodendrocytes, providing a dose-response reduction of brain injury after 7 days of recovery. Conversely, ventricular injection of tPA increased HI-induced brain damage. Together, these results suggest that tPA/plasmin induction, which may contribute to acute fibrinolysis, is a critical component of extravascular proteolytic damage in immature brains, representing a new therapeutic target for the treatment of HI encephalopathy.     

Immunomodulatory effects of plasminogen activators on hepatic fibrogenesis

Tissue-type plasminogen activators (tPA) and urokinase-type plasminogen activators (uPA) are involved in liver repair. We examined the potential immunomodulatory actions of uPA, tPA and uPA-receptor (uPAR) in carbon-tetrachloride-induced hepatic fibrosis in wild-type (WT), tPA-/-, uPA-/- and uPAR-/- mice. Carbon-tetrachloride treatment increased fibrosis in four groups but significantly less in three knock-out models. Serum cytokines and intrahepatic T cells elevated significantly following fibrosis process in WT animals but not in the knock-out groups. In culture, uPA increased lymphocyte proliferation significantly in WT and uPA-/- but not uPAR-/- animals. Following uPA exposure in vivo, there was CD8 predominance. To isolate uPA's effect on lymphocytes, WT mice were irradiated sublethally and then reconstituted with WT or uPA-/- lymphocytes. In these animals fibrosis was decreased and T cells were reduced in the uPA-/- recipients. Based on these data we postulate that plasminogen activators affect fibrosis in part by liver-specific activation of CD8 subsets that govern the fibrogenic activity of hepatic stellate cells.     

Expression of urokinase-type plasminogen activator by rat pulmonary alveolar epithelial cells

Intra-alveolar fibrin deposition accompanies many forms of inflammatory lung injury. Appropriate clearance of this fibrin matrix is important for normal healing and remodeling. The local generation of plasmin by the action of plasminogen activators (PAs) represents a pivotal step in the fibrinolytic process. To investigate whether the alveolar epithelium plays a role in the modulation of intra-alveolar fibrinolysis, we have studied PA regulation by rat pulmonary alveolar epithelial cells. We have found large quantities of PA activity both in conditioned media and cell lysates from epithelial monolayers in culture. Casein-plasminogen zymography reveals that this PA activity migrates as a tight doublet with an apparent mol wt of 45 kD, clearly distinct from rat tissue-type PA (tPA, greater than 68 kD). Analysis of freshly isolated type II alveolar epithelial cells demonstrates readily measurable PA activity in cell lysates, as well as expression of urokinase-type PA (uPA) mRNA on Northern blot analysis. Upregulation of PA activity occurs progressively with time in culture as the alveolar epithelial cells lose type II cell characteristics and become more flattened. Stimulation of alveolar epithelial cell monolayers with lipopolysaccharide or tumor necrosis factor increases levels of secreted PA activity. The relative abundance of uPA mRNA was shown to change in parallel with PA activity during in vitro differentiation or after exposure to inflammatory mediators. Thus, alveolar epithelial cells are likely an important source of uPA in the lung, the expression of which is influenced by the state of cellular differentiation as well as the presence of inflammatory mediators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasminogen mediates the pathological effects of urokinase-type plasminogen activator overexpression

Increased expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) is associated with different pathological conditions. Both uPAR-mediated signaling and plasmin-catalyzed extracellular proteolysis may contribute to pathogenesis. To evaluate the involvement of plasminogen in such circumstances, we have taken advantage of transgenic mouse models in which overexpression of uPA and/or uPAR in enamel epithelium, basal epidermis, and hair follicles leads to a pathological phenotype; uPA transgenic mice have chalky-white incisors and, when uPAR is co-expressed, develop extensive alopecia, epidermal thickening, and subepidermal blisters. We report here that when these transgenic mice were backcrossed into a plasminogen-deficient (Plg-/-) background, the dental and skin phenotypes appeared completely normal. Heterozygous Plg+/- transgenic mice exhibited a haplo-insufficiency, with an intermediate or normal phenotype. These results do not argue in favor of a role for uPAR-mediated signaling in our experimental model; rather, they demonstrate an essential, dose-dependent, requirement for plasminogen in uPA-mediated tissue alterations. They also support the hypothesis that plasminogen could play a part in certain skin diseases.     

A study of the activation of fibrin-bound plasminogen by tissue-type plasminogen activator,single chain urokinase and sequential combinations of the activators

The efficacy of tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) to activate fibrin-bound plasminogen was determined. With the use of a solid-phase model of intact and plasmin-degraded fibrin, the initial rate, Vi (fmol of plasmin/min), of plasmin generation by t-PA, scu-PA and two chain u-PA (tcu-PA) and the ratio, R (mol of plasmin generated/mol of activator) were determined as indicators of the efficiency of plasminogen activation. [Glu)t-plasminogen bound to intact fibrin was most efficiently activated by t-PA (Vi=0.843 and R=4.08), while scu-PA and tcu-PA produced markedly lower activation coefficients (Vi=0.011 and 0.023, and R=0.13 and 0.50 respectively). In contrast, when [Glu]¹-plasminogen was bound to the degraded surface, it became activatable with equivalent efficiencies by both t-PA and scu-PA (R=4.95 and 4.72, respectively). No evidence of Lys-plasminogen formation was observed. Rather, the selective activation of plasminogen bound to degraded fibrin by scu-PA is consistent with a particular interaction of Glu-plasminogen with carboxy-terminal lysine residues on the degraded surface. The consequences of this interaction of scu-PA with plasminogen bound to degraded fibrin on sequential activations of plasminogen by both t-PA and scu-PA were further explored. While the activation by scu-PA and then t-PA produced only an additive effect on plasmin generation, the opposite combination induced a greater than additive affect; the concentration of activators needed to produce 50% of plasmin generation was 70pM for the scu-PA/t-PA combination; in contrast, the amount required to produce a similar degree of activation with the opposite combination (t-PA/scu-PA) was only 17 pM. These results indicate that the potentiation of the scu-PA activity requires both the previous generation of plasmin by t-PA, and plasminogen bound to carboxy-terminal lysine residues of the degraded fibrin surface as a substrate.     

Expression of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen activator inhibitor-1 and -2 in hepatocellular carcinoma

It has become more and more clear in recent decades that the plasminogen activation system, which includes urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), plasminogen activator inhibitor (PAI)-1 and PAI-2, plays a very important role in the aggressiveness of cancer. Using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), the expression of these four components of the uPA system was analyzed in 19 cases of hepatocellular carcinoma (HCC) and 18 cases of the adjacent non-cancer tissues which all had chronic active hepatitis with liver fibrosis or liver cirrhosis. Four cases of normal liver tissues, as controls for immunohistochemical stains, were obtained from the hepatectomized liver of patients with metastatic cancer in the liver. The positive rates of uPA, uPAR, PAI-1 and PAI-2 for immunohistochemical stains in cancer tissues were 78.9, 68.4, 57.9 and 31.6%, respectively. Positive signals were mainly distributed in the cytoplasm of the cancer and in stromal cells. Moreover, the strong stains were chiefly located in the invasive front of the cancer cells. No specific stain was detected in four cases of normal liver tissues. In ELISA, there were significant differences between cancer and non-cancer tissues in concentration of uPA, uPAR and PAI-1 (P < 0.0003, 0.0024 and 0.01, respectively), but there was no significant difference in that of PAI-2 (P = 0.37). These results suggest that uPA, uPAR and PAI-1 are related to invasion of HCC.     

Interaction of tissue-type plasminogen activator with fibronectin and fibronectin fragments

In this paper we present data on the interaction, in solution, of the tissue-type plasminogen activator (t-PA) with fibronectin (FN) and its degradation products (FNdp). A cross radial caseinolytic assay (CRACA) was developed for the evaluation of the effect of the FN and/or FNdp on t-PA and urokinase plasminogen activator (u-PA) activity. A directional caseinolysis was observed when t-PA or u-PA were tested in the proximity of FNdp; no directionality was observed when intact FN or BSA were used. After incubation of t-PA, but not u-PA, with FNdp, PA activity at 170, 150, 100 and 30 kDa was detected by SDS-PAGE followed by zymography. The incubation of intact FN with t-PA gives rise to two forms of 500 and 150 kDa after prolonged incubation of the zymograms; no higher MW forms appear when u-PA substitutes t-PA.The immunoblotting analysis of the mixtures of t-PA and FN or FNdp with anti-t-PA or anti-FN sera showed that intact FN and some of its fragments interact with t-PA, giving rise to complexes recognized by both antisera and resistant to SDS-PAGE. Similar complexes are also evident, in vivo, in biological fluids like human plasma cryoprecipitates (cryos).     

组织型纤溶酶原激活物在兔髂动脉损伤后平滑肌细胞内的表达

为进一步探讨动脉粥样硬化形成及血管成形术后再狭窄的发生机制，应用光镜、显微-微机图像分析、发色底物显色法活性测定、原位分子杂交等技术，研究兔血管损伤后不同时间组织型纤溶酶原激活物（t-PA）在平滑肌细胞（SMC）的表达。研究发现：血管损伤后4天t-PA活性明显增加[（7．50±0．05）×10-2IU／mg蛋白]，此时SMC开始从中膜向内膜迁移。损伤后7天，t-PA活性降至近于正常水平[（2．50±0．37）×10-2IU／mg蛋白]。正常血管壁t-PAmRNA杂交阴性，血管损伤后4天，内弹力板下及内膜SMC有大量t-PAmRNA表达，7天时仅表达少量。说明t-PA可能在血管损伤后的SMC增生和迁移过程中起重要作用。     

Factors responsible for the differential procoagulant effects of diverse plasminogen activators in plasma

This study was designed to define the procoagulant effects of different plasminogen activators in recalcified plasma. Accordingly, a two-stage assay procedure was developed in which citrated plasma was recalcified and incubated for 1–7 min with streptokinase (SK), urokinase (UK), or tissue-plasminogen activator (t-PA). An aliquot of this first-stage plasma was added to second-stage plasma; procoagulant activity was measured as the acceleration of the clotting time compared with that in control first-stage plasma to which plasminogen activators had not been added. With this procedure, second-stage clotting times were significantly accelerated after activation of plasminogen compared with those in control plasma and were shortest with 1000 i.u./ml SK. The presence of increased thrombin activity in the recalcified citrated plasma was verified by measurement of increased concentrations of fibrinopeptide A, which were inhibited by addition of I U/ml hirudin or or U/ml heparin to plasma during incubation with the plasminogen activators. Activation of factor X was induced in plasma incubated with SK. The rate of activation of factor X was increased when thrombin was elaborated. Induction of procoagulant activity was found to be dependent on depletion of α₂-antiplasmin and on the presence of plasmin activity in plasma, and could be inhibited by aprotinin. Thus, procoagulant activity in response to pharmacologic activation of plasminogen is dependent on plasmin-mediated activation of factor X, and subsequent activation of prothrombin.     

The role of plasminogen activators in metastasis

It is proposed the tumour plasminogen activators play an essential role in the invasive and metastic process of malignancy.     

Effect of tissue-type plasminogen activator from calf kidney cell culture on hemostasis and fibrinolysis in experimental nephritis

Laboratory of Enzymic Fibrinolysis, Biological Faculty, M. V. Lomonosov Moscow University. Research Institute of Biomedical Technology, Ministry of Health of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR I. P. Ashmarin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 106, No. 10, pp. 424–426, October, 1988.     

Treatment of acute myocardial infarction with recombinant tissue-type plasminogen activator

In Ireland, to date, coronary thrombolytic therapy has been confined almost exclusively to the use of streptokinase. However, a large body of evidence suggests that, in comparison to streptokinase, the agent recombinant tissue-type plasminogen activator (rt-PA) may be more effective in lysing coronary thrombi and achieving coronary reperfusion and causes fewer disturbances of the coagulation system. With these considerations in mind, we undertook a study to explore the future potential role of rt-PA in our particular clinical practice. Sixteen patients presenting to our centre with clinical and ECG features suggestive of acute myocardial infarction were treated with rt-PA and heparin infusion within 3.8 +/- 1.3 (mean +/- SD) [range 0.6 - 5.3] hours of the onset of their symptoms. Reperfusion, as assessed by clinical, electrocardiographic and biochemical criteria, was achieved in 15 of these 16 patients. One patient developed reocclusion that was successfully treated with repeat thrombolytic therapy. Follow up coronary angiography, performed in eight patients, confirmed successful reperfusion in seven. One patient developed an intracranial haemorrhage. The result of this pilot study highlight the importance of considering thrombolytic therapy in all patients presenting with suspected acute myocardial infarction (AMI). Our observations also suggest that rt-PA is very effective in restoring myocardial perfusion in patients with AMI who present at an early stage. As with all thrombolytic agents, it may be associated with haemorrhagic complications. Determination of the precise role of rt-PA, as opposed to other thrombolytic agents, awaits the results of ongoing clinical trials.